BDBM573947 US11453697, Example 132 2-amino-9-[(5S,7R,8R,12aR,14R,15S,15aR)- 14-(6-amino-9H-purin-9-yl)-15-fluoro-2,10- dihydroxy-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-oe (Diastereomer 3)
- Moser, PC; Bergis, OE; Jegham, S; Lochead, A; Duconseille, E; Terranova, JP; Caille, D; Berque-Bestel, I; Lezoualc'h, F; Fischmeister, R; Dumuis, A; Bockaert, J; George, P; Soubrié, P; Scatton, B J Pharmacol Exp Ther 302: 731-41 (2002)
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- Harada, K; Mizukami, J; Kadowaki, S; Matsuda, I; Watanabe, T; Oe, Y; Kodama, Y; Aoki, K; Suwa, K; Fukuda, S; Yata, S; Inaba, T Bioorg Med Chem Lett 28: 1228-1233 (2018)
- ChEMBL_1363651 (CHEMBL3293393) Inhibition of TRKB (unknown origin) by off-chip mobility shift assay
- ChEMBL_1363660 (CHEMBL3293402) Inhibition of ROS1 (unknown origin) by off-chip mobility shift assay
- ChEMBL_1451317 (CHEMBL3363336) Inhibition of Mer (unknown origin) by Off-chip Mobility Shift Assay
- ChEMBL_1451318 (CHEMBL3363337) Inhibition of Flt3 (unknown origin) by Off-chip Mobility Shift Assay
- ChEMBL_1451337 (CHEMBL3363356) Inhibition of Axl (unknown origin) by Off-chip Mobility Shift Assay
- ChEMBL_1451338 (CHEMBL3363357) Inhibition of Tyro3 (unknown origin) by Off-chip Mobility Shift Assay
- ChEMBL_1451339 (CHEMBL3363358) Inhibition of TRKA (unknown origin) by Off-chip Mobility Shift Assay
- ChEMBL_1451340 (CHEMBL3363359) Inhibition of TRKC (unknown origin) by Off-chip Mobility Shift Assay
- ChEMBL_1451341 (CHEMBL3363360) Inhibition of QIK (unknown origin) by Off-chip Mobility Shift Assay
- ChEMBL_1451342 (CHEMBL3363361) Inhibition of SLK (unknown origin) by Off-chip Mobility Shift Assay
- ChEMBL_1451343 (CHEMBL3363362) Inhibition of Nuak1 (unknown origin) by Off-chip Mobility Shift Assay
- ChEMBL_1451344 (CHEMBL3363363) Inhibition of Kit (unknown origin) by Off-chip Mobility Shift Assay
- ChEMBL_1451345 (CHEMBL3363364) Inhibition of Met (unknown origin) by Off-chip Mobility Shift Assay
- ChEMBL_1457242 (CHEMBL3370286) Inhibition of human JNK2 kinase by Off-chip mobility shift assay
- ChEMBL_1457243 (CHEMBL3370287) Inhibition of human JNK3 kinase by Off-chip mobility shift assay
- ChEMBL_1457239 (CHEMBL3370283) Inhibition of human p38beta MAP kinase by Off-chip mobility shift assay
- ChEMBL_1721260 (CHEMBL4136260) Inhibition of recombinant FGFR1 (unknown origin) by off-chip mobility shift assay
- ChEMBL_1721261 (CHEMBL4136261) Inhibition of recombinant PDGFRbeta (unknown origin) by off-chip mobility shift assay
- ChEMBL_1721262 (CHEMBL4136262) Inhibition of recombinant VEGFR2 (unknown origin) by off-chip mobility shift assay
- ChEMBL_1721295 (CHEMBL4136295) Inhibition of recombinant RET (unknown origin) by off-chip mobility shift assay
- ChEMBL_590261 (CHEMBL1052796) Inhibition of human sphingosine kinase 1 by off chip mobility shift assay
- H6PD Activity Assay HEK293 H6PD-OE and HEK293 pellets were lysed and the lysate was quantified with the Bradford protein assay. Reaction mix containing galactose-6-phosphate (Gal6P) was combined with increasing concentrations of PARPi or DMSO. As an additional control, reaction mix without substrate was combined with HEK293 H6PD-OE lysate. Lysate from HEK293 or HEK293 H6PD-OE was added to each well to start the reactions. The absorbance at 340 nM (NADPH) was measured for 90 min at 10 min intervals. The 60 min data, which were within the linear range, was used for analysis. Background absorbance was subtracted and the H6PD activity of theHEK293 H6PD-OE lysates treated with PARPi was normalized to the DMSO control.
- ChEMBL_1721296 (CHEMBL4136296) Inhibition of recombinant c-KIT (unknown origin) by off-chip mobility shift assay
- ChEMBL_1457234 (CHEMBL3370278) Inhibition of full length human p38alpha MAP kinase by Off-chip mobility shift assay
- ChEMBL_1789491 (CHEMBL4261225) Inhibition of human ERG expressed in CHO-S1 cells by automated planar chip-based electrophysiology
- ChEMBL_2129136 (CHEMBL4838565) Binding affinity to FGF2 (unknown origin) by sensor chip immobilization based surface plasmon resonance analysis
- ChEMBL_2307700 Binding affinity to CM5 sensor chip immobilized human HSP90alpha assessed as dissociation constant by SPR analysis
- ChEMBL_2426653 Binding affinity to CM5 sensor chip immobilized human B3GAT3 assessed as dissociation constant by SPR analysis
- ChEMBL_2476529 Binding affinity to CM5 chip-immobilized NLRP3 (unknown origin) assessed as dissociation constant by SPR analysis
- ChEMBL_2476531 Binding affinity to CM5 chip-immobilized LC3B (unknown origin) assessed as dissociation constant by SPR analysis
- ChEMBL_2501537 Binding affinity to CM5 chip immobilized NLRP3 (unknown origin) assessed as dissociation constant by SPR analysis
- ChEMBL_2423597 Binding affinity to CM5 chip immobilized recombinant human PD-L1 assessed as dissociation constant by SPR analysis
- ChEMBL_2457174 Binding affinity to CM5 sensor chip immobilized human recombinant TMPRSS2 assessed as dissociation constant by SPR analysis
- ChEMBL_2015966 (CHEMBL4669544) Binding affinity to CM5 chip immobilized beta tubulin (unknown origin) assessed as thermodynamic constants by SPR assay
- ChEMBL_2127693 (CHEMBL4837038) Binding affinity to chip-immobilized recombinant human SIRT1 measured after 300 secs by surface plasmon resonance method
- ChEMBL_2127694 (CHEMBL4837039) Binding affinity to chip-immobilized recombinant human SIRT2 measured after 300 secs by surface plasmon resonance method
- ChEMBL_2127695 (CHEMBL4837040) Binding affinity to chip-immobilized recombinant human SIRT3 measured after 300 secs by surface plasmon resonance method
- ChEMBL_2127696 (CHEMBL4837041) Binding affinity to chip-immobilized recombinant human SIRT5 measured after 300 secs by surface plasmon resonance method
- ChEMBL_2163234 (CHEMBL5048095) Inhibition of streptavidin sensor chip immobilized biotinylated human PRMT5/MEP50 assessed as dissociation constant by SPR analysis
- ChEMBL_2326260 Binding affinity to CM5 chip immobilised recombinant mouse perforin expressed in baculovirus-infected Sf21 cells by SPR analysis
- ChEMBL_2358457 Binding affinity to PD-L1 (unknown origin) immobilized on carboxy chip assessed as dissociation constant by SPR analysis
- ChEMBL_2425070 Binding affinity to CM3 chip-immobilized full-length human galectin-1 assessed as dissociation constant by SPR analysis
- ChEMBL_2425071 Binding affinity to CM3 chip-immobilized full-length human galectin-3 assessed as dissociation constant by SPR analysis
- ChEMBL_2425072 Binding affinity to CM3 chip-immobilized full-length mouse galectin-1 assessed as dissociation constant by SPR analysis
- ChEMBL_2425073 Binding affinity to CM3 chip-immobilized full-length mouse galectin-3 assessed as dissociation constant by SPR analysis
- ChEMBL_2466588 Binding affinity to CM5 sensor chip-immobilized recombinant PRDX1 (unknown origin) assessed as dissociation constant by SPR analysis
- ChEMBL_2064148 (CHEMBL4719401) Binding affinity to STAT3 (unknown origin) expressed in Escherichia coli by CM5 sensor chip immobilization based SPR assay
- ChEMBL_2135955 (CHEMBL4845565) Inhibition of CM5 chip immobilized Influenza A virus (A/Viet Nam/1203/2004(H5N1)) HA by SPR method
- ChEMBL_2434473 Binding affinity to CM5 sensor chip immobilized MYC bHLHZip domain (unknown origin) assessed as dissociation constant by SPR analysis
- ChEMBL_2457177 Binding affinity to CM5 sensor chip immobilized SARS-CoV-2 3CL protease assessed as dissociation constant by SPR analysis
- ChEMBL_2457625 Binding affinity to His-tagged PCSK9 (unknown origin) coupled to CM5 chip assessed as dissociation constant by SPR analysis
- ChEMBL_2457626 Binding affinity to His-tagged LC3B (unknown origin) coupled to CM5 chip assessed as dissociation constant by SPR analysis
- ChEMBL_2492480 Binding affinity to CM5 chip-immobilized recombinant ROR-gamma LBD (unknown origin) assessed as dissociation constant by SPR analysis
- ChEMBL_2498946 Binding affinity to CM5 chip immobilized KEAP1 Kelch domain (unknown origin) assessed as dissociation equilibrium constant by SPR analysis
- ChEMBL_2579517 Binding affinity to BTZO-12 sensor chip immobilized human MIF expressed in Escherichia coli BL21 (DE3) by SPR analysis
- ChEMBL_1462508 (CHEMBL3398981) Binding affinity to basic fibroblast growth factor (unknown origin) using sugar chip immobilized compound by surface plasmon resonance method
- ChEMBL_1556909 (CHEMBL3771934) Inhibition of recombinant human KDAC8 using FAM-labelled substrate B incubated for 60 mins by microfluidic chip-based assay
- ChEMBL_1556911 (CHEMBL3771936) Inhibition of recombinant human KDAC1 using FAM-labelled substrate A incubated for 60 mins by microfluidic chip-based assay
- ChEMBL_2153278 (CHEMBL5037825) Binding affinity to CM5 sensor chip immobilized SARS-CoV-2 spike glycoprotein assessed as dissociation constant by SPR analysis
- ChEMBL_2317262 Binding affinity to CM-5 sensor chip immobilized Cdc42 (unknown origin) assessed as dissociation constant by surface plasmon resonance assay
- ChEMBL_2360355 Binding affinity to CM5 sensor chip immobilized human recombinant extracellular domain PD-L1 assessed as dissociation constant by SPR analysis
- ChEMBL_1556910 (CHEMBL3771935) Inhibition of recombinant human KDAC3 using FITC-p53 acetylated peptide substrate incubated for 60 mins by microfluidic chip-based assay
- ChEMBL_2147108 (CHEMBL5031454) Binding affinity to NTA sensor chip immobilized recombinant human TLR4/MD2 assessed as dissociation constant by surface plasmon resonance analysis
- ChEMBL_2155219 (CHEMBL5039879) Binding affinity to CM5 sensor chip immobilized recombinant full-length TRBP (unknown origin) assessed as dissociation constant by SPR analysis
- ChEMBL_611883 (CHEMBL1070838) Binding affinity to mouse MAGd1-3Fc expressed in CHO-Lec 3.2.8.1 cells under HEPES condition by CM5 chip SPR analysis
- ChEMBL_611884 (CHEMBL1070839) Binding affinity to mouse MAGd1-3Fc expressed in CHO-Lec 3.2.8.1 cells under NaCl condition by CM5 chip SPR analysis
- ChEMBL_752073 (CHEMBL1786237) Inhibition of human Pim1 using 5-FAM-RSRHSSYPAGT-CONH2 as substrate after 45 mins by off-chip mobility shift method
- ChEMBL_752074 (CHEMBL1786238) Inhibition of human Pim2 using 5-FAM-RSRHSSYPAGT-CONH2 as substrate after 90 mins by off-chip mobility shift method
- ChEMBL_1556908 (CHEMBL3771933) Inhibition of recombinant human KDAC6 using FITC-histone 4 acetylated peptide substrate incubated for 60 mins by microfluidic chip-based assay
- ChEMBL_2196561 (CHEMBL5109077) Binding affinity to CM5 sensor chip immobilised EED (unknown origin) assessed as dissociation constant incubated for 270 secs by SPR analysis
- ChEMBL_2436694 Inhibition of wild type N-terminal His tagged ABL kinase (unknown origin) using fluorescence-labelled substrate by off-chip mobility shift assay
- ChEMBL_611882 (CHEMBL1070837) Binding affinity to mouse MAGd1-3Fc expressed in CHO-Lec 3.2.8.1 cells under HBS-EP condition by CM5 chip SPR analysis
- ChEMBL_611886 (CHEMBL1070841) Binding affinity to mouse MAGd1-3Fc expressed in CHO-Lec 3.2.8.1 cells under HBS-EP condition by CM4 chip SPR analysis
- ChEMBL_2106831 (CHEMBL4815506) Binding affinity to CM5 sensor chip immobilized recombinant human AChE assessed as dissociation constant at 298.15 K by surface plasmon resonance assay
- ChEMBL_2234096 (CHEMBL5147868) Binding affinity to CM5 sensor chip-immobilized mouse Piezo1 (1 to 2190 residues) assessed as dissociation constant by real-time SPR assay
- ChEMBL_2298935 Binding affinity to CMS sensor chip immobilized MMP-14 catalytic domain (unknown origin) assessed as equilibrium dissociation constant by surface plasmon resonance analysis
- ChEMBL_2324716 Binding affinity to Keap1 (unknown origin) assessed as dissociation constant incubated for 200 sec by sensor chip immobilization based surface plasmon resonance analysis
- ChEMBL_2353672 Binding affinity to His-tagged recombinant human TIM3 expressed in HEK293 cells assessed as dissociation constant by sensor chip immobilization based SPR analysis
- ChEMBL_2357335 Binding affinity to biotinylated VHL/Elongin B/Elongin C (unknown origin) immobilized in SA sensor chip assessed as dissociation constant by SPR analysis
- ChEMBL_2360362 Binding affinity to CM5 sensor chip immobilized recombinant human PD-L1 ectodomain (18 to 239 residues) assessed as dissociation constant by SPR assay
- ChEMBL_2371091 Binding affinity to CMS sensor chip immobilized His-SUMO-tagged mouse NLRP3 expressed in Escherichia coli assessed as dissociation constant by SPR analysis
- ChEMBL_2372976 Binding affinity to CM5 sensor chip immobilized C-terminal FLAG tagged full length human HAT1/Rbap46 assessed as dissociation constant by SPR analysis
- ChEMBL_2547122 Inhibition of PAK1 (unknown origin) using 5-FAM-KPDRKKRYTVVGNPY-amide as substrate incubated for 120 mins by caliper off-chip mobility shift assay
- ChEMBL_2547123 Inhibition of PAK4 (unknown origin) using 5-FAM-AhxKKRNRRLSVA-amide as substrate incubated for 90 mins by caliper off-chip mobility shift assay
- ChEMBL_940961 (CHEMBL2330651) Inhibition of ROCK1 (unknown origin) using FITC-labeled peptide as substrate after 1 hr by caliper off-chip incubation mobility shift assay
- ChEMBL_1466072 (CHEMBL3404455) Inhibition of GST-His6-tagged recombinant human c-Met kinase domain (956 to 1390) after 60 mins by anoff-chip mobility shift assay
- ChEMBL_2172134 (CHEMBL5057268) Binding affinity to CM-5 sensor chip immobilized beta-amyloid monomer (1 to 42) (unknown origin) assessed as dissociation constant by SPR analysis
- ChEMBL_2248299 (CHEMBL5162509) Binding affinity to CM5 sensor chip immobilized human NPR-C (27 to 541 residues) assessed as dissociation constant by surface plasmon resonance analysis
- ChEMBL_611885 (CHEMBL1070840) Binding affinity to mouse MAGd1-3Fc expressed in CHO-Lec 3.2.8.1 cells under carboxymethyl-dextran sodium salt condition by CM5 chip SPR analysis
- ChEMBL_992394 (CHEMBL2445614) Binding affinity to human Arno Sec7 domain immobilized on CAP sensor chip after 15 mins by 1H 1D-NMR-700 MHz spectra analysis
- ChEMBL_1475747 (CHEMBL3424980) Inhibition of human recombinant N-terminal His6-tagged IGF-1R using fluorescence substrate after 80 mins by caliper off-chip incubation mobility shift assay
- ChEMBL_1552123 (CHEMBL3760460) Inhibition of GST-tagged human CK2alpha (1 to 391 residues) using CK2tide as substrate incubated for 1 hr by off-chip mobility shift assay
- ChEMBL_1552124 (CHEMBL3760461) Inhibition of GST-tagged human CK2alpha' (1 to 350 residues) using CK2tide as substrate incubated for 1 hr by off-chip mobility shift assay
- ChEMBL_2135950 (CHEMBL4845560) Binding affinity to CM5 chip immobilized Influenza A virus (A/Michigan/45/2015) (H1N1)) hemagglutin assessed as dissociation constant by surface plasmon resonance assay
- ChEMBL_2262113 (CHEMBL5217124) Binding affinity to human Sirt3 (118 to 399 residues) assessed as dissociation constant by short DNA nano levers based switchSENSE microfluid bio-chip analysis
- ChEMBL_2349714 Binding affinity to SA sensor chip immobilized N-terminal avi-tagged biotinylated TEAD2 YAP binding domain (unknown origin) assessed as binding constant by SPR analysis
- ChEMBL_2475936 Binding affinity to streptavidin sensor chip-immobilized biotinylated recombinant human NLRP3 NACHT domain extracted from Sf9 insect cells assessed as dissociation constant by SPR analysis
- ChEMBL_941085 (CHEMBL2330882) Inhibition of recombinant ROCK2 (unknown origin) using 5-FAM-labeled peptide as substrate after 1 hr by caliper off-chip incubation mobility shift assay
- ChEMBL_941087 (CHEMBL2330884) Inhibition of recombinant Akt3 (unknown origin) using 5-FAM-labeled peptide as substrate after 1 hr by caliper off-chip incubation mobility shift assay
- ChEMBL_941088 (CHEMBL2330885) Inhibition of recombinant Akt2 (unknown origin) using 5-FAM-labeled peptide as substrate after 1 hr by caliper off-chip incubation mobility shift assay
- ChEMBL_941089 (CHEMBL2330886) Inhibition of recombinant Akt1 (unknown origin) using 5-FAM-labeled peptide as substrate after 1 hr by caliper off-chip incubation mobility shift assay
- ChEMBL_1619578 (CHEMBL3861747) Inhibition of N-terminal GST tagged human TrkC catalytic domain (456 to 825 amino acids) using fluorescence-labeled substrate by off-chip mobility shift assay
- ChEMBL_2262111 (CHEMBL5217122) Binding affinity to human Sirt3 (118 to 399 residues) assessed as dissociation constant (Kon) by short DNA nano levers based switchSENSE microfluid bio-chip analysis
- ChEMBL_2262112 (CHEMBL5217123) Binding affinity to human Sirt3 (118 to 399 residues) assessed as dissociation constant (Koff) by short DNA nano levers based switchSENSE microfluid bio-chip analysis
- ChEMBL_2444314 Binding affinity to GST-tagged human FKBP51 expressed in Escherichia coli BL21 (DE3) immobilized in CM7 sensor chip assessed as equilibrium dissociation constant by SPR analysis
- ChEMBL_2444315 Binding affinity to GST-tagged human FKBP52 expressed in Escherichia coli BL21 (DE3) immobilized in CM7 sensor chip assessed as equilibrium dissociation constant by SPR analysis
- Mobility-Shift kinase assay Compounds of the examples provided herein were assessed for their ability to inhibit Syk kinase by utilizing Caliper Life Sciences' proprietary LabChip technology. The off-chip incubation mobility-shift kinase assay uses a microfluidic chip to measure the conversion of a fluorescent peptide substrate to a phosphorylated product. The reaction mixture, from a microtiter plate well, is introduced through a capillary sipper onto the chip, where the nonphosphorylated substrate and phosphorylated product are separated by electrophoresis and detected via laser induced fluorescence. The signature of the fluorescence signal over time reveals the extent of the reaction. The phosphorylated product migrates through the chip faster than the non-phosphorylated substrate, and signals from the two forms of the peptide appear as distinct peaks.
- ChEMBL_1792101 (CHEMBL4264020) Inhibition of full length human BTK (8 to 80 residues) using FITC-AHA-EEPLYWSFPAKKK-NH2 as substrate after 90 mins by off-chip mobility shift assay
- ChEMBL_2070088 (CHEMBL4725622) Inhibition of IKKbeta (unknown origin) assessed as substrate phosphorylation using IkappaBalpha as substrate incubated for 1 hr in presence of ATP by chip-based fluorescence assay
- ChEMBL_756735 (CHEMBL1805281) Binding affinity to eEF1A2 expressed in human MDA-MB-231 cells using 1 uM biotinylated compound immobilized on streptavidin sensor chip by surface plasmon resonance method
- ChEMBL_756745 (CHEMBL1805291) Binding affinity to eEF1A1 expressed in human MDA-MB-231 cells using 1 uM biotinylated compound immobilized on streptavidin sensor chip by surface plasmon resonance method
- ChEMBL_944710 (CHEMBL2340956) Inhibition of HDAC9 (unknown origin) assessed as fluorescence intensity measured after 60 mins incubation at room temperature by trypsin-free microfluidic lab-on-a-chip assay
- ChEMBL_944711 (CHEMBL2340957) Inhibition of HDAC7 (unknown origin) assessed as fluorescence intensity measured after 60 mins incubation at room temperature by trypsin-free microfluidic lab-on-a-chip assay
- ChEMBL_944712 (CHEMBL2340958) Inhibition of HDAC5 (unknown origin) assessed as fluorescence intensity measured after 60 mins incubation at room temperature by trypsin-free microfluidic lab-on-a-chip assay
- ChEMBL_944713 (CHEMBL2340959) Inhibition of HDAC3 (unknown origin) assessed as fluorescence intensity measured after 60 mins incubation at room temperature by trypsin-free microfluidic lab-on-a-chip assay
- ChEMBL_944714 (CHEMBL2340960) Inhibition of HDAC1 (unknown origin) assessed as fluorescence intensity measured after 60 mins incubation at room temperature by trypsin-free microfluidic lab-on-a-chip assay
- ChEMBL_944719 (CHEMBL2340965) Inhibition of HDAC8 (unknown origin) assessed as fluorescence intensity measured after 60 mins incubation at room temperature by trypsin-free microfluidic lab-on-a-chip assay
- ChEMBL_944720 (CHEMBL2340966) Inhibition of HDAC6 (unknown origin) assessed as fluorescence intensity measured after 60 mins incubation at room temperature by trypsin-free microfluidic lab-on-a-chip assay
- ChEMBL_944721 (CHEMBL2340967) Inhibition of HDAC4 (unknown origin) assessed as fluorescence intensity measured after 60 mins incubation at room temperature by trypsin-free microfluidic lab-on-a-chip assay
- ChEMBL_944722 (CHEMBL2340968) Inhibition of HDAC2 (unknown origin) assessed as fluorescence intensity measured after 60 mins incubation at room temperature by trypsin-free microfluidic lab-on-a-chip assay
- ChEMBL_2211562 (CHEMBL5124511) Binding affinity to CM5 sensor chip immobilized human PARP14 MD2 expressed in Escherichia coli BL21 (DE3) assessed as dissociation constant measured after 6 mins by SPR analysis
- ChEMBL_2337254 Binding affinity to CM5 chip-immobilised full-length recombinant SARS-CoV-2 3CL protease expressed in Escherichia coli BL21 (DE3) cells assessed as dissociation constant by SPR analysis
- ChEMBL_1799440 (CHEMBL4271732) Inhibition of human N-terminal GST-tagged FGFR1 (398 to 822 residues) cytoplasmic domain expressed in baculovirus expression system after 60 mins by off-chip mobility shift assay
- ChEMBL_1799441 (CHEMBL4271733) Inhibition of human N-terminal GST-tagged FGFR2 (399 to 821 residues) cytoplasmic domain expressed in baculovirus expression system after 60 mins by off-chip mobility shift assay
- ChEMBL_1799442 (CHEMBL4271734) Inhibition of human N-terminal GST-tagged FGFR3 (436 to 806 residues) cytoplasmic domain expressed in baculovirus expression system after 60 mins by off-chip mobility shift assay
- ChEMBL_1799443 (CHEMBL4271735) Inhibition of human N-terminal GST-tagged VEGFR2 (790 to 1356 residues) cytoplasmic domain expressed in baculovirus expression system after 60 mins by off-chip mobility shift assay
- ChEMBL_2045429 (CHEMBL4700128) Inhibition of BMX (unknown origin) preincubated with enzyme for 15 mins followed by substrate/ATP challenge for 90 mins by ProfilerPro plate based lab-chip EZ reader analysis
- ChEMBL_2214816 (CHEMBL5127948) Binding affinity to CM7 sensor chip immobilized full-length KDM3B (unknown origin) expressed in Sf9 insect cells assessed as dissociation constant incubated for 60 secs by SPR analysis
- ChEMBL_2251460 (CHEMBL5165670) Binding affinity to CM5 chip immobilized human MKK3 (20 to 347 residues) expressed in Escherichia coli BL21 (DE3) assessed as dissociation constants, KD by surface plasmon resonance analysis
- ChEMBL_2251495 (CHEMBL5165705) Binding affinity to CM5 chip immobilized human MKK3 (20 to 347 residues) expressed in Escherichia coli BL21 (DE3) assessed as dissociation constant, Kd by surface plasmon resonance analysis
- ChEMBL_2349719 Binding affinity to CM5 chip immobilised N-terminal histidine -tagged human TEAD4 (217 to 434 residues) expressed in Escherichia coli (DE3) cells assessed as dissociation constant by SPR assay
- ChEMBL_2045430 (CHEMBL4700129) Inhibition of Aurora A (unknown origin) preincubated with enzyme for 15 mins followed by substrate/ATP challenge for 90 mins by ProfilerPro plate based lab-chip EZ reader analysis
- ChEMBL_2211437 (CHEMBL5124386) Inhibition of BTK C481S mutant (unknown origin) expressed in baculovirus infected Sf9 insect cells incubated for 30 mins in presence of ATP by Microfluidic chip based mobility shift assay
- ChEMBL_2193370 (CHEMBL5105730) Binding affinity to CM-5 sensor chip immobilized His-tagged beta-catenin (134 to 668 residues) (unknown origin) expressed in Escherichia coli BL21 assessed as dissociation constant by SPR analysis
- ChEMBL_2211435 (CHEMBL5124384) Inhibition of BTK T474M mutant (unknown origin) expressed in baculovirus infected Trichoplusia ni pro cells incubated for 30 mins in presence of ATP by Microfluidic chip based mobility shift assay
- ChEMBL_2211438 (CHEMBL5124387) Inhibition of BTK C481R mutant (unknown origin) expressed in baculovirus infected Trichoplusia ni pro cells incubated for 30 mins in presence of ATP by Microfluidic chip based mobility shift assay
- ChEMBL_2211439 (CHEMBL5124388) Inhibition of BTK T474I mutant (unknown origin) expressed in baculovirus infected Trichoplusia ni pro cells incubated for 30 mins in presence of ATP by Microfluidic chip based mobility shift assay
- ChEMBL_2240949 (CHEMBL5155159) Inhibition of N-terminal GST-tagged human CK1epsilon (1 to 348 residues) expressed in baculovirus expression system in presence of peptide substrate and ATP by off-chip mobility shift assay
- Surface Plasmon Resonance (SP1R) As the device, Biacore T200 (manufactured by GE Healthcare) was used. VHL was obtained by co-expressing Elongin B and Elongin C with a Twin-Strep-tag added to the N-terminus. As the sensor chip, Sensor chip CM5 (manufactured by GE Healthcare) was used. First, Strep-Tactin XT (manufactured by GE Healthcare) was immobilized on the sensor chip by amine coupling. Thereafter, VHL was immobilized using the affinity of Strep-Tactin XT and Twin-Strep-tag, and VHL was further immobilized on the sensor chip by amine coupling. As the running buffer, a mixed solution of 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20, and 2% DMSO was used. Solutions of various compound concentrations of the test compounds (compounds described in Examples 1 to 162 above) were flowed on the sensor chip with VHL immobilized thereon at a flow rate of 30 uL/min under 25° C. conditions, and changes in mass on the sensor chip were observed. The Contact time and the Dissociation time were determined according to the properties of the compounds. For many compounds, 60 seconds and 120 seconds were respectively used. The observed data was analyzed using Biacore T200 Evaluation software Version 2.0 (manufactured by GE Healthcare) and the Kd value for VHL of each compound was calculated.
- ChEMBL_2064177 (CHEMBL4719430) Inhibition of 6xHis-tagged human FUBP1 expressed in HEK293T cells assessed as reduction in FUBP1 interaction with biotinylated NLC chip immobilized FUSE p21 oligonucleotide incubated for 21 hrs by SPR analysis
- ChEMBL_2214815 (CHEMBL5127947) Binding affinity to CM7 sensor chip immobilized partial-length JMJD1C (2274 to 2498 residues) (unknown origin) expressed in Escherichia coli assessed as dissociation constant incubated for 60 secs by SPR analysis
- ChEMBL_2235868 (CHEMBL5149640) Inhibition of MSK2 (unknown origin) FAM-PSKPAATRKRRWSAPESR-NH2 as substrate preincubated for 1 hr followed by activation with ERK2 and substrate addition measured after 2 hrs by microfluidic chip based electrophoresis
- ChEMBL_2434809 Binding affinity to CM5 chip immobilized N-terminal GST-tagged NSD3 PWWP1 domain (263 to 398 residues) (unknown origin) expressed in Escherichia coli BL21(DE3) assessed as dissociation constant by SPR analysis
- ChEMBL_1475748 (CHEMBL3424981) Inhibition of human recombinant full length C-terminal His6-tagged CDK2 expressed in baculovirus infected Sf21 insect cells using fluorescence substrate after 60 mins by caliper off-chip incubation mobility shift assay
- ChEMBL_2487484 Binding affinity to CM5 sensor chip immobilized N-terminal His tagged human SHIP2 Sam domain (1194 to 1258 residues) extracted from Escherichia coli BL21 (DE3) cells assessed as dissociation constant by SPR analysis
- ChEMBL_2352483 Binding affinity to CM5 chip immobilized GST-tagged mouse KEAP1 Kelch domain (322 to 624 residues) expressed in Escherichia coli BL21 (DE3) cells assessed as dissociation constant incubated for 60 sec by SPR assay
- ChEMBL_2444298 Inhibition of GST-tagged human CHIP expressed in Escherichia coli BL21 (DE3) using NH2-EDASRMEEVD-COOH peptide as substrate preincubated for 15 mins followed by substrate addition measured after 15 mins by Alpha Screen assay
- ChEMBL_2528044 Inhibition of human N-terminal GST-fused NPM1-ALK (1 to 680 residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_1683746 (CHEMBL4034225) Inhibition of recombinant N-terminal GST-tagged human SphK1 (1 to 384 residues) expressed in baculovirus expression system using sphingosine as substrate after 1 hr in presence of ATP by off-chip mobility shift assay
- ChEMBL_1683747 (CHEMBL4034226) Inhibition of recombinant N-terminal GST-tagged human SphK2 (1 to 618 residues) expressed in baculovirus expression system using sphingosine as substrate after 1 hr in presence of ATP by off-chip mobility shift assay
- ChEMBL_1910044 (CHEMBL4412490) Inhibition of N-terminal His-tagged human TAK1 (1 to 303 amino acids)/human TAB1 (437 to 504 amino acids) expressed in baculovirus expression system using fluorescence-labeled substrate by off-chip mobility shift assay
- ChEMBL_2069343 (CHEMBL4724596) Inhibition of human N-terminal GST-tagged FGFR3 (436 to end residues) expressed in baculovirus expression system using CSKtide as substrate incubated for 30 mins in presence of ATP by off-chip mobility shift assay
- ChEMBL_2069349 (CHEMBL4724602) Inhibition of human N-terminal GST-tagged FGFR1 (398 to end residues) expressed in baculovirus expression system using CSKtide as substrate incubated for 30 mins in presence of ATP by off-chip mobility shift assay
- ChEMBL_2069350 (CHEMBL4724603) Inhibition of human N-terminal GST-tagged FGFR2 (399 to end residues) expressed in baculovirus expression system using CSKtide as substrate incubated for 30 mins in presence of ATP by off-chip mobility shift assay
- ChEMBL_2069351 (CHEMBL4724604) Inhibition of human N-terminal GST-tagged FGFR4 (460 to end residues) expressed in baculovirus expression system using CSKtide as substrate incubated for 30 mins in presence of ATP by off-chip mobility shift assay
- ChEMBL_2069352 (CHEMBL4724605) Inhibition of human N-terminal GST-tagged VEGFR2 (790 to end residues) expressed in baculovirus expression system using CSKtide as substrate incubated for 30 mins in presence of ATP by off-chip mobility shift assay
- ChEMBL_2243518 (CHEMBL5157728) Inhibition of HDAC1 (unknown origin) using FAM-RHKK(Ac)-NH2/FAM-RHKK(trifluoroacetyl)-NH2 as substrate preincubated for 15 mins followed by substrate addition and measured after 3 hrs by microfluidic chip based fluorescence assay
- ChEMBL_2243519 (CHEMBL5157729) Inhibition of HDAC3 (unknown origin) using FAM-RHKK(Ac)-NH2/FAM-RHKK(trifluoroacetyl)-NH2 as substrate preincubated for 15 mins followed by substrate addition and measured after 3 hrs by microfluidic chip based fluorescence assay
- ChEMBL_2243520 (CHEMBL5157730) Inhibition of HDAC8 (unknown origin) using FAM-RHKK(Ac)-NH2/FAM-RHKK(trifluoroacetyl)-NH2 as substrate preincubated for 15 mins followed by substrate addition and measured after 3 hrs by microfluidic chip based fluorescence assay
- ChEMBL_2243521 (CHEMBL5157731) Inhibition of HDAC6 (unknown origin) using FAM-RHKK(Ac)-NH2/FAM-RHKK(trifluoroacetyl)-NH2 as substrate preincubated for 15 mins followed by substrate addition and measured after 3 hrs by microfluidic chip based fluorescence assay
- ChEMBL_2243522 (CHEMBL5157732) Inhibition of HDAC4 (unknown origin) using FAM-RHKK(Ac)-NH2/FAM-RHKK(trifluoroacetyl)-NH2 as substrate preincubated for 15 mins followed by substrate addition and measured after 3 hrs by microfluidic chip based fluorescence assay
- ChEMBL_2254475 (CHEMBL5168685) Inhibition of recombinant AKT1 (104 to 480 end residues) (unknown origin) catalytic domain expressed in Sf21 cells using peptide substrate incubated for 1 hr in presence of ATP by caliper off-chip mobility shift assay
- ChEMBL_2254476 (CHEMBL5168686) Inhibition of recombinant AKT2 (120 to 481 end residues) (unknown origin) catalytic domain expressed in Sf21 cells using peptide substrate incubated for 1 hr in presence of ATP by caliper off-chip mobility shift assay
- ChEMBL_2528054 Inhibition of human N-terminal GST-fused LTK catalytic domain (498 to 796 residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_1792777 (CHEMBL4264696) Inhibition of recombinant human full length N-terminal GST-tagged BTK (2 to 659 residues) expressed in baculovirus expression system using FITC-AHA-EEPLYWSFPAKKK-NH2 substrate measured after 90 mins by off-chip mobility shift assay
- ChEMBL_2211436 (CHEMBL5124385) Inhibition of His-tagged full-length recombinant wild-type human BTK (Ala2 to Ser659 residues) expressed in baculovirus infected insect cells incubated for 30 mins in presence of ATP by Microfluidic chip based mobility shift assay
- ChEMBL_2528041 Inhibition of human N-terminal GST-fused ALK cytoplasmic domain (1058 to 1620(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528045 Inhibition of human N-terminal GST-fused ACK catalytic domain (110 to 476 residues) expressed in baculovirus expression system using WASP peptide as substrate measured after 5 hrs by off-chip mobility shift assay relative to control
- ChEMBL_2528046 Inhibition of human N-terminal GST-fused AXL cytoplasmic domain (464 to 885(end) residues) expressed in baculovirus expression system using CSKtide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528051 Inhibition of human N-terminal GST-fused FRK catalytic domain (223 to 505(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528052 Inhibition of human N-terminal His-tagged JAK2 catalytic domain (826 to 1132(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528053 Inhibition of human full-length N-terminal GST-fused LCK (1 to 509(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528055 Inhibition of human N-terminal GST-fused MER cytoplasmic domain (528 to 999(end) residues) expressed in baculovirus expression system using CSKtide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528056 Inhibition of human N-terminal GST-fused MET cytoplasmic domain ([956 to 1390(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528057 Inhibition of human N-terminal GST-fused MUSK catalytic domain (527 to 869(end) residues) expressed in baculovirus expression system using CSKtide as substrate measured after 5 hrs by off-chip mobility shift assay relative to control
- ChEMBL_2528058 Inhibition of human N-terminal GST-fused RON cytoplasmic domain (979 to 1400(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528059 Inhibition of human N-terminal GST-fused ROS cytoplasmic domain (1883 to 2347(end) residues) expressed in baculovirus expression system using IRS1 as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528061 Inhibition of human N-terminal GST-fused TRKA cytoplasmic domain (436 to 790(end) residues) expressed in baculovirus expression system using CSKtide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528062 Inhibition of human N-terminal GST-fused TRKB cytoplasmic domain (456 to 822(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528063 Inhibition of human N-terminal GST-fused TRKC cytoplasmic domain (456 to 825(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528064 Inhibition of human N-terminal GST-fused TYRO3 cytoplasmic domain (453 to 890(end) residues) expressed in baculovirus expression system using CSKtide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528065 Inhibition of human full-length N-terminal GST-fused YES (1 to 543(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_1873903 (CHEMBL4375192) Covalent inhibition of N-terminal GST-fused human BTK (2-659(end) amino acids) expressed in baculovirus expression system using FITC-AHA-EEPLYWSFPAKKK-NH2 as substrate incubated for 90 mins by microfluidic off-Chip Mobility Shift Assay
- ChEMBL_2226371 (CHEMBL5139884) Binding affinity to CM5 chip immobilized full-length wild-type SARS-CoV-2 papain-like protease (1564 to 1878 residues) expressed in Escherichia coli BL21 (DE3) assessed as dissociation constant incubated for 90 secs by SPR analysis
- ChEMBL_2262116 (CHEMBL5217127) Binding affinity to human Sirt3 (118 to 399 residues) assessed as dissociation constant of ternary complex using acetylated MnSOD peptide as substrate in presence of OAADPr by short DNA nano levers based switchSENSE microfluid bio-chip analysis
- ChEMBL_2528048 Inhibition of human N-terminal GST-fused EPHA2 cytoplasmic domain (572 to 976(end) residues) expressed in baculovirus expression system using Blk/Lyntide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528049 Inhibition of human N-terminal GST-fused EPHA6 cytoplasmic domain (683 to 1130(end) residues) expressed in baculovirus expression system using Blk/Lyntide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528050 Inhibition of human N-terminal GST-fused EPHB4 cytoplasmic domain (577 to 987(end) residues) expressed in baculovirus expression system using Blk/Lyntide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528060 Inhibition of human N-terminal GST-fused TNK1 isoform 2 catalytic domain (106 to 390 residues) expressed in baculovirus expression system using CSKtide as substrate measured after 5 hrs by off-chip mobility shift assay relative to control
- ChEMBL_2076029 (CHEMBL4731563) Binding affinity to N-terminal FLAG-tagged human PRMT5 (1 to 637 residues)/N-terminal thrombin His-tagged MEP50 (2 to 342 residues) expressed in baculovirus infected Sf21 cells by streptavidin coated sensor chip based surface plasmon resonance analysis
- ChEMBL_2254477 (CHEMBL5168687) Inhibition of recombinant N-terminal GST tagged AKT2 (108 to 479 end residues) (unknown origin) catalytic domain expressed in Sf21 cells using peptide substrate incubated for 1 hr in presence of ATP by caliper off-chip mobility shift assay
- ChEMBL_2262114 (CHEMBL5217125) Binding affinity to human Sirt3 (118 to 399 residues) assessed as dissociation constant (Kon) of ternary complex using acetylated MnSOD peptide as substrate in presence of OAADPr by short DNA nano levers based switchSENSE microfluid bio-chip analysis
- ChEMBL_2262115 (CHEMBL5217126) Binding affinity to human Sirt3 (118 to 399 residues) assessed as dissociation constant (Koff) of ternary complex using acetylated MnSOD peptide as substrate in presence of OAADPr by short DNA nano levers based switchSENSE microfluid bio-chip analysis
- ChEMBL_2528042 Inhibition of human N-terminal GST-fused ALK F1174L mutant cytoplasmic domain (1058 to 1620(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528043 Inhibition of human N-terminal GST-fused ALK R1275Q mutant cytoplasmic domain (1058 to 1620(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2528047 Inhibition of human N-terminal GST-fused EGFR L858R mutant cytoplasmic domain (669 to 1210(end) residues) expressed in baculovirus expression system using Srctide as substrate measured after 1 hr by off-chip mobility shift assay relative to control
- ChEMBL_2193371 (CHEMBL5105731) Inhibition of His-tagged beta-catenin (134 to 668 residues)/CM-5 sensor chip immobilized N-terminal GST-tagged TCF4 interaction (unknown origin) preincubated with beta-catenin for 15 mins followed by TCF4 addition measured after 60 secs by SPR analysis
- ChEMBL_2242606 (CHEMBL5156816) Binding affinity to CM5 sensor chip immobilized C-terminal His6/TEV fused-GST-tagged human TTBK1 (14 to 313 residues) expressed in baculovirus infected Sf9 insect cells assessed as dissociation constant measured after 180 secs by surface plasmon resonance analysis
- ChEMBL_1995343 (CHEMBL4629238) Inhibition of N-terminal GST-tagged human FGFR3 cytoplasmic domain (436-806 end amino acids residues) expressed in baculovirus expression system preincubated for 30 to 120 mins followed by incubation with substrate and ATP for 30 mins by off-chip mobility shift assay
- ChEMBL_1995344 (CHEMBL4629239) Inhibition of N-terminal GST-tagged human VEGFR2 cytoplasmic domain (790-1356 end amino acid residue) expressed in baculovirus expression system preincubated for 30 to 120 mins followed by incubation with substrate and ATP for 30 mins by off-chip mobility shift assay
- ChEMBL_1995346 (CHEMBL4629241) Inhibition of N-terminal GST-tagged human FGFR1 cytoplasmic domain (398-822 end amino acids residues) expressed in baculovirus expression system preincubated for 30 to 120 mins followed by incubation with substrate and ATP for 30 mins by off-chip mobility shift assay
- ChEMBL_1995347 (CHEMBL4629242) Inhibition of N-terminal GST-tagged human FGFR2 cytoplasmic domain (399-821 end amino acids residues) expressed in baculovirus expression system preincubated for 30 to 120 mins followed by incubation with substrate and ATP for 30 mins by off-chip mobility shift assay
- ChEMBL_1995348 (CHEMBL4629243) Inhibition of N-terminal GST-tagged human FGFR4 cytoplasmic domain (460-802 end amino acids residues) expressed in baculovirus expression system preincubated for 30 to 120 mins followed by incubation with substrate and ATP for 30 mins by off-chip mobility shift assay
- ChEMBL_2208907 (CHEMBL5121856) Inhibition of wild type N-terminal GST tagged FGFR3 (436 to 806 residues) (unknown origin) expressed in sf21 insect cells preincubated for 30 mins followed by substrate addition and measured after 30 mins in presence of ATP by off-chip mobility shift assay
- ChEMBL_2235866 (CHEMBL5149638) Inhibition of full-length N-terminal His-TEV-tagged inactive form of MSK1 (2 to 802 residues) (unknown origin) expressed in HEK293 cells using FAM-PSKPAATRKRRWSAPESR-NH2 as substrate measured after 2 hrs in presence of ERK2 by microfluidic chip based electrophoresis
- ChEMBL_2357331 Binding affinity to VHL (54 to 213 residues)/N-terminal Avi-tagged biotinylated-Elongin B (1 to 104 residues)/Elongin C (17 to 112 residues) (unknown origin) expressed in Escherichia coli cells immobilized in SA sensor chip assessed as dissociation constant by SPR analysis
- In Vitro ATR Kinase Inhibition Assay The inhibitory activity of the compounds on ATR kinase in vitro was determined by Mobility Shift Assay at an ATP concentration of Km using Caliper EZ Reader as a mobility shift assaying technology based on microfluidic chip technology.
- ChEMBL_2378133 Inhibition of N-terminal GST-fusion protein tagged full length human CK2alpha1 (1 to 391 residues) expressed in baculovirus expression system co-expressing human His-tagged CK2beta using CK2tide as substrate incubated for 1 hrs in presence of ATP by off-chip mobility shift assay
- MEK-RAF Binding Assay Table 3: Compound effects on binding affinity of MEK to BRAF or CRAF were followed by surface plasmon resonance (SPR) with single-cycle kinetic analysis. GST-BRAF or GST-CRAF was immobilized onto a chip and increasing concentrations of His-tagged MEK1 were flowed over the sensor chip. 500 mM ATP was added to the running buffer. The dissociation constant for MEK1 binding to either BRAF or CRAF was calculated in the absence and presence of compound. Analyses were carried out on a Biacore 8K instrument. Sensorgrams were double-reference and DMSO corrected.
- ChEMBL_1835637 (CHEMBL4335770) Reversible inhibition of FAM-labeled peptide binding to full-length human N-terminal GST-tagged BTK (2 to 659 residues) expressed in baculovirus infected Sf21 insect cells at preincubated for 10 mins followed by FAM-labeled peptide addition and measured by off-chip mobility shift assay
- ChEMBL_1835638 (CHEMBL4335771) Reversible inhibition of FAM-labeled peptide binding to full-length human N-terminal GST-tagged BTK C481S mutant (2 to 659 residues) expressed in baculovirus infected Sf21 insect cells preincubated for 10 mins followed by FAM-labeled peptide addition and measured by off-chip mobility shift assay
- SPR Assay Surface Plasmon Resonance (SPR) assays have been developed to test the affinity (KD) of VHL or CRBN-based compounds to respective recombinant ligase complex/domain.The assays are based on surface plasmon resonance (SPR), which enables to measure the changes of the local refractive index due to changes of molecular mass on a gold chip surface in the case of a binding event and in a flowing system. To detect binding between both partners, the respective E3 ligase is immobilized to the chip surface, while the test compounds are flown over the chip surface at a steady velocity. The detected changes in the RU response are indicative of the binding event and are concentration dependent.For the SPR experiments, either a commercially available VHL complex (Merck, 23-044; composed of 5 units: VHL, Elongin B, Elongin C, Cul2, and Rbx1) exhibiting a his-tag at the Cul2 subunit or an internally produced biotin-tagged mouse CRBN thalidomide binding domain (mCRBN-TBD) was used. These tags provide the anchor for the capturing process to either an NTA or Streptavidin coated chip surface (immobilization level of 3,000-5,000 RU). Because of the rather complex structure of the VHL complex, it was additionally coupled to the chip surface by amino coupling to prevent any protein loss by disruption of the complex in the flowing system.To detect binding of compounds and extract dissociation constants KD for the tested compounds to the immobilized E3 ligase, concentration response curves of the compounds were recorded. Compounds were usually tested in 10-pt dilutions up to 20 μM final concentration in assay buffer and were flown over the chip at 30 μL/min. The contact time for each cycle includes 90 s for association and 200 s for dissociation of compounds. Every test cycle was read out as a sensorgram that was referenced to the sensor surface that does not present the target protein.
- ChEMBL_2235865 (CHEMBL5149637) Inhibition of full-length N-terminal His-TEV-tagged inactive form of MSK1 (2 to 802 residues) (unknown origin) expressed in HEK293 cells using FAM-PSKPAATRKRRWSAPESR-NH2 as substrate preincubated for 1 hr followed by activation with ERK2 and substrate addition measured after 2 hrs by microfluidic chip based electrophoresis
- ChEMBL_2235867 (CHEMBL5149639) Inhibition of full-length N-terminal His-TEV-tagged inactive form of MSK1 (2 to 802 residues) (unknown origin) expressed in HEK293 cells using FAM-PSKPAATRKRRWSAPESR-NH2 as substrate preincubated for 3 hrs followed by activation with ERK2 and substrate addition measured after 2 hrs by microfluidic chip based electrophoresis
- Inhibition of Histone Deacetylase Enzymatic Activity Below is a standardized protocol for running HDAC selectivity panel on Caliper LabChip EZ-Reader Instrument.The Caliper HDAC Assay Buffer (acronym HAB, 1 liter) was prepared as follows:Final Components: Concentration: Catalog #s:100 mL 1M KCL 100 mM Sigma #9541-500G 50 mL 1M HEPES, 50 mM Sigma #H3375-1KGpH 7.4 1 mL 10% BSA 0.01% SeraCare #AP-4510-80-100G 20 μL 50% Tween-20 0.001% Zymed #00-3005-20mLThe components were added to 1 liter Milli-Q water and store at 4° C.The substrate (stock conc.) was prepared as follows:Substrate A was prepared as 2 mM in DMSO. Its final concentration in the assay for HDACs 1,2,3,6 is 2 μM.Substrate B was prepared as 2 mM in 100% DMSO. Its final concentration in the assay for HDACs 4,5,7,8,9 is 2 μM.LBH was used as quench inhibitor to stop the reaction at the end point. The instrument buffer was ProfilerPro Separation Buffer (e.g., Caliper #760367). The instrument chip was LabChip EZ Reader II 12-Sipper Off-Chip Mobility Shift Chip (e.g., Caliper #760404).
- Surface Plasmon Resonance (SPR) SPR measurements were performed with a Biacore X100 instrument (GE Healthcare Life Sciences, Piscataway, NJ) at 15.0 °C. The biotinylated PTHRct-22 ligand was immobilized on an SA streptavidin sensor chip (GE Healthcare Life Sciences) usingthe manufacturer's protocol until the desired response target was reached. PDZ1 or PDZ2 analyte, dissolved in 10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant polysorbate 20, was injected on the PTHRct-coated sensor surface at increasing concentrations from 10 nM to 100 μM. At the end of each ligand injection−dissociation cycle, the sensor chip was regenerated with 4.0 M MgCl2, 50 mM triethylamine (pH 9.15), and HBS-EP buffer.
- ChEMBL_2235869 (CHEMBL5149641) Inhibition of full-length N-terminal His-TEV-tagged inactive form of MSK1 (2 to 802 residues) (unknown origin) expressed in HEK293 cells using FAM-PSKPAATRKRRWSAPESR-NH2 as substrate preincubated for 1 hr followed by activation with ERK2 and substrate addition measured after 2 hrs in presence of 1 mM ATP by microfluidic chip based electrophoresis
- Surface Plasmon Resonance (SPR) Assay The direct binding kinetics of JBZ-4 to fentanyl and related analogues was thoroughly characterized by surface plasmon resonance (SPR) single cycle kinetics in which the antibody was immobilized by amide coupling to the sensor chip surface and various dilutions of compound (40, 20, 10, 5, 2.5 nM) were flowed across the sensor.
- Surface Plasmon Resonance (SPR) Assay The kinetic and affinity parameters of protein-exemplary compound interactions were evaluated by SPR. hsTEAD1 (209-426) was immobilized onto a CM7 (Series S) sensor chip via the standard amine coupling procedure, at 15° C. Prior to immobilization, the carboxymethylated surface of the chip was activated with 400 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 100 mM N-hydroxysuccinimide for 10 min. hsTEAD1 (209-426) was diluted to 11 μg/mL in 10 mM Bis-Tris at pH 6.5 and immobilized on the activated surface chip for 8 and 10 min, in order to reach 6000 to 9000 response units (RU). The remaining activated carboxymethylated groups were blocked with a 7 min injection of 1 M ethanolamine pH 8.5 after which the surface chip was washed with 0.5% (w/v) sodium dodecyl sulphate and 50 mM glycine. HBS-N, which consists of 10 mM HEPES pH 7.4 and 150 mM NaCl, was used as the background buffer during immobilization. Exemplary compounds were prediluted in DMSO, diluted 1:50 in running buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1 mM DTT, 5 mM MgCl2, 0.1 mM EGTA, 0.05% CHAPS) and injected at ten different concentrations using two-fold dilution series, from 100 μM to 0.2 μM. A DMSO solvent correction (1%-3%) was performed to account for variations in bulk signal and to achieve high-quality data. Interaction analysis cycles consisted of a 240 sec sample injection (30 μL/min; association phase) followed by 600 sec of buffer flow (dissociation phase).
- HSP70 Biacore Direct Binding Assay All experiments were performed on a Biacore T100 at 25 deg C with a flow rate of 30 uL per minute. Double His-tagged HSP70 was immobilised on the surface of a NTA sensor chip; approximately 2000 RU of protein were immobilised. Compounds were injected for 90 sec, and the KDs were determined from equilibrium binding at 80 sec.
- Patch Clamp Assay The patch-clamp measurement was carried out at room temperature in whole-cell configuration on human embryonic kidney cells (HEK293) which have been transfected in a stable manner with the hERG gene.The whole-cell configurations were carried out using an automated patch clamp device (Patchliner, Nanion Technologies, Munich). This is a glass chip-based system with which automated whole-cell measurements on up to 8 cells simultaneously are possible. The glass chip has a hole of defined size to which the cell is transferred into the Gigaseal by application of a reduced pressure and brought into the whole-cell configuration. Buffer, cell suspension and test substances were added to microchannels of the chip using a Teflon-coated pipette. The cells were clamped to a holding potential of -80 mV. For measurement of substance-promoted inhibition of the Kv11.1 channel, the following voltage protocol was applied at 10-second intervals: 51 ms/-80 mV, 500 ms/+40 mV, 500 ms/-40 mV, 200 ms/-80 mV. The leakage current is subtracted by means of the P4 method. The cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cell had been collected, the seal was improved by addition of a seal enhancer buffer. As soon as the whole-cell configuration had been reached, the seal enhancer buffer was washed out and replaced by extracellular buffer. The measurement started in EC for 1.5 min. DMSO (vehicle control, 0.1% of DMSO) was then applied, and the control current was recorded for 3 min. The test substance was subsequently added twice in the same concentration, and the potassium current was measured for 3.5 min in each case.
- CYPA Binding Analysis by Surface Plasmon Resonance (SPR) Assay All SPR experiments were conducted on a Biacore 8K+ instrument (Cytiva). Recombinant biotinylated CYPA protein with a C-terminal AVI tag was purified and immobilized on flow cell 2 of SA chip (Cytiva, BR100531), with the flow cell 1 as the blank reference. The running buffer was HBS-P+ Buffer (Cytiva, BR100827, 0.01 M HEPES pH 7.4,0.15 M NaCl, 0.05% v/v Surfactant P20), and the flow rate was 10 μL/min. The immobilization level was achieved to about 1800 response units (RU). Compounds were 3-fold serially diluted to 1.5-3333 nM using HBS-P+Buffer with a final DMSO concentration of 1.5%, and then loaded to flow through the chip surface at a rate of 30 μL/min with HBS-P+Buffer containing 1.5% DMSO as the running buffer. Experiments were performed at 25° C. The contact time and dissociation time were set to 60 s and 200 s, respectively. After binding and dissociation of each sample, the chip was regenerated using 1 M NaCl with a short contact time of 19 s at a rate of 60 μL/min. The sensorgrams were analyzed using Biacore Insight Evaluation software with 1:1 kinetics binding model to determine the association rate constant (kon), dissociation rate constant (koff) and equilibrium dissociation constant (KD).
- Chromatin Immunoprecipitation (CHIP) ChIP assay was performed using EZ ChIP Kit (Millipore, Billerica, Mass., USA) as per manufacturer's instruction. Briefly, after crosslinking with formaldehyde (1%), cell were lysed using SDS Lysis Buffer containing Protease Inhibitor Cocktail and nuclear extract were obtained. Then, crosslinked DNA was sheared using sonication; sheared DNA size was checked on agarose gel and ranged, in all cases, between 300-500 bp. An amount of 1/20 of the shared chromatin was kept as an input. Sheared chromatin was incubated with rabbit polyclonal Phospho-Stat5 (Tyr694) Antibody (Cell Signaling, Danvers, Mass., USA) at dilution 1:50 or rabbit polyclonal IgG at appropriate concentration. Incubation was performed at 4° C. overnight. The antibody/chromatin complex was precipitated using Protein G Agarose beads. After elution, the crosslink of protein/DNA was reversed, and DNA was purified using Spin Columns. PCR was performed using platinum Taq (Invitrogen Canada, Burlington, ON, Canada). Two sets of primers were designed to amplify two DNA segments that contain STAT5-binding sites located 1672 bp and 428 bp upstream the start sites of C-Myc and Cyclin-D1, respectively. The STAT5-binding site in the C-MYC promoter is characterized by a 4N spacer and has the sequence (ttcccccgaa), whereas the one in the Cyclin D1 promoter is characterized by a 3N spacer and has the sequence (ttcttggaa).
- Electrophysiological Assay (EP) (In Vitro Assay) Sodium currents are measured in the whole-cell configuration using Syncropatch 384PE (Hanlon Technologies, Germany). 1NPC®-384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110CsCl, 10CsCl, 20EGTA, and 10Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60NMDG, 80NaCl, 4 KCl, 1MgCl2, 2CaCl2, 2D-Glucose monohydrate, 10Hepes (pH adjusted to 7.4 with NaOH).After system flushing, testing compounds are dissolved in external solution containing 0.1% Pluronic F-127. The chip is moved into the measuring head and the instrument primes the chip with external and internal solutions. 10 μl cells are added to the chip from a cell hotel, and a negative pressure of −50 mBar is applied to form a seal. Following treatment with seal enhancer solution and wash-off with external solution, negative pressure of −250 mbar is applied for 1 second to achieve the whole-cell configuration, followed by three washing steps in external solution. 20 μl of compounds is added to 40 μl in each well (1:3 dilution of compounds), and after mixing, 20 μl is removed so the volume is retained at 40 ul. After approximately 13 minutes recordings, 20 μl/well of 2 uM TTX, or 333 uM Tetracaine (for Nav1.5) is added to achieve full block.
- SPR Protocol to Measure Binding of a Compound to FKBP12 This protocol utilizes Surface Plasmon Resonance (SPR) as a method to determine kinetics (KD, Ka, Kd) for the binding of compound (analyte) to immobilized FKBP12 (ligand).Reagents:Compound in 100% DMSO (in-house), 10×HBS-P+ buffer (GE Healthcare BR-1006-71), Assay buffer (1×HBS-P+ buffer, 1% DMSO), 12×HIS tagged FKBP12 (in-house).Equipment:BIACORE X100 (GE Healthcare)Supplies:NTA Sensor chip (GE Healthcare BR-1000-34)Experimental Protocol:Experiments are performed at 25° C. Stock solution of 12×HIS tagged FKBP12 is diluted to 100 nM in assay buffer (1% DMSO final). Approximately 500-600 RU of FKBP12 is immobilized on one of two flow cells of an activated NTA chip. The second flow cell is not activated as a reference for non-specific interaction of the analyte to the sensor chip. Various concentrations of compound (1 nM-1 μM range), serially diluted into the same assay buffer (1% DMSO final), are injected onto the FKBP12 surface and reference surface at a flow rate of 10 μl/min. The surface is regenerated between analyte injections with 350 mM EDTA.Data Fitting:The BiaEvaluation software program is used for data fitting. All data is reference subtracted against both the reference flow cell and a buffer injection. For kinetic analyses, data is locally fit to a 1:1 interaction model.
- Enzymatic Activity Assay The compounds were tested for serum and glucocorticoid-regulated kinase 1 (SGK-1) inhibitory activity in a substrate phosphorylation assay designed to measure the ability of the isolated enzyme to catalyze the transfer of phosphate from ATP to serine/threonine residues in a fluorescein-labeled substrate peptide, using recombinant human SGK-1 enzyme produced in a baculovirus system (Biomol, Hamburg, Germany, Cat. No. 4-331). The synthesized fluorescent labeled peptide substrate contained (5(6)-Carboxyfluorescein)-RPRAATF-NH2. The phosphorylated substrate peptide and non-phosphorylated substrate peptide were separated with caliper life science's lab-chip technology based on a micro fluidics method. All fluid flow was established on the chip by applying a vacuum of a few psi to the waste well transporting fluid from various sources through interconnecting channels. Because the phosphoryl group is doubly negatively charged, under the pressure-driven hydrodynamic flow.
- Surface Plasmon Resonance (SPR)Assay The interactions between compounds and ABAD were performed using the dual flow cell BIACORE 3000 instrument. Surface Plasmon Resonance (SPR) studies were performed on a BIACORE 3000 at 25° C. SPR binding experiments with ABAD were performed in phosphate-buffered saline (PBS, pH 7.4, 0.005% surfactant P20) as the running and the sample buffer. The surface of the sensor chip was first activated with mixtures of N-hydroxysuccinimide (NHS, 115 mg/ml) and N-(3-dimethyl-aminopropyl)-N'-ethyl-carbodiimide-hydrochloride (EDC, 750 mg/ml) for 7 minutes. ABAD was dissolved in PBS buffer (pH 5.0) at a concentration of 10 μg/ml. The protein was immobilized directly and covalently on the hydrophilic carboxymethylated dextran matrix of the CM5 sensor chip (BIACORE) by using the standard primary amine coupling reaction on a CM5 sensor chip according to standard procedures. After the immobilization of the protein, excess activated carboxylic acid groups were quenched with ethanolamine (1 M, pH 8.5). Special care was taken during injection of samples because of carryover effects. Special washing routines were used to clean the system before injection of new samples. In addition, predipping of needles was performed. The sample flow was 40 μl/minute in experiments performed for the determination of the kinetic and equilibrium constants. Regeneration of the surfaces between subsequent binding experiments was achieved by washing the surface extensively (>>1 hour) with buffer solution.
- Surface Plasmon Resonance (SPR) SPR studies were performed using a Biacore T200 (GE Health Sciences Inc.) at 20 °C. Approximately 8,400 response units (RU) of biotinylated EED was captured onto one flow cell of a streptavidin-conjugated SA chip (according to manufacturer's protocol) while another flow cell was left empty for reference subtraction. A-395 was dissolved in 100% DMSO (50 mM Stock) and diluted to 400 nM before triplicate two-fold serial dilutions were prepared in 100% DMSO.
- AKT Enzyme Assay A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
- AKT Enzyme Assay A TTP Mosquito liquid handling instrument is used to place 125 ul of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.50 0.1 ng/uL His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 uM substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338) 0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25° C, and then stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
- Electrophysiological Assay (EP) (In Vitro Assay) The following voltage clamp electrophysiology studies are performed on representative compounds using cells heterologously expressing Nav1.7 or Nav1.5 channels. cDNAsfor Nav1.7 (NM_002977) and Nav1.5 (AC137587) are stably expressed in Chinese Hamstr Ovary (CHO) cells and CHL (Chinese Hamster Lung) cells respectively. Sodium currents are measured in the whole-cell configuration using Syncropatch 384PE (Nanlon Technologies, Germany). 1NPC -384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110 CsCl, 10 CsCl, 20 EGTA, and 10 Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60 NMDG, 80 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2), 2 D-Glucose monohydrate, 10 Hepes (pH adjusted to 7.4 with NaOH). After system flushing, testing compounds are dissolved in external solution containing 0.1% Pluronic F-127. The chip is moved into the measuring head and the instrument primes the chip with external and internal solutions. 10 ul cells are added to the chip from a cell hotel, and a negative pressure of +-50 mBar is applied to form a seal. Following treatment with seal enhancer solution and wash-off with external solution, negative pressure of +-250 mbar is applied for 1 second to achieve the whole-cell configuration, followed by three washing steps in external solution. 20 ul of compounds is added to 40 ul in each well (1:3 dilution of compounds), and after mixing, 20 ul is removed so the volume is retained at 40 ul. After approximately 13 minutes recordings, 20 ul/well of 2 uM TTX, or 333 uM Tetracaine (for Nav1.5) is added to achieve full block.
- SPR Binding Affinity Assay (Single Cycle) Single cycle kinetics (SCK): five increasing concentrations were injected successively without allowing for the dissociation of the protein-ligand complex. SPR experiments were performed at 25 °C using a Biacore T200 instrument (GE Healthcare). PBS (pH 7.4) supplemented with 0.05% Tween 20 was used as running buffer. Human FD and FB were immobilized covalently to a Series S Sensor Chip CM5 (GE Healthcare) at a flow rate of 10 μL/min using an amine-coupling protocol. Reagents for the immobilization were purchased from GE Healthcare (Amine Coupling Kit; BR-1000-50). The sensor-chip surface was activated by a 5 min injection of a 1:1 (v/v) mixture of a 100 mM N-hydroxysuccinimide (NHS) solution and a 390 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) hydrochloride salt solution in water. Both proteins were diluted to0.05 mg/mL in 20 mM HEPES pH 6.0 for immobilization onto different flow cells of the chip. After a 5 min injection of the protein, remaining reactive groups were deactivated by injecting a 1 M ethanolamine hydrochloride solution in aqueous NaOH (pH 8.5) for 5 min. Different chips were used with immobilization levels ranging between 3,000 and 5,000 response units (RU). To determine kinetic parameters for the binding of compound 6 to human FD and FB, several independent experiments were run. Threefold serial dilutions of compound 6 were prepared ranging from 0.4 to 900 nM. Each solution was injected for 60 s at a flow rate of 30 to 60 μL/min with a dissociation time of at least 1,200 s (parameters identical within one experiment).
- SPR Binding Affinity Assay (Standard) Standard kinetics: an independent association-dissociation cycle was run for each concentration by injecting compound solution and waiting for dissociation before the injection of the following concentration. SPR experiments were performed at 25 °C using a Biacore T200 instrument (GE Healthcare). PBS (pH 7.4) supplemented with 0.05% Tween 20 was used as running buffer. Human FD and FB were immobilized covalently to a Series S Sensor Chip CM5 (GE Healthcare) at a flow rate of 10 μL/min using an amine-coupling protocol. Reagents for the immobilization were purchased from GE Healthcare (Amine Coupling Kit; BR-1000-50). The sensor-chip surface was activated by a 5 min injection of a 1:1 (v/v) mixture of a 100 mM N-hydroxysuccinimide (NHS) solution and a 390 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) hydrochloride salt solution in water. Both proteins were diluted to0.05 mg/mL in 20 mM HEPES pH 6.0 for immobilization onto different flow cells of the chip. After a 5 min injection of the protein, remaining reactive groups were deactivated by injecting a 1 M ethanolamine hydrochloride solution in aqueous NaOH (pH 8.5) for 5 min. Different chips were used with immobilization levels ranging between 3,000 and 5,000 response units (RU). To determine kinetic parameters for the binding of compound 6 to human FD and FB, several independent experiments were run. Threefold serial dilutions of compound 6 were prepared ranging from 0.4 to 900 nM. Each solution was injected for 60 s at a flow rate of 30 to 60 μL/min with a dissociation time of at least 1,200 s (parameters identical within one experiment).
- Surface Plasmon Resonance Each PLpro enzyme was immobilized on a CM5 sensor chip using standard aminecoupling with running buffer HBS-P (10 mM HEPES, 150 mM NaCl, 0.05% surfactant P-20, pH 7.4) using a Biacore T200 instrument. The MERS-PLpro enzyme was immobilized to flow channels 2 and 3, and immobilization levels of flow channels 2 and 3 were ∼16 900 RU and ∼16 700 RU, respectively. SARS-PLpro was immobilized to flow channel 4 at the immobilization level of ∼14 600 RU to be compared with MERS-PLpro.
- Measurement of Dissociation Constants (Kd) by SPR SPR experiments were performed using the P4SPR from Affinité Instruments using Ni-NTA immobilization chips and his-tagged protein. Ni-NTA coated surfaces allow the immobilization of his-tagged proteins by chelation of histidine residues to the nickel ion. The sensor chip was inserted into a quad inlet model P4SPR (with 4 independent channels). Once the instrument was turned on, the baseline was stabilized by deionized (DI) water, followed by signal stabilization by the running buffer. His-tagged protein at 10 μg/mL was injected into all 4 channels of the P4SPR and was left to react for 20 min. The sensor chip was then washed with DI water. The lowest concentration of the ligand was injected into the channels of the P4SPR and was left to react for 10 min. The SPR shift was saved. Then, a higher concentration of the ligand was injected, and the sample injection steps were repeated until all 5 concentrations have been added. The KD of the binding interaction between the ligand and protein was determined by using the affinity curve fitting function in the P4SPR Control software.
- Patch clamp assay HEK293 cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (D-MEM/F-12) supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium, 100 ug/mL streptomycin sulfate and 500 ug/mL G418. Before testing, cells in culture dishes were washed twice with Hank's Balanced Salt Solution, treated with Accutase and re-suspended in the culture media ( 20 106 cells in 20 mL). Cells in suspension were allowed to recover for 10 minutes in tissue culture incubator set at 37 C. in a humidified 95% air: 5% CO2 atmosphere. Immediately before use in the SyncroPatch 384PE system (SP384PE), the cells were washed twice in extracellular buffer (HB-PS) to remove the culture medium and re-suspended in 20 mL of HB-PS. Extracellular buffer was loaded into the wells of Nanion 384-well Patch Clamp (NPC-384, 4 M) chip (60 uL per well). Then, cell suspension was pipetted into the wells (20 uL per well) of the chip. After establishment of the whole-cell configuration, membrane currents were recorded using patch clamp amplifier in the SP384PE system.
- SPR Assay Recombinant ezrin (Hypermol) was diluted to 70 mg/mL in sodium acetate buffer (pH 5.5, 10 mM) immobilized for 350 s on a CM5 Sensor Chip (Biacore) by standard amine coupling to yield around 10,000 response units. The reference flow cell was prepared exactly as the ligand flow cell, although sodium acetate buffer only (pH 5.5, 10 mM) was used instead of recombinant ezrin. Binding interactions were monitored at 25 °C with a flow rate of 30 mL/min in HBSEP/1% DMSO as running buffer. Disorazole A was injected at 0, 10, 20, 40, 60, and 80 mM. Disorazole Z was injected at 0, 20, 40, 60, 80, and 100 mM. The contact time for each compound was 120 s and the dissociation time 500 s. To rule out non-specific binding, a negative control protein was immobilized on a CM5 Sensor Chip (Biacore) by standard amine coupling: BSA (Sigma) was diluted to 70 mg/mL in sodium acetate buffer (pH 4.5, 10 mM) and immobilized for 90 s to yield around 5,000 response units. The reference flow cell was prepared with sodium acetate buffer (pH 4.5).
- Surface Plasmon Resonance (SPR) Assay Surface Plasmon Resonance (SPR) Experiments. SPR experiments were performed on a Biacore S200 instrument at 25° C. Biotinylated Hsp90 Nuclear Binding Domain (NBD) was diluted to 40 μg/mL and immobilized on a streptavidin chip (Sensor Chip SA, GE Healthcare) at a density of 2000-2500 response units (RU) on the biosensor surface.Recombinant Hsp90 NBDs were expressed and purified as previously described (Whitesell et al., Nat Commun 10, 402 (2019)) with the following modification; Hsp90 NBD expression constructs were modified to encode a C-terminal AviTag for site-specific on-column biotinylation with a BirA biotin-ligase kit (Avidity LLC; BirA-500). Stock protein solutions in 50% glycerol were stored at −20° C. until dilution into relevant buffers. Binding experiments were done in HBS-P (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% v/v surfactant P20, GE Healthcare) with 2% DMSO at flow rate of 40 μL/min. Test compounds (dilution series) were injected with a 60 s association time and 600 s dissociation time. Resulting sensorgrams were analyzed with a fit to a 1:1 binding model, using BIA evaluation software.
- The stabilization effect of I-2 and avutometinib (VS6766) on MEK1-CRAF complex were tested and compared using SPR. Table 11: The experiments were performed on Biacore S200 (Cytiva). Biotinylated, unphosphorylated MEK1 was diluted to 0.1 uM by SPR buffer C (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20, 10 mM MgCl2) and immobilized on sensor chip SA to reach an immobilization level of 60-100 RU. Tag-free CRAF (306-648, Y340D, Y341D) were diluted to a concentration of 3.75, 7.5, 15, 30, 60, 120 nM, and I-2 and avutometinib (VS6766) were diluted to 50 nM in SPR buffer D (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20, 10 mM MgCl2, and 1% DMSO). I-2 or avutometinib (VS6766) was first injected over the chip surface at 30 μL/min with a 60 second on-time and 0 second off-time, followed by injection of CRAF in a single cycle kinetic (SCK) mode, at a flow rate of 30 μL/min and with a 60 second on-time and a 900 second off-time.
- Biological Assay The QPatch HT system (Sophion Bioscience A/S, Denmark) was used with the conventional whole-cell configuration. This system is an automated, chip-based planar patch clamp device allowing for up to 48 parallel independent experiments in one experimental assay run. Cells are added to each well and drawn by suction onto a small aperture to obtain a Gigaohm seal between the cell membrane and treated silicon surface, and whole-cell recordings initiated after access is achieved by suction and/or voltage pulses. The QPatch HT uses static perfusion, whereby a small volume of recording solution or drug is added to a reservoir on the chip and the solution perfuses across the cell through quartz-lined microfluidic channels; this solution is removed by capillary action when the next sample application is made.CHO-Kv1.3 cells were prepared for experiments by dissociation from T175 cell culture flasks using trypsin-EDTA (0.05%), cells were kept in serum free media in the cell hotel on board the QPatch HT. These cells were sampled, washed and re-suspended in extracellular recording solution by the QPatch HT immediately before application to the recording well site on the chip. Experiments were performed using the following solutions; extracellular solution contained (in mM); 150 NaCl, 10 KCl, 1 MgCl2, 3 CaCl2), 10 Glucose and 10 HEPES (pH 7.4, NaOH) and intracellular solution contained (in mM); 20 KCl, 120 KF, 10 HEPES, 10 EGTA, 5 ATP (pH 7.2, KOH).The potency (Inhibitory Concentration 50%, IC50) of synthesized compounds against Kv1.3 was determined from concentration-response relationships established by cumulatively applying four escalating concentrations of test compound to an individual cell and a minimum of N≥3 individual cells of data per compound were used to generate the IC50 value.
- Surface Plasmon Resonance (SPR) Assay Surface plasmon resonance (SPR) assays were carried out on a BIAcore 3000 instrument (Biacore, Inc). Briefly, purified hTNFα was dissolved at 320 μg/ml in PBS, pH 7.0, and coupled to a carboxyl surface (HR-5 chip from HRBio, Inc.) by following the manufacturer's instructions. For the equilibrium-binding experiments, the flow rate was set to 10 μl min^-1, and 50μl of the compound sample was injected. For the kinetic experiments, the flow rate was also 10 μl min^-1, and 50 μl was injected for kinetic assay.
- TrkC Activity Human TrkC, catalytic domain [456-825 (end) amino acids of accession number NP_002521.2] was expressed as N-terminal GST-fusion protein (69 kDa) using baculovirus expression system. GST-TRKC was purified by using glutathione sepharose chromatography and stored in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Brij35, 1 mM DTT, 10% glycerol, pH7.5 at −80 C. The kinase activity was measured by off-chip mobility shift assay. The enzyme was incubated with fluorecence-labeled substrate, Srctide, in the presence of 100 uM of ATP (Mg/or Mn)/ATP). The phosphorylated and unphosphorylated substrates were separated and detected by LabChip 3000.
- TrkC Activity Human TrkC, catalytic domain [456-825(end) amino acids of accession number NP_002521.2] was expressed as N-terminal GST-fusion protein (69 kDa) using baculovirus expression system. GST-TRKC was purified by using glutathione sepharose chromatography and stored in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Brij35, 1 mM DTT, 10% glycerol, pH7.5 at −80 C. The kinase activity was measured by off-chip mobility shift assay. The enzyme was incubated with fluorecence-labeled substrate, Srctide, in the presence of 100 uM of ATP (Mg/or Mn)/ATP). The phosphorylated and unphosphorylated substrates were separated and detected by LabChip™3000.
- Enzyme Assay AKT: A TTP Mosquito liquid handling instrument was used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components were added to a final volume of 12.5 μl: 0.1 ng/μl His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 μM substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338) 0.015% Brij-35 (Sigma, B4184)The reaction was incubated for 90 min at 25 C., and then stopped by the addition of 70 μl of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate was read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure −2.3 psi, upstream voltage −500, and downstream voltage −3000. These conditions caused unphosphorylated substrate and phosphorylated product peptide to resolve as separate peaks allowing direct measurement of percentage of conversion of substrate to product.
- Chip Based Microfluidic Mobility Shift Assay All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, as well as four 8-point serial dilutions of staurosporine as reference compound, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on a Thermo CatX workstation equipped with a Innovadyne Nanodrop Express. Between pipetting steps, tips were cleaned in wash cycles using wash buffer. The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 μl per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 1 mM MgCl2, 3 mM MnCl2, 4 μM ATP, 4 μM peptide (5-Fluo-Ahx-GAPDYENLQELNKK-Amid) and 4.5 μl per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 1 mM MgCl2, 3 mM MnCl2, 4 nM SYK (SYK(2-635), produced in-house from insect cells). Kinase reactions were incubated at 30 °C. for 60 minutes and subsequently terminated by addition of 16 μl per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology. Briefly, samples from terminated kinase reactions were applied to the chip. Analytes are transported through the chip by constant buffer flow and the migration of the substrate peptide is monitored by the fluorescence signal of its label. Phosphorylated peptide (product) and unphosphorylated peptide (substrate) are separated in an electric field by their charge/mass ratio. Kinase activities were calculated from the amounts of formed phospho-peptide.
- In Vitro TrkC Activity Human TrkC, catalytic domain [456-825(end) amino acids of accession number NP_002521.2] was expressed as N-terminal GST-fusion protein (69 kDa) using baculovirus expression system. GST-TRKC was purified by using glutathione sepharose chromatography and stored in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Brij35, 1 mM DTT, 10% glycerol, pH7.5 at −80 C. The kinase activity was measured by off-chip mobility shift assay. The enzyme was incubated with fluorecence-labeled substrate, Srctide, in the presence of 100 uM of ATP (Mg/or Mn)/ATP). The phosphorylated and unphosphorylated substrates were separated and detected by LabChip 3000.
- Inhibition Assay Human TrkC, catalytic domain [456-825(end) amino acids of accession number NP-002521.2] was expressed as N-terminal GST-fusion protein (69 kDa) using baculovirus expression system. GST-TRKC was purified by using glutathione sepharose chromatography and stored in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Brij35, 1 mM DTT, 10% glycerol, pH7.5 at -80 C. The kinase activity was measured by off-chip mobility shift assay. The enzyme was incubated with fluorecence-labeled substrate, Srctide, in the presence of 100 uM of ATP (Mg/or Mn)/ATP). The phosphorylated and unphosphorylated substrates were separated and detected by LabChip 3000.
- Surface Plasmon Resonance (SPR) Binding Assay Surface plasmon resonance (SPR) binding assay. All SPR studies were performed on a Biacore X100 plus or T200 instrument (GE Healthcare). Immobilization of purified His-DDB1 was carried out at 25° C. using a CM5 sensor chip. The surface was pre-equilibrated in HBS-EP running buffer (10 mM HEPES, pH7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20), then activated with EDC/NHS. His-DDB1 proteins were immobilized by amine coupling to a density of 11,000-13,000 resonance units (RUs) on flow cell FC2, whereas flow cell FC1 was used as reference. Both DDB1 immobilized and reference surfaces were deactivated with 1M ethanolamine.
- AKT Enzyme Assay A TTP Mosquito liquid handling instrument was used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components were added to a final volume of 12.5 μl:0.1 ng/μl His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C).160 uM ATP (Fluka, 02055)1 mM DTT (Sigma, D0632)1 mM MgCl2 (Sigma, M1028)1 μM substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2),synthesized by Tufts Peptide Synthesis service.100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction was incubated for 90 min at 25° C., and then stopped by the addition of 70 μl of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate was read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure −2.3 psi, upstream voltage −500, and downstream voltage −3000. These conditions caused unphosphorylated substrate and phosphorylated product peptide to resolve as separate peaks allowing direct measurement of percentage of conversion of substrate to product. The percent conversion was plotted against concentration of inhibitor to produce a sigmoidal dose response curve, from which an IC50 was calculated.
- Affinity Test Between the Compounds of the Invention and hPD-L1 Protein The test compound was dissolved in DMSO to make a stock solution with a final concentration of 10 mM, then diluted to 1× with 1.05× PBS-P+, and then diluted by 4 times with 1× PBS-P+ buffer containing 5% DMSO; the Fc-PD-L1 protein was diluted to 10 μg/mL with 1× PBS-P+ solution containing 5% DMSO, and was then captured on the Protein A chip at a flow rate of 10 μl/min for 120 s. Each experiment used dual channels, one of which captured the ligand and the other served as a reference channel; the analyte was ready for use, and at least five concentrations were set for each analyte. The concentration selection varied with the analyte. The analyte flowed through the two channels at 30 μl/min for 90 seconds; solvent calibration was used for exclusion of the influence of DMSO, 1× PBS-P+ in 50% DMSO solution was used to wash the flow path with 30 μl/min; 10 mM glycine pH 1.5 for 30 s was used for chip surface regeneration, the experimental temperature is 25° C.; Biacore T200 Evaluation software was used, 1:1 binding model was used to calculate the KD value.
- Disrupting KRAS-PI3Kα Interaction by SPR Inhibition Assay SPR binding experiments were collected on a Biacore 8K Instrument (Cytiva). Neutravidin (Pierce) was amine coupled to the carboxymethylated dextran surface of a CM5 sensor chip (Cytiva) using standard amine coupling chemistry. The CM5 chip surface was first activated with 0.1 M N-hydroxy succinimide and 0.4 M N-ethyl-N′-(3-dimethyl aminopropyl) carbodiimide at a flow rate of 20 μL/min using 20 mM HEPES pH 7.4, 150 mM NaCl as the running buffer. Next, neutravidin was diluted to 20 μg/mL in 10 mM sodium acetate (pH 4.5) and injected on all flow cells until a density of approximately 10,000 response units (RU) was immobilized. Activated amine groups were quenched with an injection of 1 M ethanolamine (pH 8.0). 300-500 RU of avi-tagged full length PI3Kα was captured on all flow cells in 20 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM TCEP, 0.05% tween 20, 5% DMSO buffer. KRAS-Q25A at 20 μM was mixed with 8 concentrations of compound (50 nM-100 μM) and injected over the full-length PI3Kα at 30 μL/min and 25° C. Steady-state levels of KRAS binding were recorded and fit with a 4-parameter inhibition model to determine the IC50 values.
- Microfluidic Off-Chip Mobility Shift Assay Protocol for Potency Assessment Against BTK Enzyme Briefly, 2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2; developed in-house) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM HEPES, pH 7.5, and 10 mM DTT. 5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27° C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM HEPES, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorilation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel, Switzerland).
- Microfluidic Off-Chip Mobility Shift Assay Protocol for Potency Assessment Against BTK Enzyme Briefly, 2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2; developed in-house) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM HEPES, pH 7.5, and 10 mM DTT. 5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27° C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM HEPES, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorilation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel. Switzerland).
- Surface Plasmon Resonance (SPR) Binding Surface plasmon resonance (SPR) STING agonist binding studies were carried out using a Biacore T200 instrument (GE Healthcare) at 4° C. in a 150 mM KCl, 25 mM Hepes (pH 7.5), 1 mM TCEP, 2.5 mM MgCl2, 5% (v/v) glycerol, 0.005% (v/v) P20, 1% (v/v) DMSO running buffer. The recombinant protein immobilized on the streptavidin chip was either human WT or H232R STING. A truncated construct of STING was used in all studies. The STING constructs were comprised of residues 155-341 with both N- and C-terminal truncations; the N-terminal transmembrane domains were removed (1-154), as well as the C-terminal tail (342-379). A highly specific N-terminal biotinylation was achieved enzymatically with the E. coli biotin ligase (BirA) and inclusion of the high-affinity biotinylation peptide AviTag . A Carboxymethylated dextran pre-immobilized with streptavidin (series S Streptavidin CM5 Sensor Chip) was used to capture the biotinylated STING protein. Test compound injections were made at a flow rate of 100 μl per minute with a 60 second association time and variable dissociation time. A three-fold dilution series from a 10 μM starting concentration was used for all test compounds. Data analysis was performed using the BiacoreT200 data evaluation software package (GE Healthcare).
- Activity Assay Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
- Biacore SPR Assay Binding parameters of compounds were determined using a Biacore S51 sensor. An anti-GST antibody was immobilized onto a CM5 chip by primary amine-coupling in accordance with the manufacturer's recommendations.In running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20, mM MgCl2, and 1% DMSO)N-terminally GST-fused purified FAK enzyme was captured on both spot 1 and 2. Spot 1 was subsequently blocked by loading with 30 nM PF-562,271 at the beginning of each cycle. Concentration series' of the test compounds were injected over the spots at 25 C. The specific binding was calculated as difference between spot 2 and 1 signals followed by solvent correction.
- SPR Assay to Determine Binding Affinity to FK506-Binding Proteins (FKBP) N-terminal avi-his6 tagged FKBP fusions to FKBP12, FKBP51 and FKBP52 were expressed in E. coli and purified using standard chromatography. Each protein was subsequently immobilized on a streptavidin chip in a Biacore 8K SPR instrument (GE Healthcare). Using single-cycle kinetics, compound titrations were flowed at 45 uL/min over each surface using 2-minute association and 30-minute dissociation phases in a buffer containing 50 mM Tris pH 7.5/150 mM NaCl/0.01% Tween 20/1 mM DTT/2% DMSO. The data was fit using low molecular weight (LMW) single-cycle kinetics. The equilibrium dissociation constants (KD) are reported.
- AKT/PKB Kinase Assay In order to measure AKT inhibition in the Caliper Life Sciences LC3000, a TTP Mosquito liquid handling instrument was used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction, the following components were added to a final volume of 12.5 μl: 0.1 ng/μl His-AKT (Full Length) (Invitrogen, Part # P2999, Lot #641228C); 160 μM ATP (Fluka, 02055); 1 mM DTT (Sigma, D0632); 1 mM MgCl2 (Sigma, M1028); 1 μM substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service; 100 mM HEPES pH 7.5 (Calbiochem, 391338); and 0.015% Brij-35 (Sigma, B4184).The reaction was incubated for 90 min at 25° C., and then stopped by the addition of 70 μl of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)). The plate was read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure −2.3 psi, upstream voltage −500, and downstream voltage −3000. These conditions cause unphosphorylated substrate and phosphorylated product peptide to resolve as separate peaks allowing direct measurement of percentage of conversion of substrate to product. The percent conversion was plotted against concentration of inhibitor to produce a sigmoidal dose response curve, from which an IC50 was calculated.
- SPR Binding Assay The His-tagged SARS-CoV-2 PLpro enzyme was initially prepared in phosphate buffer and diluted to 50 pg/mL with 10 mM sodium acetate (pH 5.5) and immobilized on a CM5 sensor chip by standard amine-coupling with running buffer PBSP (10 mM phosphate, pH 7.4, 2.7 mM KCl, 137 mM NaCl, 0.05% Tween-20). The CM5 sensor chip surface was first activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxy succinimide (NHS) mixture using a Biacore 8K instrument (Cytiva). SARS-CoV-2 PLpro enzyme was immobilized to flow channels 1 through 4 followed by ethanolamine blocking on the unoccupied surface area, and immobilization levels for all four channels were similar at 12,000 RU. Each flow channel has its own reference channel, and blank immobilization using EDC/NHS and ethanolamine was done for all reference channels. Compound solutions with a series of increasing concentrations (0.049-30 uM at 2.5-fold dilution) were applied to all active and reference channels in SPR binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.05% Tween-20, 0.5 mM TCEP, and 2% DMSO) at a 30 uL/min flow rate at 25 C. The data were double referenced with a reference channel and zero concentration (2% DMSO) responses, and reference subtracted sensorgrams were fitted with 1 to 1 Langmuir kinetic model using a Biacore Insight evaluation software, producing two rate constants (ka and kd).
- Surface Plasmon Resonance (SPR) (1) Screening of Macrolide and Cyclic Peptide Drugs by Taking SelH as a TargetA surface plasmon resonance (SPR) method was adopted to detect the interaction condition of a small molecule and a protein, so that the screening of drugs targeting SelH was carried out.1. Protein ImmobilizationPurified SelH was diluted into a 50 μg/mL protein solution by using 10 mM sodium acetate with a pH value of 5.5, the purified SelH was fixed onto a CM5 chip by an amino coupling method by using Biacore 8K (GE Healthcard, Sweden), and an RU value was recorded.2. Sample PreparationThe macrolide compound or cyclic peptide compound (the compounds specifically include carrimycin, isovalerylspiramycin I, spiramycin, carbomycin, azithromycin, erythromycin and thiostrepton) was dissolved in 100% DMSO to be prepared into solutions containing 5% DMSO of different concentrations (0, 31.25, 62.5, 125, 250 and 500 μM) by using a 1.05×PBS-P+ buffer solution (GE Healthcare, obtained by diluting 10×PBS-P+).3. Binding ExperimentA single-cycle kinetics method was adopted, the 1.05×PBS-P+ buffer solution containing 5% DMSO was used as a Running Buffer, different compounds of different concentrations flowed through the SelH fixed onto the chip, wherein the binding time was 120 s, and the change condition of the RU value was recorded. An equilibrium dissociation constant (KD) was calculated by using software in Biacore 8K so as to evaluate the binding affinity between the protein and the compounds.
- Target Residence Time of Factor B Inhibitors Determined by Surface Plasmon Resonance (SPR) Table 2: Biacore 8 k instrument was primed using 1× PBS-P+ buffer before docking a Cytiva NTA chip. Recombinant human Factor B catalytic domain were immobilized on a NTA chip to a level of about 5000 resonance units (RU) using 1× PBS-P+ buffer [20 mM phosphate buffer with 2.7 mM KCl, 137 mM NaCl, and 0.05% (v/v) Tween-20]. The protein ligand was further crosslinked to sensorchip surface by amine coupling kit. Immobilization and binding experiment were performed at room temperature.After changing buffer to 1× PBS-P+ buffer with 2% (v/v) DMSO, a pre-run was performed for a period of at least 30 min at a flow rate of 30 μl/min to obtain a stable surface. The kinetic constants of the compounds were determined by single-cycle kinetics with six consecutive injections (or multi-cycle kinetics with eight consecutive injections) with an increasing compound concentration in ranges of 0.8-200 nM, 12.5-400 nM, 4.1-1,000 nM or 41-10,000 nM depending on the potency. Single-cycle kinetics experiments were performed with an association time of 60 s per concentration and a dissociation time of 300 s (or a dissociation time of 120 s for multi-cycle kinetics experiments). A flow rate of 30 μl/min was used. A blank run with the same conditions was performed before the compound was injected.
- Kv11.1 (hERG) ION CHANNEL ACTIVITY (Patch Clamp Assay) Method for the detection and characterisation of test substances which interfere with the Kv11.1 (hERG) channel: Kv11.1 (hERG, human ether a-go-go related gene) is a potassium channel which plays a central role for repolarisation of the cells in the ventricular cardiomyocytes.The patch-clamp measurement was carried out at room temperature in whole-cell configuration on human embryonic kidney cells (HEK293) which have been transfected in a stable manner with the hERG gene.The whole-cell configurations were carried out using an automated patch clamp device (Patchliner, Nanion Technologies, Munich). This is a glass chip-based system with which automated whole-cell measurements on up to 8 cells simultaneously are possible. The glass chip has a hole of defined size to which the cell is transferred into the Gigaseal by application of a reduced pressure and brought into the whole-cell configuration. Buffer, cell suspension and test substances were added to microchannels of the chip using a Teflon-coated pipette. The cells were clamped to a holding potential of −80 mV. For measurement of substancepromoted inhibition of the Kv11.1 channel, the following voltage protocol was applied at 10-second intervals: 51 ms/−80 mV, 500 ms/+40 mV, 500 ms/−40 mV, 200 ms/−80 mV. The leakage current is subtracted by means of the P4 method. The cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cell had been collected, the seal was improved by addition of a seal enhancer buffer. As soon as the whole-cell configuration had been reached, the seal enhancer buffer was washed out and replaced by extracellular buffer. The measurement started in EC for 1.5 min. DMSO (vehicle control, 0.1% of DMSO) was then applied, and the control current was recorded for 3 min. The test substance was subsequently added twice in the same concentration, and the potassium current was measured for 3.5 min in each case.If the measurement result of a test substance at an initial concentration of 10 μM was smaller than (−)50% effect (threshold value) (for example (−)60% effect), the test substance was, in order to determine a dose/action relationship, added cumulatively in increasing concentration, where each concentration was measured for 5 min.The reference substance used was the Kv11.1 (hERG) ion channel blocker quinidine. The effects of test substances and quinidine were standardised to the associated vehicle control. The effect on the Kv11.1 (hERG) channel activity was assessed from the potassium current at −40 mV. For the calculation, the current was evaluated for the respective final trace. A test-substance-induced inhibition of the Kv11.1(hERG) channel was standardised to the vehicle control (0.1% of DMSO).During the measurement, an aliquot of the test substance was taken for concentration determination. The sample was measured immediately by HPLC, and the final concentration was determined from a calibration curve.
- Biacore Binding of DAAO Inhibitors Binding affinity and kinetics were measured using biotinylated recombinant human DAAO bound to a Neutravidin surface in a Biacore binding assay. A custom Neutravidin surface was generated using standard amine coupling methods from Biacore on a CM5 sensor chip. An enzymatically biotinylated human DAAO was immobilized onto the Neutravidin surface to a level of ~9000 Ru. Binding studies were performed in a Biacore 3000 (GE Healthcare) instrument. Compounds were injected at three concentrations, and binding responses were processed using Scrubber 2 (BioLogic Software, Inc.) to align and double reference the data. The kinetic data were fit globally to a simple 1:1 interaction model using Biaeval (GE Healthcare) to obtain the rate constants and affinity.
- PHGDH SPR Assay The binding affinities of compounds towards PHGDH were assayed using the SPR-based Biacore T200 instrument (GE Healthcare). PHGDH was immobilized on a CM5 sensor chip by using standard amine-coupling at 25℃ with running buffer PBS-P (20 mM phosphate buffer, 2.7 mM NaCl, 137 mM KCl, 0.05% surfactant P-20, pH 7.4), respectively, as described previously. A reference flow cell was activated and blocked in the absence of PHGDH. In the direct binding experiments between PHGDH and compounds, PHGDH immobilization level was fixed at 800 response units (RU), and then different concentrations of compounds containing 5% DMSO were serially injected into the channel to evaluate binding affinity. Regeneration was achieved by extended washing with the running buffer after each sample injection.
- Caliper Assay In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples ( 12-sipper chip , Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 μm Na3VO4, 10 mM β-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
- Caliper Assay The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 μL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 μM ATP, 4 μM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 μL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 μL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology. Briefly, samples from terminated kinase reactions were applied to the chip. Analytes are transported through the chip by constant buffer flow and the migration of the substrate peptide is monitored by the fluorescence signal of its label. Phosphorylated peptide (product) and unphosphorylated peptide (substrate) are separated in an electric field by their charge/mass ratio.
- Determination of TET2 Binding by Surface Plasmon Resonance The binding of compounds of the examples was determined by Surface Plasmon Resonance (SPR) using a Biacore T200 SPR biosensor instrument (GE Healthcare, Uppsala, Sweden) at 25° C. The human TET2 construct was immobilized on a CM5 sensor chip using a standard covalent immobilization via amine coupling following the activation of the carboxymethyl dextran matrix of the sensor chip (injection of a solution containing 0.1 M N-hydroxysuccinimide and 0.4 M 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride at a flow rate of 15 μL/min for 7 min). The protein immobilization was achieved after the injection of 5 μg/mL TET2 in 10 mM sodium acetate (pH 4.0) at a flow rate of 5 μL/min. Unreacted activated groups of the dextran matrix were deactivated by injection of 1 M ethanolamine hydrochloride for 7 min at a flow rate of 15 μL/min. The corresponding matrix activation and protein immobilization were performed using as a running buffer a phosphate buffered saline solution (1.05× PBS: 10 mM phosphate, pH 7.4, 150 mM NaCl). The screened compounds were prepared in a 20 mM stock solution in DMSO and diluted with 1.05× PBS to achieve a final 5% (v/v) DMSO concentration. The running buffer of the interaction assays consisted of 1×PBS, 5% (v/v) DMSO. The flow rate used for the screening was 60 μL/min and the ligand association and dissociation times set were 60 s and 120 s, respectively.
- Surface Plasmon Resonance (SPR) Biosensor Assay The SPR experiments were performed using a Biacore S200 instrument and CM5 biosensor chips (Cytiva, Uppsala, Sweden) at 25 C. Streptavidin (Sigma) was immobilized by amine coupling. The CM5 chip surface was activated by an injection of a 1:1 mixture of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (Cytiva, Uppsala, Sweden) or 7 min at a flow rate 10 uL/min. Streptavidin (Sigma) was diluted to 250 ug/mL in sodium acetate buffer (pH 5.0) and injected over the activated surface at a flow rate 2 uL/min for 10 min. The surface was then deactivated by the injection of 1 M ethanolamine (Cytiva, Uppsala, Sweden) for 7 min. Subsequently, the biosensor chip was conditioned with four pulse injections of 1 M NaCl/50 mM NaOH solution. Mpro was diluted to 100 ug/mL in 1.02 running buffer (50 mM TrisHCl, pH 7.5, 0.05% Tween-20) and injected at the flow rate of 2 uL/min, reaching a typical immobilization level of 8000-9000 RU. After immobilization, compounds were injected over the surface using a 10-point concentration series, at a flow rate 30 uL/min in 50 mM TrisHCl, pH 7.5, 0.05% Tween-20. An association phase was monitored for 60 s and a dissociation phase for 120 s. Sensorgrams were double-referenced by subtracting the signals from a reference surface and the signal from one blank injection. A solvent correction accounting for 2% DMSO was performed.
- Folate Receptor SPR Assay Table 16: Folate receptor SPR binding assays were conducted on a Biacore 8K instrument (Cytiva) using biotinylated folate receptor beta (Acro Biosystems). Briefly, folate receptor was immobilized at -550 RU on a streptavidin chip (Cytiva) in 10 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween 20. Multi-cycle kinetic studies were conducted with 8 compound concentrations using 2-fold dilutions in 10 mM HEPES pH 7.5, 150 mM NaCl, 0.05 % Tween 20, 2% DMSO at 25° C. A flow rate or 75 μl/mmn with an association time of 120 s and a dissociation time of 600 s were used. Surfaces were regenerated with 4 M MgCl2 at 30 μl/min for 30 s. Data were fit to a 1:1 model to determine the reported binding parameters.
- SPR assay SPR measurements wereperformed on BIAcore T100 instrument (BIAcore GE Healthcare), at25 C on series S NTA chips (certified) according to provider's protocolswith 10mMHEPES, pH 7.4, 150mMNaCl, 500 μMEDTA, 0.05%Tween-20, and 1% DMSO as a running buffer. Histidine-tagged Grp78was immobilized on the sensor surface; reference surfaces withoutimmobilized Ni2+ served as controls for nonspecific binding and refractiveindex changes. Concentrations of inhibitors (0--200 μM) weretypically injected over the sensor chip at 35 μL/min. Zero concentrationsamples were used as blanks. The sensor surface was regenerated betweenexperiments by injections of 0.1 mg/mL trypsin and 50% DMSO. Dataprocessing was performed using BIAevaluation 2.1 software (BIAcore GEHealthcare Bio-Sciences Corp.) by globally fitting the entire inhibitorconcentration series data set to the steady state affinity model.
- Microfluidic Off-Chip Mobility Shift Assay Protocol for Potency Assessment Against BTK C481S Enzyme The protocol below describes a microfluidic, off-chip mobility shift kinase assay to measure inherent potency of compounds against BTK C481S enzyme. The mechanics of the assay platform are described by the vendor (PerkinElmer, Hopkinton, Mass.) on their website at the following URL: http://caliperls.com.Briefly, 2.5× stocks of His-TEV-hsBTK(328-659)(C481S) from the Merck Serono Protein Purification Laboratory in Darmstadt, Germany (PCS, Q27/234), 1.6×ATP and appropriate KinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2; Tufts University Core Facility custom synthesis) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM HEPES, pH 7.5, and 10 mM DTT.5 uL of enzyme buffer and 7.5 uL of ATP/KinKDR peptide substrate mix were added to Matrix (#4315) 384-well, sterile, flat-bottom polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 25° C. Following the incubation period, reactions were terminated by adding 65 uL quench buffer consisting of 100 mM HEPES, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (PerkinElmer, Mountain View, Calif.), 5% DMSO. Terminated reactions were monitored at −2 PSI, −3000 V/−700 Volts in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was quantified by laser-induced fluorescence measuring the charge/mass difference between substrate and product resulting from peptide phosphorylation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel, Switzerland).
- SPR Assay Surface plasmon resonance data was collected on a Biacore T200 or 3000 system (GE Healthcare) at 25° C. Streptavidin was immobilized on a CM5 (GE Healthcare) or CMD500d sensor chip (XanTec Bioanalytics) using standard amine-coupling chemistry at 25° C. with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 12 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 μL/min. For capture of streptavidin, protein was diluted to 0.2 mg/mL in 10 mM sodium acetate (pH 4.5) and captured by injecting 100 μL onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5). Avi-tagged PCSK9 protein was captured on the streptavidin surface by injection of 150 μL of protein diluted to 16 pg/mL in FIBS-N, 0.05% tween-20, 0.1 mM CaCl2. Typical surface densities obtained were 8000-10000 RU. SPR binding data were obtained using an appropriate dilution series of each compound at a flow rate of 30 μL/min, with a capture time of 100 s and dissociation times of 300 s. Running buffer for compound binding studies was FIBS-N, 0.05% tween-20, 0.1 mM CaCl2), 4% DMSO. Data were corrected for DMSO excluded volume effects. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software).
- kinetic binding analysis Dual histidine and Avi tagged human EIF4E (His6-3C-avi-eIF4E) was expressed in 8 L of TB Media. Induction by 0.4 mM IPTG occurred at 2.0 OD600, and cells were harvested at 15 OD600. 225 gram pellet was diluted in buffer (50 mM Tris, 500 mM NaCl, 2 mM MgCl2, 1 mM TCEP, pH 7.5 containing 10% glycerol, protease inhibitors, and DNase) to volume of 600 mL and passed once over the microfluidizer. Sample was run on 5 mL HisTrap HP IMAC at 4.0 mL/min IMAC with a 25 mM to 500 mM imidazole gradient for one column volume. GST-PreScission Protease (2 mg, made in-house) was added to sample and allowed to react overnight at 4° C. The cleaved pool was passed over 0.5 mL GST and 0.5 IMAC resin in a gravity column. Sample volume was increased to 800 mL using 50 mM Tris pH 7.5, 1 mM TCEP and passed over a 5 mL hiTrap SP FF at 4.0 mL/min with a 0 to 1 M NaCl gradient over 20 column volumes. Sample was then injected onto a 124 mL S75 Gel Filtration Column at 20 mg/mL. Final Avi-eIF4E (26931 Da) was diluted to 1 mg/mL in 1× Bicine buffer to a volume of 2.5 mg and mixed with ATP/Biotin Mix (10 mM ATP, 10 mM Mg(OAc)2, 50 μM d-biotin final). Biotin Ligase (25 μg BirA produced in house) was added to reaction. Reactions were performed with mixing (500 rpm) on Eppendorf ThermoMixer R at 30° C. for 60 minutes and checked for completeness using LC-MS. To the sample, 100 μl of immobilized glutathione (1:1 with buffer) was add and mixed for 15 min at 4° C. to bind C3 and Bir3 and removed by centrifugation. The sample was buffer exchanged using two consecutive PD-10 columns equilibrated with 20 mM HEPES, 100 mM KCl, 1 mM DTT, pH 7.5.Due to low eIF4E stability, the streptavidin coated chip was prepared and run at 10° C. on the Biacore T200. The eIF4E (0.04 mg/mL, 150 μl) was bound to the sample channel of Series S Sensor Chip SA (GE Life Sciences, BR-1005-31) to surface density of 5000 to 7000 RU (Response Units). Buffer flowed over the chip at 30 μL/min, using 1×PBS, 50 mM NaCl, 0.1% Glycerol, 0.1% CHAPS, and 1% DMSO. Samples of various cap analogs were diluted to various concentrations in a range of 100 μM to less than 1 nM. Samples were injected into the Biacore chip with a two minute association time and a five minute dissociation time. Several buffer injections were done for each sample for blank subtraction.Analysis was done for all sets using Biacore T200 evaluation software. Binding analysis for all compounds is reported as response units at 1 micromolar compound where a higher value for RU is interpreted as greater ligand binding to the surface immobilized eIF4E protein. A subset of compounds was further characterized to determine dissociation constants using kinetic binding. Steady State Affinity fits were done with default settings (4 seconds before injection stop with 5 second window). Kinetic fits were normally done with 1:1 binding model, with constant RI=0 and all other variables set to fit globally.
- Electrophysiological Assay (EP) (In Vitro Assay) Patch voltage clamp electrophysiology allows for the direct measurement and quantification of block of voltage-gated sodium channels (NaV's), and allows the determination of the time- and voltage-dependence of block which has been interpreted as differential binding to the resting, open, and inactivated states of the sodium channel (Hille, B., Journal of General Physiology (1977), 69: 497-515).The following voltage clamp electrophysiology studies were performed on representative compounds using cells heterologously expressing Nav1.7 or Nav1.5 channels. cDNAs for Nav1.7 (NM_002977) and Nav1.5 (AC137587) were stably expressed in Chinese Hamstr Ovary (CHO) cells and CHL (Chinese Hamster Lung) cells respectively. Sodium currents were measured in the whole-cell configuration using Syncropatch 384PE (NanIon Technologies, Germany). 1NPC -384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110 CsCl, 10 CsCl, 20 EGTA, and 10 Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60 NMDG, 80 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 2 D-Glucose monohydrate, 10 Hepes (pH adjusted to 7.4 with NaOH).After system flushing, testing compounds are dissolved in external solution containing 0.1% Pluronic F-127. The chip is moved into the measuring head and the instrument primes the chip with external and internal solutions. 10l cells are added to the chip from a cell hotel, and a negative pressure of −50 mBar is applied to form a seal. Following treatment with seal enhancer solution and wash-off with external solution, negative pressure of −250 mbar is applied for 1 second to achieve the whole-cell configuration, followed by three washing steps in external solution. 20 μl of compounds is added to 40 μl in each well (1:3 dilution of compounds), and after mixing, 20 μl is removed so the volume is retained at 40 μl. After approximately 13 minutes recordings, 20 μl/well of 2 uM TTX, or 333 uM Tetracaine (for Nav1.5) is added to achieve full block.For voltage protocol, an holding potential of −50 mV is applied during the whole experiment. A depolarizing step is applied to −10 mV for 10 ms, followed by a hyperpolarization step to −150 mV for 20 ms to allow channel recovery from inactivation. A second depolarizing step is applied from −150 mV to −10 mV for 10 ms, where currents were measured to derive blocking effects of compounds. Inhibition is determined based on 7.5 min of compound incubation.
- Electrophysiological Assay (EP) (In Vitro Assay) Patch voltage clamp electrophysiology allows for the direct measurement and quantification of block of voltage-gated sodium channels (NaV's), and allows the determination of the time- and voltage-dependence of block which has been interpreted as differential binding to the resting, open, and inactivated states of the sodium channel (Hille, B., Journal of General Physiology (1977), 69: 497-515).The following voltage clamp electrophysiology studies were performed on representative compounds using cells heterologously expressing Nav1.7 or Nav1.5 channels. cDNAs for Nav1.7 (NM_002977) and Nav1.5 (AC137587) were stably expressed in Chinese Hamstr Ovary (CHO) cells and CHL (Chinese Hamster Lung) cells respectively. Sodium currents were measured in the whole-cell configuration using Syncropatch 384PE (Nanlon Technologies, Germany). 1NPC -384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110 CsCl, 10 CsCl, 20 EGTA, and 10 Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60 NMDG, 80 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 2 D-Glucose monohydrate, 10 Hepes (pH adjusted to 7.4 with NaOH).After system flushing, testing compounds are dissolved in external solution containing 0.1% Pluronic F-127. The chip is moved into the measuring head and the instrument primes the chip with external and internal solutions. 10 l cells are added to the chip from a cell hotel, and a negative pressure of −50 mBar is applied to form a seal. Following treatment with seal enhancer solution and wash-off with external solution, negative pressure of −250 mbar is applied for 1 second to achieve the whole-cell configuration, followed by three washing steps in external solution. 20 l of compounds is added to 40 l in each well (1:3 dilution of compounds), and after mixing, 20 l is removed so the volume is retained at 40 ul. After approximately 13 minutes recordings, 20 l/well of 2 uM TTX, or 333 uM Tetracaine (for Nav1.5) is added to achieve full block.For voltage protocol, an holding potential of −50 mV is applied during the whole experiment. A depolarizing step is applied to −10 mV for 10 ms, followed by a hyperpolarization step to −150 mV for 20 ms to allow channel recovery from inactivation. A second depolarizing step is applied from −150 mV to −10 mV for 10 ms, where currents were measured to derive blocking effects of compounds. Inhibition is determined based on 7.5 min of compound incubation.
- SPR Method for Detecting Affinity of Compounds Disclosed Herein for KRAS Protein Subtype G12D or WT Biotinylated Avi-KRAS-WT or Avi-KRAS-G12D was diluted to 20 μg/mL with 1x HBS-P+(Cat. #BR1006-71) buffer containing 100 mM MgCl2, and then flowed through SA (Cat. #BR1005-31) biosensing chip channel 2 for 420 s to obtain a coupling level of approximately 5000-7000 RU. Then samples of small molecular compounds were injected for 120 s in an ascending order, and then were dissociated for 720 s. The experiment employed a single-cycle kinetic mode. Reaction signals were detected in real time using a Biacore 8K instrument to obtain association and dissociation curves. After the experiment ended, data analysis was performed using Biacore 8K evaluation software, and affinity data were obtained by performing data fitting using a 1:1 model.
- Surface Plasmon Resonance (SPR) Assay Pure His-cyclophilins were immobilized and covalently stabilized on the NTA sensor chip according to the protocol described in Thermo-kinetic analysis space expansion for cyclophilin-ligand interactions—identification of a new nonpeptide inhibitor using Biacore T200. Using 200 nM concentrations of each protein, in Running Buffer (PBS, pH 7.4; 0.05% surfactant P20, 2% v/v ethanol; 50 μM EDTA), at 30 μl min−1 with 60 second contact times on the activated NTA surfaces. This gave signals of 1,921 RU for His-CypA, 1932 RU for His-CypB and 1,397 RU for His-CypD. Specific surface protein activity was assayed by passing saturating amounts of CsA (2 μM) in Running Buffer over these surfaces; values of 94.1%, 95.5% and 95.6% activity were obtained for His-CypA, -B and -D, respectively.
- Enzymatic Activity Assay The compounds were tested for serum and glucocorticoid-regulated kinase 1 (SGK-1) inhibitory activity in a substrate phosphorylation assay designed to measure the ability of the isolated enzyme to catalyze the transfer of phosphate from ATP to serine/threonine residues in a fluorescein-labeled substrate peptide, using recombinant human SGK-1 enzyme produced in a baculovirus system (Biomol, Hamburg, Germany, Cat. No. 4-331). The synthesized fluorescent labeled peptide substrate contained (5(6)-Carboxyfluorescein)-RPRAATF-NH2. The phosphorylated substrate peptide and non-phosphorylated substrate peptide were separated with caliper life science's lab-chip technology based on a micro fluidics method. All fluid flow was established on the chip by applying a vacuum of a few psi to the waste well transporting fluid from various sources through interconnecting channels. Because the phosphoryl group is doubly negatively charged, under the pressure-driven hydrodynamic flow and the voltage-driven flow within the electric field, the fluorescent labeled peptide substrate and its phosphorylation product appear at different times in the detection window to the detection point. Substrate turnover can thus be determined as the ratio of the product peak area and the sum of substrate peak area and product peak area.The enzyme reaction was carried out in a buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM MnCl2, 2 mM DTT, and 0.03% bovine serum albumine. The enzyme was pre-incubated with the test compound for 30 min at 24° C. The kinase reaction was initiated by addition of the substrate mixture containing the peptide substrate (final concentration 1 μM) and ATP (final concentration 10 μM). After 60 min incubation at 37° C., the enzyme reaction was terminated by adding a buffer containing 100 mM Hepes (pH 7.4) and 35 mM EDTA.
- Enzyme Assay The following describes a microfluidic, off-chip mobility shift kinase assay used to measure inherent potency of compounds against BTK enzyme. Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent BTK Enzyme Assay IC50. These IC50 values are reported in ranges wherein: A<100 nM, B<1 uM, and C>1 uM.2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM Hepes, pH 7.5, and 10 mM DTT.5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27 C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM Hepes, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorylation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel, Switzerland). Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent PBMC BTK Enzyme Assay IC50.
- KRas G12D Surface Plasmon Resonance (SPR) Binding Assay Briefly, 1L of 1.05X HBS-Mg buffer (262.5 mM Bioultra Hepes, pH 7.5, 157.5 mM NaCl, 105 mM MgCl2, 0.525 mM TCEP, 0.0305% Brij-35) was prepared and filter sterilized using a 0.22 m bottle top filter. Approximately 50 mL of 1.05X HBS-Mg buffer was removed and saved for future dilutions. A 50 mL aliquot of DMSO (Sigma Aldrich DMSO Lot. #SHBK2079) was added and continued to stir for 10 minutes, creating the final 1.0X HBS-Mg buffer (250 mM Bioultra Hepes pH 7.5, 150 mM NaCl, 100 mM MgCl2, 0.5 mM TCEP, 0.03% Brij-35). Biacore T200 instrument was primed using 1.0X HBS-Mg buffer before docking a GE Streptavidin (SA) chip and then primed two additional times prior to beginning the immobilization step. All immobilized protein mixtures were created using 3-5 mg/mL Biotinylated Avidin-tagged KRAS protein using the following immobilization settings: SA chip type, 1 flow cells per cycle, 720 second contact time, and 5 ul/min flow rate. Normalization of the detector was also performed during the immobilization step using the GE BiaNormalize solution. All compounds were diluted to 10 mM in 100% DMSO prior to being diluted 20X in 1.05X buffer. Another 10X dilution was created using 1.0X buffer prior to performing a series of 3X dilutions to create a compound concentration curve using the following assay settings: 20C analysis temperature, General Settings=10 Hz data collection rate and multi-detection; Assay Steps=all set to LMW kinetics; Cycle Types=LMW kinetics (60 s contact time, 120 s dissociation time, 100 ul/min flow rate, extra wash after injection with 50% DMSO, flow path 1,2,3,4); Flow path detection=2-1, 4-3).
- Secondary Binding Analysis by Surface Plasmon Resonance The His-tagged SARS-CoV-2 PLpro enzyme was initially prepared in phosphate buffer and diluted to 50 ug/mL with 10 mM sodium acetate (pH 5.5) and immobilized on a CM5 sensor chip by standard amine coupling with running buffer PBSP (10 mM phosphate, pH 7.4, 2.7 mM KCl, 137 mM NaCl, 0.05% Tween-20). The CM5 sensor chip surface was first activated by 1-ethyl-3-(3- (dimethylamino)propyl)carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) mixture using a Biacore 8K instrument (Cytiva). SARS-CoV-2 PLpro enzyme was immobilized to flow channels 1−4 followed by ethanolamine blocking on the unoccupied surface area, and immobilization levels for all four channels were similar at 12,000 RU. Each flow channel has its own reference channel, and blank immobilization using EDC/NHS and ethanolamine was done for all reference channels. Compound solutions with a series of increasing concentrations (0.049−30 uM at 2.5-fold dilution) were applied to all active and reference channels in SPR binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.05% Tween-20, 0.5 mM TCEP, and 2% DMSO) at a 30 uL/min flow rate at 25 °C. The data were double referenced with a reference channel and zero concentration (2% DMSO) responses, and reference subtracted sensorgrams were fitted with 1:1 Langmuir kinetic model using a Biacore Insight evaluation software, producing two rate constants (ka and kd) (Figure S1). The equilibrium dissociation constants (KD) were determined from two rate constants (KD = kd/ ka). For steady-state affinity fittings, response units at each concentration were measured during the equilibration phase, and the KD values were determined by fitting the data to a single rectangular hyperbolic curve equation, where y is the response, ymax is the maximum response, and x is the compound concentration.
- Surface Plasmon Resonance (SPR) Assay Surface plasmon resonance data was collected on a Biacore T200 or 3000 system (GE Healthcare) at 25° C. Streptavidin was immobilized on a CMS (GE Healthcare) or CMD500d sensor chip (XanTec Bioanalytics) using standard amine-coupling chemistry at 25° C. with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 12 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 μL/min. For capture of streptavidin, protein was diluted to 0.2 mg/mL in 10 mM sodium acetate (pH 4.5) and captured by injecting 100 μL onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5). Avi-tagged PCSK9 protein was captured on the streptavidin surface by injection of 150 μL of protein diluted to 16 μg/mL in HBS-N, 0.05% tween-20, 0.1 mM CaCl2. Typical surface densities obtained were 8000-10000 RU. SPR binding data were obtained using an appropriate dilution series of each compound at a flow rate of 30 μL/ min, with a capture time of 100 s and dissociation times of 300 s. Running buffer for compound binding studies was HBS-N, 0.05% tween-20, 0.1 mM CaCl2, 4% DMSO. Data were corrected for DMSO excluded volume effects. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software).
- Enzyme Assay P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of −1.4 psi, and upstream and downstream voltages of −3000 and −700 respectively.
- STING SPR Binding Assay Compounds were analyzed on an 5200 biacore SPR instrument (GE Healthcare). E.coli produced truncated STING protein was immobilized on a series S streptavidin chip via biotin capture (GE Healthcare #BR100531) with. Compounds were screened at 1:2 dilutions from 100 uM to 0.195 uM in run buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% P20, 1 mM TECEP). Steady state affinity and kinetic evaluations were carried out using 1:1 binding model (STING was treated as a dimer). Run parameters were as follows: 60 sec on, 300 sec off for the IFM compounds, cyclic-di-GMP (60 sec on/60 sec off), thiol isomer 1 (60 sec on/300 sec off) and cGAMP (60 sec on/1200 sec off) with a flow rate of 50 μL/min and data collection at 40 Hz at 25 □C.
- STING SPR Binding Assay Compounds were analyzed on an S200 biacore SPR instrument (GE Healthcare). E. coli produced truncated STING protein was immobilized on a series S streptavidin chip via biotin capture (GE Healthcare #BR100531) with. Compounds were screened at 1:2 dilutions from 100 uM to 0.195 uM in run buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% P20, 1 mM TECEP). Steady state affinity and kinetic evaluations were carried out using 1:1 binding model (STING was treated as a dimer). Run parameters were as follows: 60 sec on, 300 sec off for the IFM compounds, cyclic-di-GMP (60 sec on/60 sec off), thiol isomer 1 (60 sec on/300 sec off) and cGAMP (60 sec on/1200 sec off) with a flow rate of 50 μL/min and data collection at 40 Hz at 25° C.
- Surface Plasmon Resonance (SPR)-Based Assay All measurements were performed on a Biacore T100 instrument (GE Healthcare). The purified HIS6-tagged NS5BΔC21 polymerases were immobilized using non-covalent capturing to an NTA sensor chip (GE Healthcare) in immobilization buffer (20 mM MOPS pH 7.4, 500 mM NaCl, 0.005% Tween-P20, 1 mM DTT, 50 μM EDTA). Interaction studies were all performed at 25° C. Inhibitors were serially diluted in running buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 50 μM EDTA, 1 mM DTT, 0.005% Tween-P20) containing 5% dimethyl sulfoxide (DMSO). Single-cycle kinetics were used, in which 5 increasing concentrations of compound were injected for a period of 300 s each in 1 single cycle, and dissociation was monitored for a period of 1200 s. The sensor surface was completely regenerated in between the cycles.
- hFAP Binding Assay Table 1: Compounds were tested in a direct binding assay using 8K surface plasmon resonance biosensor (GE Healthcare) at 20° C. Immobilization of hFAP (M39-A757) on a CMD200M sensor chip (Xantec) was performed using standard amine coupling procedure in immobilization buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween20, pH 7.4). The surface was washed with 10 mM NaOH, 1M NaCl before being activated with EDC/NHS (GE Healthcare), followed by immobilization of hFAP (in 10 mM Acetate pH 5.0). Finally, the surface was deactivated by ethanolamine. Immobilization levels of hFAP were around 4000-6000 RU. The reference spot was treated as described, omitting the injection of hFAP. Compound concentration series were injected over the immobilized protein in increasing concentrations (2-500 nM) using single cycle kinetics in running buffer (20 mM TRIS, 150 mM NaCl, 0.05% Tween20, 1% DMSO, pH 7.4).
- Surface Plasmon Resonance (SPR) Binding interactions of ligands with STING proteins were quantified using Surface Plasmon Resonance (SPR) with a minimally biotinylated STING protein immobilized on Streptavidin chip surface. In this manner highly active STING protein surfaces were obtained that were not compromised by a low pH required for amine coupling methods. Minimal biotinylation of purified huSTING protein was performed using a previously described methodology (Chabbra 2012). Briefly, approximately 20 nmol of recombinant STING protein in 1×TBS buffer (25 mM Tris-HCl, 150 mM NaCl, 5 mM DTT) was mixed with of EZ-LinkH Sulfo-NHS-LC-LC-Biotin (Thermofisher Scientific, cat #21338) at a molar ratio of 1 to 0.6 and then incubated on ice for 2 hours. To remove any unreacted biotin reagent, protein/biotin mixture was passed through a Superdex 75 (10/300 GL) column equilibrated with 10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM DTT, 5% (v/v) glycerol. A protein peak containing biotinylated huSTING protein was collected and stored in aliquots at −80° C. Streptavidin was simultaneously immobilized in all four channels of a CM5 sensor chip docked in a Biacore instrument (either Biacore S200 or Biacore T200, GE Healthcare) as described previously (Zender 2013). Minimally biotinylated STING protein was captured onto Streptavidin coated chip surface at 25° C. in SPR binding buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 2.5 mM TCEP, 2% (v/v) DMSO) gradually injected in a single channel at a constant flow-rate of 2 μL/min until SPR response increases were no longer observed resulting in typical capture levels of 5000 to 7000 RU (1 RU=1 μg/mm2). To determine binding affinity, compound interaction with immobilized STING protein was analysed using dose-response experiments. All binding experiments were performed at 25° C. in SPR binding buffer. Fresh 10 mM DMSO solutions of compound were diluted directly into SPR binding buffer to a final concentration of 10 μM and then further diluted either 2-fold or 3-fold aiming for either a 5- or 7-point concentration series range. Each ligand concentration series was injected at a constant flow rate of 60 μL/min with a 90 second association and a 180 second dissociation time. Scrubber 2 (www.biologic.com.au) was utilized for data processing and analysis. Thus, SPR signals were referenced against the blank surface (streptavidin+D-biotin) and further corrected for DMSO refractive index change and then double-referenced using a buffer-blank injection (Papalia 2006). To determine binding affinities (KD values) from dose-response experiments, binding responses at equilibrium were fit to either a 1:1 steady state affinity or to 1:1 binding kinetic models (both available within Scrubber software).
- Biacore Assay The amount of a ligand immobilized on a sensor chip was adjusted within the range of 1608 RU to 8042 RU, so as to suppress mass transport limitation. As analytes, biocytin and Compound 29 (the structure thereof is shown below) synthesized in Example 1 were used. It is to be noted that biocytin is a main existence form of biotin. With regard to the concentrations of the analytes, in the case of biocytin, eight 2-fold dilution series were prepared from 1 μM, and in the case of Compound 29, twelve 2-fold dilution series were prepared from 16 μM. The measurement was carried out at a flow rate of 30 μl/min, for a contact time of 120 seconds, and for a dissociation time of 600 seconds. For the analysis of the data, sensorgrams for all concentrations were incorporated into analysis software, the equilibrium value analysis was then carried out, and the dissociation constant KD was then calculated.
- Biacore Assay The evaluation of the binding affinity (KD) of the compounds of formula (I) to the HSP90 protein was done using Biacore T100.His-HSP90 N-term domain was immobilized by capture of his tag by the Ab previously covalently bound to the chip (CM5) surface. A short cross-linking procedure after HSP90 binding was necessary in order to obtained a more stable signal.The compounds were analyzed using the following running buffer: 20 mM Tris/HCl pH 7.6, 150 mM KCl, 5 mM MgCl2, 0.05% P20 and 1% DMSO. The flow rate was 50 ul/min with and association time of 60 seconds.The cpds were diluted in running buffer from 100x stock solution; the different conc injected were obtained from a sequential dilution in order to have the same DMSO conc. in each sample and avoid any solvent correction.A series of 5 sequential injections of increasing concentrations of compound were performed and the results were analyzed using a method reported in literature (Robert Karlsson et al).
- Binding Assay Binding measurements were performed in parallel sets of either WT/G12D/G12C/G12V KRAS or WT K/H/N RAS proteins in GDP and/or GTPγS-loaded forms.Biacore instrument was desorbed and docked with a Series S Sensor Chip SA. The proteins were diluted to 50 μg/mL with the assay buffer (50 mM HEPES, 150 mM NaCl, 10 μM GDP for GDP-loaded proteins or 10 μM GTPγS for GTPγS-loaded proteins, 5 mM MgCl2, 0.5 mM TCEP, 5% glycerol, 0.02% Tween-20, 2% DMSO, pH 7.2) and immobilized at a flow rate of 3 μL/min at 10° C. with a contact time of 3-10 min. to capture ˜3000-4000 RUs of proteins on the surface. The functionalized surface was then equilibrated with assay buffer for approximately 1 hour. Un-functionalized SA surfaces with no immobilized protein served as reference for binding kinetic analysis. Compound binding kinetics were measured in either multi-cycle or single-cycle kinetic format.
- Caliper's mobility shift assay (MSA) FLT3 is a receptor tyrosine kinase involved in survival and proliferation of leukemic cells. Constitutively activating FLT3 mutations has been found in about 30% of all patients with acute myeloid leukemia (AML). The tested compounds were screened for their ability to inhibit FLT3 kinase activity using Caliper's mobility shift assay (MSA). The assay uses a microfluidic chip to measure the conversion of a fluorescent peptide substrate to a phosphorylated product following separation by electrophoresis. The signature of the fluorescence signal over time reveals the extent of the reaction. Protein tyrosine kinase assays were carried out in a final volume of 25 ul containing 0.9 nM purified FLT3 (Carna, Cat 08-154) enzyme protein, 50 mM HEPES [pH=7.5], 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT, 2% DMSO, 97 uM ATP, 1.5 uM peptide 2. Each Compound was added into the reaction to final concentrations from 300 nM to 0.015 nM (10 concentration points in 3 fold serial dilution).
- Enzyme Assay P70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components was then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide was analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of −1.4 psi, and upstream and downstream voltages of −3000 and −700 respectively. Product peaks were resolved before substrate peaks on the resulting chromatograms.
- Enzyme Assay p70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components were then added to the compound plate to initiate the enzyme reaction: p70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide was analyzed on a Caliper Life Sciences Lab Chip 3000, using a pressure of −1.4 psi, and upstream and downstream voltages of −3000 and −700, respectively. Product peaks were resolved before substrate peaks on the resulting chromatograms.
- Gel-Based Assay Tdp1 reactions were performed as recently described. Briefly, a 5'-[32P]-labeled single-stranded DNA oligonucleotide containing a 3'-phosphotyrosine (N14Y) incubated with 5 pM recombinant Tdp1 in the absence or presence of inhibitor for 15 min at room temperature in a buffer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM EDTA, 1 mM DTT, 40 ug/ml BSA and 0.01% Tween-20. Reactions were terminated by the addition of 1 volume of gel loading buffer [99.5% (v/v) formamide, 5 mM EDTA, 0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol blue]. Samples were subjected to a 16% denaturing PAGE and dried gels were exposed to a PhosphorImager screen (GE Healthcare). Gel images were scanned using a Typhoon 8600 (GE Healthcare) and densitometric analyses were performed using the ImageQuant software (GE Healthcare).Binding experiments were performed as recently described. Briefly, Tdp1 was amine coupled to a CM5 sensor chip (GE Healthcare, Piscataway N.J.).
- In-vitro HDAC Enzymatic Endpoint Assay Purified HDAC1-9 (0.5~5 nM) were incubated with 2 μM carboxyfluorescein (FAM)-labeled acetylated peptide substrate A or B and test compound for 60 min at room temperature, in HDAC assay buffer that contained 50 mM HEPES (pH 7.4), 100 mM KCl, 0.01% BSA and 0.001% Tween-20. Reactions were terminated by the addition of the known non-selective HDAC inhibitor LBH-589 (panobinostat) with a final concentration of 1.4 μM. Acetylated peptide substrate and deacetylated peptide product were separated on a Caliper LabChip EZ Reader II equipped with a 12-sipper chip in ProfilerPro separation buffer and fluorescence intensity in the substrate and product peaks was determined and analyzed using EZ Reader software (Caliper Life Sciences; Hopkinton, MA). The separation of substrate and product was performed under the following optimized conditions: upstream voltage = −500 V, downstream voltage = −1500V and pressure = −1.3 psi. The reactions were performed in duplicate for e
- Inhibition Assay Inhibition constants of MerTK, Flt3, Tyro3 and Axl kinase activity by an active compound as described herein is determined at the Km for ATP using a microfluidic capillary electrophoresis (MCE) assay in which phosphorylated and unphosphorylated substrate peptides were separated and analyzed using a LabChip EZ Reader.Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μl, of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 mM incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
- Interaction between Compound and Human PD-L1 Protein Example 40: In the experiment, the instrument OctectRED of Fortebio Company based on biofilm interferometry (BLI) technology was used to capture human PD-L1/AVI with SA chips. The concentration of antigen PD-L1/AVI was diluted to 10 μg/mL with running buffer (PBS+0.02% Tween-20+2% DMSO), and the loading time was 300 s; similarly, the analyte was also diluted to the corresponding concentrations (20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.3125 nM) with running buffer in gradient manner, and a buffer blank control group was set at the same time. The binding time of human PD-L1/AVI to the analyte was 180 s, and the dissociation time was 300 s; the chip was regenerated with 10 mM glycine HCl, pH 1.7 solution and repeated 5-second pulse for 3 times. The data were fitted to a 1:1 binding model to determine the equilibrium dissociation constant KD.
- P70S6K Enzyme Assay P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
- P70S6K Enzyme Assay P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
- P70S6K Enzyme Assay P70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components was then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide was analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of −1.4 psi, and upstream and downstream voltages of −3000 and −700 respectively. Product peaks were resolved before substrate peaks on the resulting chromatograms.
- SPR Analysis The interaction of PSMA-binding variants with human FOLH1 was measured with Surface Plasmon Resonance (SPR) technology. Biotinylated FOLH1 was captured at a concentration of 30 μg/mL onto a Streptavidin chip. Binding kinetics of the analytes were measured with a Biacore 8K instrument in two-fold serial dilutions starting at 500 nM down to 1.95 nM in single cycle kinetic measurements. After each cycle, needles were washed with 50% DMSO. To measure the association to the FOLH1 protein, the samples were injected with a flow rate of 30 μL/min for 90 s, followed by 1200 s buffer only to detect the dissociation. The used running buffer was 1×PBS with 0.05% Tween20 and 2% DMSO. The relative response units (RU, Y-axis) are plotted against time (s, X-axis) and analyzed in a kinetic 1:1 binding model. In a different setup, biotinylated FOLH1 was captured at a concentration of 0.8 μg/mL and the running buffer was 1×PBS with 0.05% Tween20.
- Surface Plasmon Resonance (SPR) Assay Binding of inhibitors to expressed cyclophilins was determined using surface plasmon resonance (SPR) experiments. Briefly, avi-tagged cyclophilin proteins with mono-biotinylation were immobilized onto a Biotin CAPture chip (GE Healthcare, cat. #28920234). SPR experiments were carried out on an upgraded Biacore T200 system using a running buffer containing the inhibitor, 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% P20, and 3% DMSO. Single-cycle kinetics measurements were used to study the interactions between cyclophilin inhibitors and different cyclophlins. Accordingly, compound at various concentrations was injected to the flow cells with a contact time of 1 min each at a flow rate of 50 μL/min. The final dissociation phase lasts for 10 min following the sample injection. Data was analyzed using Biacore T200 Evaluation Software Version 1.0 and a 1:1 binding model was applied to fit the data to obtain kon, koff, and KD.
- Surface Plasmon Resonance (SPR) Binding kinetics were determined by surface plasmon resonance (SPR) using a Biacore S51 (Biacore, Uppsala, Sweden). MPO (50 μg/ml dissolved in 10 mM sodium acetate, pH 5.0) was immobilized onto the surface of CM5 sensor chips (Biacore) using surface amine coupling. One of the spots on the sensor surface was left without MPO to control for nonspecific binding. The signal observed in response to analyte binding was as expected linearly related to the amount of immobilized ligand, and 10,000 response units was routinely used to characterize compoundbinding (data not shown). Compounds were dissolved in binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM Na2EDTA, 0.005% (w/v) surfactant P20, 1% (v/v) DMSO final), and association was assessed during 60-210-s injections. After this time, analyte injection was terminated, and the chip surface was perfused with binding buffer at 30 μl/min for 4-12 min to monitor compound dissociation.
- Surface plasmon resonance (SPR) The SPR assay is configured to examine solution inhibition of BCL-2 binding to peptide derivatized sensor surfaces to generate IC50 values as a measure of inhibitor potency. Solution Inhibition Assay Format: Biacore TM A100 (GE Healthcare, Uppsala, Sweden) was used to conduct all experiments reported herein. Sensor surface preparation and all interaction analyses experiments were performed at 25° C. Reagents were purchased from GE Healthcare. Running buffer containing 10 mM Hepes, pH7.4, 150 mM sodium chloride, 1.25 mM Dithiothreitol, 3% Dimethyl sulfoxide and 0.05% polysorbate 20 were utilized throughout all analyses. Biotinylated BAK, BAD and NOXA peptides were diluted to 10 nM in running buffer and captured onto a sensor surface pre-derivatized with streptavidin (sensor chip SA) to peptide surface densities in the range 50-100 R.U. Peptide captured surfaces were blocked with 50004 PEO2Biotin. A blank detection spot in each flowcell was similarly blocked with PEO2-biotin and served as a reference spot in the competition assay.
- hFAP Binding Assay Table 1B: Compounds were tested in a direct binding assay using 8K surface plasmon resonance biosensor (GE Healthcare) at 20° C. Immobilization of hFAP (M39-A757) on a CMD200M sensor chip (Xantec) was performed using standard amine coupling procedure in immobilization buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween20, pH 7.4). The surface was washed with 10 mM NaOH, 1M NaCl before being activated with EDC/NHS (GE Healthcare), followed by immobilization of hFAP (in 10 mM Acetate pH 5.0). Finally, the surface was deactivated by ethanolamine. Immobilization levels of hFAP were around 4000-6000 RU. The reference spot was treated as described, omitting the injection of hFAP. Compound concentration series were injected over the immobilized protein in increasing concentrations (2-500 nM) using single cycle kinetics in running buffer (20 mM TRIS, 150 mM NaCl, 0.05% Tween20, 1% DMSO, pH 7.4). Interaction models were fitted globally to the experimental traces, enabling determination of kon, koff and Kd.
- Caliper Assay All assays were performed in 384-well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, as well as four 8-point serial dilutions of staurosporine as a reference compound, plus 16 high and 16 low controls. Liquid handling and incubation steps were done on a Thermo CatX workstation equipped with Innovadyne Nanodrop Express. Between pipetting steps, tips were cleaned in wash cycles using wash buffer. The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 μL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 μM ATP, 4 μM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 μL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30 °C. for 60 minutes and subsequently terminated by addition of 16 μL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology. Briefly, samples from terminated kinase reactions were applied to the chip. Analytes are transported through the chip by constant buffer flow and the migration of the substrate peptide is monitored by the fluorescence signal of its label. Phosphorylated peptide (product) and unphosphorylated peptide (substrate) are separated in an electric field by their charge/mass ratio.
- Caliper Assay The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
- Caliper Assay Tyk2 & JAK2: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
- Surface Plasmon Resonance (SPR) Assay Using a GE Biacore 8K SPR instrument, avi-tagged SOS1 catalytic domain protein was immobilized to a level of approximately 6000 response units (RU) on a streptavidin-coated SPR sensor chip in assay buffer containing 0.01 M HEPES, 0.15 M NaCl and 0.05% v/v Surfactant P20. In assay buffer containing 2% DMSO, concentration series of test compounds were generated spanning 5 μM to 4.9 nM over ten 2-fold dilutions. For each test compound, a separate 0 μM sample was generated for use during subsequent double reference subtraction. Serially for each test compound, individual dilution samples were flowed over the immobilized SOS1 protein at a flow rate of 50 μL/minute to monitor the association with SOS1. Dissociation of bound test compound from the SOS1 protein was immediately monitored by flowing assay buffer over the sensor surface and monitoring the decrease in binding signal back to the baseline level seen in the absence of compound. This was repeated for all compound dilutions in each series.
- Surface Plasmon Resonance (SPR)-Based Biosensor Measurements (KD) and Enzyme Inhibition Assay (IC50) SPR experiments where performed with a Biacore A100 using Series S sensor chip CM5 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The proteins were amine coupled to the sensor. The immobilization levels were 5000-6000 RU (resonance units) for wt-thrombin and blocked thrombin (thrombin-DFP). The response used for affinity calculations was the differential steady-state binding signal between wt-thrombin and the blocked thrombin (the wt-thrombin signal minus that of the blocked thrombin). For equilibrium binding responses of compounds binding to thrombin (wt minus blocked), the affinity (KD) was calculated by fitting to the equation of a single-site binding model. The thrombin inhibitor potency (IC50) was measured with a chromogenic substrate method in a robotic microplate processor, using 96-well, half-volume microtiter plates. The linear absorbance increase values were determined by measurements at 405 nm during 40 min with Melagatran as control substance. The IC50 values were calculated by fitting the data to a three parameter equation by Microsoft XLfit.
- Tyk2 & JAK2 Caliper Assay The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
- p70S6K Enzyme Assay p70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components were then added to the compound plate to initiate the enzyme reaction: p70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide was analyzed on a Caliper Life Sciences Lab Chip 3000, using a pressure of −1.4 psi, and upstream and downstream voltages of −3000 and −700, respectively. Product peaks were resolved before substrate peaks on the resulting chromatograms. To assess the inhibitory potential of the compounds, IC50 values were determined, as shown above.
- Receptor Binding Assay 1. Experimental Materials1.1 Reagent MaterialsLigand: LANCL2Running buffer: 20 mM MES, 150 mM NaCl, 0.05% P20, pH 6.5, 1% DMSO1.2 EquipmentsEquipment name: Biacore S200Chip type: CM5 (29-1496-03)2. Experimental Methods2.1 Ligand CouplingLANCL2 protein was immobilized, and diluted to 50 μg/mL using sodium acetate solution at pH 4.0.Injection conditions: the CM5 chip surface was activated with a mixture of EDC/NHS with a flow rate of 10 μL/min and an injection time of 420 s; then LANCL2 injection was performed with a flow rate of 5 μL/min and an injection time of 2000 s, and the ligand coupling volume each time was about 1800 RU; finally, the chip surface was blocked with ethanolamine with a flow rate of 10 μL/min and an injection time of 420 s.Coupling buffer: 20 mM MES, 150 mM NaCl, 0.05% P20, pH 6.5.2.2 Experimental ConditionsAnalytes: All sample analytes of small molecule compounds were diluted 2-fold from a concentration of 50 μM to a concentration of 0.78 M, so as to obtain a compound solution containing 1% DMSO.Injection conditions for small molecule compounds: a flow rate of 30 μL/min, a binding time of 60 s, a dissociation time of 300 s.Running buffer: 20 mM MES, 150 mM NaCl, 0.05% P20, pH 6.5, 1% DMSO.Sample chamber temperature: 25° C.; analysis temperature: 25° C.MethodsKinetic determination of LANCL2-small molecule interactions was performed. The kinetic parameters of small molecules BT-11 and L-1 to 60 (analytes) binding to LANCL2 (ligand) were determined using BIACORE S200. Data were generated in triplicate in a dose-dependent manner (5-8 titration points) and analyzed to determine binding models (Langmuir, conformational shifts, etc.), real-time association and dissociation constants, and equilibrium dissociation constants. SPR technology allows the validation of specific LANCL2-phytochemical interactions as well as an increased insight into the gold standard of binding mechanisms and rates. Experiments were performed on carboxymethyl polydextrose (CM5) sensor chips by amine-coupled covalent attachment of LANCL2. Data were analyzed with BIACORE S200T200 evaluation software (version 1), so as to determine affinity binding constants (KD) using a 1:1 binding model.
- SPR Binding Assay All binding assays were performed on a ProteOn XPR36 SPR Protein Interaction Array System (Bio-Rad Laboratories, Hercules, CA, USA). The instrument temperature was set at 25° C. for all kinetic analyses. ProteOn GLH sensor chips were preconditioned with two short pulses each (10 s) of 50 mM NaOH, 100 mM HCl, and 0.5% sodium dodecyl sulfide. Then the system was equilibrated with running buffer (1×PBS pH 7.4, 3% DMSO and 0.005% polysorbate 20). The surface of a GLH sensor chip was activated with a 1:100 dilution of a 1:1 mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.2 M) and sulfo-N-hydroxysuccinimide (0.05 M). Immediately after chip activation, the HIV-1 CA protein constructs were prepared at a concentration of 10 μg/mL in 10 mM sodium acetate, pH 5.5, and injected across ligand flow channels for 5 min at a flow rate of 30 μL/min. Then, after unreacted protein had been washed out, excess active ester groups on the sensor surface were capped by a 5 min injection of 1M ethanolamine HCl (pH 8.0) at a flow rate of 5 μL/min. A reference surface was similarly created by immobilizing a nonspecific protein (IgG b12 anti-HIV-1 gp120; was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Anti-HIV-1 gp120 Monoclonal (IgG1 b12) from Dr. Dennis Burton and Carlos Barbas) and was used as a background to correct nonspecific binding. Serial dilutions of hACSS-2 inhibitors were then prepared in the running buffer and injected at a flow rate of 100 μL/min, for a 50 s association phase, followed by up to a 5 min dissociation phase using the “one-shot kinetics” capability of the ProteOn instrument. Data were analyzed using the ProteOn Manager Software version 3.0 (Bio-Rad). The responses from the reference flow cell were subtracted to account for the nonspecific binding and injection artifacts. Experimental data were fitted to a simple 1:1 binding model. Experiments were performed in triplicate to detect kinetic and equilibrium dissociation constants (KD).
- In Vitro AKT1 Kinase Assay This assay detects inhibitors of AKT1 (PKBα) kinase activity using Caliper LabChip LC3000. The Caliper off-chip incubation mobility shift assay uses a microfluidic chip to measure the conversion of a fluorescent labelled peptide to a phosphorylated product by a respective kinase. The complete enzyme reaction is carried out in microtitre plates and then quenched. The resulting stopped solutions are serially "sipped" through a capillary onto the chip, where the peptide substrate and phosphorylated product are separated by electrophoresis. They are then detected via laser-induced fluorescence. Substrate and product are separated into two peaks by the application of a high electric field and directly detected using fluorescence. The signature of the fluorescent signal reveals the extent of the reaction.For Echo dosing the solvent was 100% DMSO. A master plate was prepared with 40 ul of 10 mM stock from our Primary Liquid Store in quadrant 1 of a Labcyte 384 well plate. A 1 in 100 dilution was made from quadrant 1 into quadrant 2 by removing 0.4 ul and adding it to 39.6 ul of DMSO. Subsequent 1 in 100 dilutions were made into quadrant 3 from quadrant 2 and quadrant 4 from quadrant 3. Multiple 2.5 nl droplets were dispensed from each quadrant of the master plate using ECHO dosing technology (Labcyte Inc. Sunnyvale, Calif., USA) to generate the dose range that was required in the test. The dose range most commonly used was as follows: 100 uM, 30 uM, 10 uM, 3 uM, 1 uM, 0.3 uM, 0.1 uM, 0.03 uM, 0.01 uM, 0.003 uM, 0.001 uM, 0.0001 uM. Each well was backfilled with Dimethyl Sulphoxide (DMSO) to a total volume of 120 nl, such that when the enzyme and substrate mix was added the final DMSO concentration was 1%. DMSO was added to max control wells as 120 nl, minimum control wells were treated with 120 nl of compound at a concentration that inhibited the enzyme activity 100%. Following addition of compound or control to the assay plate, 6 ul peptide mix containing 3 uM substrate (5-FAM-GRPRTSSFAEG-CONH2; CRB) and 40 uM ATP in Kinase base buffer (100 mM Hepes pH 7.5, 0.015% Brij-35) and 6 ul enzyme mix containing 8 nM AKT1/PKBα active enzyme (Upstate Biotechnology, Cat No. 14-276), 8 mM DTT and 20 mM MgCl2 in kinase base buffer was added. All buffers were made up with 18M water. The plates were sealed and incubated at room temperature for 50 minutes. The reaction was stopped by the addition of 10 ul stop buffer (100 mM Hepes pH 7.5, 0.015% Brij-35 solution, 0.1% coating reagent #3, 40 mM EDTA, 5% DMSO) to each well (N.B. plates can be frozen after stopping and read later). The plates were then analysed using the Caliper LabChip LC3000 Drug Discovery System (Caliper Life Sciences, 1 Wellfield, Preston Brook, Runcorn, WA7 3AZ) using the following separation conditions; -1.8 PSI, -500 upstream voltage, -1700 downstream voltage, sample sip time of 0.2 sec, post sample sip time of 30 sec and a final delay of 120 sec. Integration of the substrate and product peaks was carried out using Caliper LabChip software and IC50 curves were calculated using Origin software (OriginLab Corporation, Northampton, Mass., USA).
- Binding Assay hERG: The purpose of the hERG binding assay is to evaluate the effects of test compounds on the voltage-dependent potassium channel encoded by the human ether go go-related gene (hERG) using a constitutively expressing CHO cell line on the Nanion Syncropatch 384PE automated patch clamp system.The assay was conducted as follows with all reagents used at room temperature unless otherwise stated.Reagent Preparations Include:1. Internal "IC700" solution used to perfuse the underside of chip (in mM), KF 130, KCl 20, MgCl2 1, EGTA 10 and HEPES 10, (all Sigma-Aldrich; pH 7.2-7.3 using 10 M KOH, 320 mOsm) and supplemented with 25 μM escin.2. External and cell buffer (in mM), NaCl 137, KCl 4, HEPES 10, D-glucose 10, CaCl2 2, MgCl2 1 (pH7.4, NaOH)3. NMDG "reference" buffer used to establish a stable baseline prior to the addition of test compounds, NaCl 80, KCl 4, CaCl2 2, MgCl2 1, NMDG Cl 60, D-Glucose monohydrate 5, HEPES 10 (pH7.4 NaOH 298 mOsm)4. Seal enhancer used to improve seal quality of cells, NaCl 80, KCl 3, CaCl2 10, HEPES 10, MgCl2 1 (pH7.4 NaOH)
- Caliper Enzyme Assay Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
- Inhibition Assay The kinase activity of purified human MAP4K4 kinase domain was measured by monitoring the phosphorylation of a peptide substrate derived from moesin protein (Leu-Gly-Arg-Asp-Lys-Tyr-Lys-Thr-Leu-Arg-Gln-Ile-Arg-Gln) fluorescently labeled on the N-terminus with 5-carboxyfluorescein using the Caliper LabChip technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (IC50), compounds were serially diluted in DMSO and added to 10 uL kinase reactions containing 1 nM purified MAP4K4 enzyme, 1 uM peptide substrate, 10 uM ATP, 10 mM MgCl2, 1 mM EGTA, 50 mM Hepes pH 7.2, 1 mM DTT, 0.01% Triton X-100, and 2% DMSO. Reactions were incubated at room temperature in Perkin Elmer Proxiplates for 45 minutes and stopped by the addition of 10 uL of an EDTA-containing solution (50 mM Hepes pH 7.2, 40 mM EDTA, 0.02% Triton X-100). The fraction of phosphorylated peptide was determined as a fraction of total peptide substrate using the Caliper Lab Chip 3000 according to the manufacturer's instructions. IC50 values were determined using the four-parameter non-linear fit model.
- Kinase (PDGFRα, PDGFRβ, ABL1 and FLT3) Inhibition Test of Compounds The method adopted in the test was Caliper Mobility Shift Assay, which was a detection platform based on the mobility detection technology of microfluidic chip technology. Test steps: 1.25 kinase reaction buffer (62.5 mmol/L HEPES, pH 7.5; 0.001875% Brij-35; 12.5 mmol/L MgCl2; 2.5 mM DTT) and kinase reaction stop solution (100 mmol/L HEPES, pH 7.5; 0.015% Brij-35; 0.2% Coating Reagent #3) were configured. 10 μL of 2.5 kinase solution (adding kinase in 1.25 kinase reaction buffer) was added into 5 μL of compound solution with 5 concentration (dissolved in DMSO, diluted 10 times with water), incubated at room temperature for 10 minutes, then added with 10 μL of 2.5 substrate peptide solution (adding FAM labeled peptide and ATP in 1.25 kinase reaction buffer), reacted at 28 C. for a specific time, and then added with 25 μL of kinase reaction stop solution. Collected data was tested on Caliper to yield that inhibition ratio to kinase activity=(max−conversion)/(max−min) 100. max was DMSO control without adding compound, and min was low control.
- MAP4K4 Inhibition Assay The kinase activity of purified human MAP4K4 kinase domain was measured by monitoring the phosphorylation of a peptide substrate derived from moesin protein (Leu-Gly-Arg-Asp-Lys-Tyr-Lys-Thr-Leu-Arg-Gln-Ile-Arg-Gln) fluorescently labeled on the N-terminus with 5-carboxyfluorescein using the Caliper LabChip technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (IC50), compounds were serially diluted in DMSO and added to 10 uL kinase reactions containing 1 nM purified MAP4K4 enzyme, 1 uM peptide substrate, 10 uM ATP, 10 mM MgCl2, 1 mM EGTA, 50 mM Hepes pH 7.2, 1 mM DTT, 0.01% Triton X-100, and 2% DMSO. Reactions were incubated at room temperature in Perkin Elmer Proxiplates for 45 minutes and stopped by the addition of 10 uL of an EDTA-containing solution (50 mM Hepes pH 7.2, 40 mM EDTA, 0.02% Triton X-100). The fraction of phosphorylated peptide was determined as a fraction of total peptide substrate using the Caliper Lab Chip 3000 according to the manufacturer's instructions. IC50 values were determined using the four-parameter non-linear fit model.
- SPR Assay to Determine Binding Affinity to FKBP51 Biotinylated avi-FKBP51 was immobilized on a streptavidin chip (Cytiva Series S SA) using a Biacore 8K or 8 k+ (Cytiva). To achieve an immobilization level of 2000 RU, 3 μg/ml biotinylated avi-FKBP51 were injected for 360 sec at a flow rate of 10 μl/min. Rapalogs were diluted in DMSO to 100× working concentration. Each Rapalog was 100-fold diluted in 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20 and a serial dilution prepared (8 concentrations, 3-fold dilutions, 0.5-1000 nM). Rapamycin was used as reference sample (8 concentrations, 3-fold dilutions, 0.5-1000 nM). The compound dilutions were then injected at 100 uL/min for 120 seconds contact time and with 3600 seconds dissociation time with increasing concentrations. 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20, 1% DMSO was used as running buffer. Multi-cycle kinetics data were fit to a 1:1 binding model to measure the association rate ka (1/Ms), the dissociation rate kd (1/s) and the affinity Kd (M).
- Surface Plasmon Resonance Assay Using a Biacore T200, Avidin tagged VHL co-expressed with Elongins B and C were immobilized to a Biacore SA chip in running buffer without DMSO. Compounds were tested individually at varying concentrations in running buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 0.5 mM TCEP, 0.001% Tween 20, 0.2% PEG3350, 2% DMSO) at 20° C. Sensorgrams were run in order from low to high concentration using a flow rate of 80 μL/min. Association and disassociation times were varied depending on the estimated potency of the compound tested. All sensor chips were monitored for loss of activity with the injection of a control compound ((2S,4R)-1-((S)-2-acetamido-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide, which is Compound 7 in C. Galdeano et al, J. Med. Chem. 2014, 57, 8657-8663, herein incorporated by reference) which retained greater than 75% of the activity over the course of the run. Analysis of the binding curves and determination of the kinetic parameters were done using evaluation software (Version 2.0, Biacore).
- WNK1 Mobility Shift Assay A mixture of fluorescein labeled OSR1 peptide substrate (Toray Research Center, Inc.) and ATP was prepared with final concentrations of 10 and 25 μM, respectively, in 7 μL of reaction buffer [20 mM HEPES Na (pH 7.5), 1 mM MnCl2, 0.01% Tween 20, and 2 mM DTT]. Compounds at final concentrations from 0.003 to 100 μM in 1 μL were added to the peptide/ATP mixture at a final DMSO concentration of 10%. The reaction was initiated by the addition of 2 μL of GST-WNK1 (1−491; Carna Biosciences) that was 86% pure at a final concentration of 25 nM in a 384-well plate. After incubation for 3 h at 25 °C, where 15% conversion of the substrate to the product was observed, 10 μL of a stop buffer containing 0.2% coating-3 reagent with 2 mM EDTA was added. For 800 μM ATP, the WNK1 assay was performed for 50 min at 25 °C, where 15% conversion of the substrate to the product was observed. Aliquots from each well were transferred onto a four-sipper chip on a Labchip3
- Caliper Assay A kinase selectivity panel which measures substrate peptide phosphorylation was set-up for wild-type ALK2 (aa172-499), ALK2 FOP mutant (aa172-499 R206H), ALK1 (aa166-493), ALK5 (aa162-503) and ALK6 (aa168-495). The technology used for the described assay is based on the separation and quantification of substrate and product in an electrical field. In the course of the kinase reaction the peptide substrate is phosphorylated by a kinase. The transfer of a phosphate residue also causes the introduction of two additional negative charges and hence to a change in the net charge of the phospho-peptide compared to the unphosphorylated peptide. Due to this difference in charge the phosphorylated und unphosphorylated peptides migrate with different velocities in an electrical field.In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples ( 12-sipper chip , Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 m Na3VO4, 10 mM If-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO. Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
- Kinase Assay A kinase selectivity panel which measures substrate peptide phosphorylation was set-up for recombinant human Jak1 (aa 866-1154), Jak2 (aa808-1132), Jak3 (aa811-1124) and Tyk2 (aa888-1187). The technology used for the described assay is based on the separation and quantification of substrate and product in an electrical field. In the course of the kinase reaction the peptide substrate is phosphorylated by a kinase. The transfer of a phosphate residue also causes the introduction of two additional negative charges and hence to a change in the net charge of the phospho-peptide compared to the unphosphorylated peptide. Due to this difference in charge the phosphorylated und unphosphorylated peptides migrate with different velocities in an electrical field.In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 3 nM of each kinase together with 2 uM of a fluorescently labeled substrate peptide specific for each enzyme (Jak1 and Jak3 substrate FITC-Ahx-KKSRGDYMTMQIG-NH2, Jak2 and Tyk2 substrate 5(6)-Carboxyfluorescein-Ahx-GGEEEEYFELVKKKK) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 uM Na3VO4, 10 mM -Glycerolphosphate, specific concentrations of MgCl2 (Jak1 12 mM, Jak2 and Tyk2 9 mM, Jak3 1.5 mM) and 45 uM ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO. Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
- Assay for Inhibition of CDK2/CyclinE1 The CDK2 enzyme assay for IC50 determination was performed as follows. Microfluidic kinase detection technology (Caliper) was used to monitor the phosphorylation of peptide substrate by CDK2/CyclinE1. The total reaction volume was 15 μL containing buffer A (100 mM HEPES (pH 7.5), 0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl2, 10 μM Sodium Orthovanadate, 10 μM Beta-Glycerophosphate), 100 μM ATP, 5 nM CDK2/CyclinE1 (SignalChem, C29-18G), 5 μM FL-18 (5-FAM-QSPKKG-NH2), and the test compound at appropriate dilutions in DMSO. All components were added to the 384-well plate (Corning, 4514), and incubate at Room Temperature for 3 hours. The reaction was terminated by addition of 15 μL Stop Buffer (180 mM HEPES (pH 7.5), 20 mM EDTA, Coating-3 reagent (PerkinElmer, 760050)). The plate was loaded on Caliper EZ Reader (EZ Reader II, PerkinElmer, HD-4HYSG2772), and the reaction mixtures including substrate and product were sipped into the microfluidic chip for separation and detection. The IC50 values of the test compound were determined by fitting the inhibition curves by 4 parameter sigmoidal dose-response model using the Xlfit5/GraphPad Prism 5 software.
- Assay for Inhibition of CDK4/CyclinD1 The CDK4 enzyme assay for IC50 determination was performed as follows. Microfluidic kinase detection technology (Caliper) was used to monitor the phosphorylation of peptide substrate by CDK4/CyclinD1. The total reaction volume was 15 μL containing buffer A (100 mM HEPES (pH 7.5), 0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl2, 10 μM Sodium Orthovanadate, 10 μM Beta-Glycerophosphate), 200 μM ATP, 1 nM CDK4/CyclinD1 (Thermofisher, PR8064A), 1 μM FL-34 (5-FAM-RRRFRPASPLRGPPK), and the test compound at appropriate dilutions in DMSO. All components were added to the 384-well plate (Corning, 4514), and incubated at Room Temperature for 3 hours. The reaction was terminated by addition of 15 μL Stop Buffer (180 mM HEPES (pH 7.5), 20 mM EDTA, Coating-3 reagent (PerkinElmer, 760050)). The plate was then loaded on Caliper EZ Reader (EZ Reader II, PerkinElmer, HD-4HYSG2772), and the reaction mixtures including substrate and product were sipped into the microfluidic chip for separation and detection. The IC50 values of the test compound were determined by fitting the inhibition curves by 4 parameter sigmoidal dose-response model using the Xlfit5/GraphPad Prism 5 software.
- Assay for Inhibition of CDK6/CyclinD3 The CDK6 enzyme assay for IC50 determination was performed as follows. Microfluidic kinase detection technology (Caliper) was used to monitor the phosphorylation of peptide substrate by CDK6/CyclinD3. The total reaction volume is 15 μL containing buffer A (100 mM HEPES (pH 7.5), 0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl2, 10 μM Sodium Orthovanadate, 10 μM Beta-Glycerophosphate), 300 μM ATP, 2 nM CDK6/CyclinD3 (Carna, 04-107), 1 μM FL-34 (5-FAM-RRRFRPASPLRGPPK), and the test compound at appropriate dilutions in DMSO. All components were added to the 384-well plate (Corning, 4514), and incubated at Room Temperature for 3 hours. The reaction was terminated by addition of 15 μL Stop Buffer (180 mM HEPES (pH 7.5), 20 mM EDTA, Coating-3 reagent (PerkinElmer, 760050)). The plate was then loaded on Caliper EZ Reader (EZ Reader II, PerkinElmer, HD-4HYSG2772), and the reaction mixtures including substrate and product were sipped into the microfluidic chip for separation and detection. The IC50 values of the test compound were determined by fitting the inhibition curves by 4 parameter sigmoidal dose-response model using the Xlfit5/GraphPad Prism 5 software.
- BTK IC50 Enzyme Assay 2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM Hepes, pH 7.5, and 10 mM DTT.5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27 C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM Hepes, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorilation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel, Switzerland).
- In Vitro Studies All assay reaction conditions for IC50 determinations were within the linear range with respect to time and enzyme concentration. In a 384 well polypropylene plate, c-FMS (0.14 nM, Carna 08-155) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 μM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 70 pM c-FMS, 1.5 μM peptide substrate and 500 μM ATP (ATP Km). The reaction was incubated at room temperature for 120 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi.
- SPR Assay The anti-His antibody (His Capture Kit, GE Healthcare) was immobilized on sensor chip CM5 (GE Healthcare), in a Biacore T200 or Biacore S200 instrument, to ∼10000 resonance units (RU) using a standard amine coupling procedure (Amine Coupling Kit, GE Healthcare). His-tagged aldose reductase (ALR2), at a concentration of 100 μg/mL, was captured on the anti-His antibody using dual injection consisting of a 3 min injection of protein followed by a 100 s activation with a mixture (1:1) of 0.2 M N-ethyl-N-(dimethylamino)propylcarbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS), and final blocking with an injection of 1.0 Methanolamine (pH 8.5) for 100 s. Activation with EDC and NHS and blocking with ethanolamine stabilized the ALR2 capture and allowed the use of a single surface for the analysis of a number of compounds with no need for regeneration. The ALR2 capture level was ∼1000 RU, and all injections were performed at a flow rate of 10 μL/min. For the interaction analysis, the compound concentrations ranged from 3 to 10000 nM with a 5-fold dilution step, using a single-cycle kinetic approach with three or five concentrations or injections per cycle.
- SPR Assay to Determine Binding Affinity to FKBP12 Biotinylated avi-FKBP12 was immobilized on a streptavidin chip (Cytiva Series S SA) using a Biacore 8K or 8 k+ (Cytiva). To achieve an immobilization level of 1000 RU, 2 μg/ml biotinylated avi-FKBP12 were injected for 100 sec at a flow rate of 10 μl/min. Rapalogs were diluted in DMSO to 100× working concentration. Each Rapalog was 100-fold diluted in 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20 and a serial dilution prepared (9 concentrations, 3-fold dilutions, 0.08-500 nM). Rapamycin was used as reference sample (9 concentrations, 3-fold dilutions, 0.02-100 nM). The compound dilutions were then injected at 100 uL/min for 120 seconds contact time in sequence with increasing concentrations. Dissociation was monitored for 3600 seconds. 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20, 1% DMSO was used as running buffer. The single-cycle kinetics data were fit to a 1:1 binding model to measure the association rate ka (1/Ms), the dissociation rate kd (1/s) and the affinity Kd (M).
- hERG Electrophysiological Assay Electrophysiological recordings (all performed at RT) from stably transfected CHO hKv11.1 cells were obtained using the Nanion Syncropatch 768PE. Test compounds, vehicle or positive controls were added with 6 compound plates each at a different concentration to allow cumulative dosing onto cells (10 mM, 3.167 mM, 1 mM, 0.3167 mM, 0.1 mM, 0.03167 mM). 600 Figure US11325906-20220510-P00001of compound is resuspended into 90 μl of reference buffer (in mM, NaCl 80, KCL 4, CaCl 5, MgCl 1, NMDG Cl 60, D-Glucose monohydrate 5, HEPES 10 (pH7.4 HCL, 298 mOsm) for a final compound concentration of 39.6 μM, 13.2 μM, 4.4 μM, 1.46 μM, 0.48 μM, 0.16 μM. For each Nanion Syncropatch 768PE run, the current amplitude in each cell in the presence of extracellular solution (in mM, NaCl 80, KCL 4, CaCl 5, MgCl 1, NMDG Cl 60, D-Glucose monohydrate 5, HEPES 10 (pH7.4 HCL, 298 mOsm) is measured with all liquid additions performed using the Syncropatch liquid handling system. Add 40 μL external solution (in mM, HBPS, CaCl2 2, MgCl2 1 (pH7.4, NaOH) to 384 well multihole medium resistance recording chip and perfuse internal buffer (in mM, KF 130, KCl 20, MgCl2 1, EGTA 10, HEPES 10, Escin 25 (all Sigma-Aldrich; pH 7.2-7.30 using 10 M KOH, 320 mOsm) to the underside of plate. Dispense 20 μL of cells at a density of 1e6 cells/ml maintained at 9° C. into each well of the chip followed by 20 μL of seal enhancer (in mM, NaCl 80, KCl 3, CaCl 10, HEPES 10, MgCl 1 (pH7.4 NaOH). Perform wash step leaving a residual volume of 40 μL. Dispense 40 μL of reference buffer to establish a stable baseline prior to the addition of test compounds, with a removal step of 40 μL after 3 min, repeat this step. Dispense 40 μL of compound concentration 1 (0.16 μM), real time recordings for 3 min exposure prior to removal of 40 μL. This step is repeated for 5 further subsequent compound plates to generate cumulative curve analysis. All data is leak subtracted, 2 pulses to −80 mV 100 ms with 100 ms delay. Outward K+ currents are then evoked by voltage step to +60 mV from a holding potential of −90 mV, Each pulse is delivered at a frequency of 2 Hz with a 15 s pulse interval.
- SPR Assay to Characterize Ternary Complex Formation with FKBP12 Biotinylated avi-FKBP12 was immobilized on a streptavidin chip (Cytiva Series S SA) using a Biacore 8K or 8 k+ (Cytiva). To achieve an immobilization level of 100 RU, 0.3 g/ml biotinylated avi-FKBP12 were injected for 80 sec at a flow rate of 10 l/min. Serial dilution of FRB was prepared (12 concentrations, 3-fold dilutions, 0.00011-20 M) and supplemented with 100 nM of rapalog. A-B-A injection mode was used to ensure saturation immobilized FKBP12 with respective rapalog. 100 nM solution of the respective rapalog was injected before FRB injection for 120 sec and during dissociation for 420 sec. The FRB dilutions were then injected 120 seconds contact time with increasing concentrations. Rapamycin was used as reference sample. 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20, 1% DMSO was used as running buffer at a flow rate of 30 l/min. The multi-cycle kinetics data were fit to a 1:1 binding model to measure the association rate ka (1/Ms), the dissociation rate kd (1/s) and the affinity Kd (M). In case of fast association and dissociation, steady state affinity analysis following the law of mass action was used to determine the affinity Kd (M).
- SPR Assay to Characterize Ternary Complex Formation with FKBP51 Biotinylated avi-FKBP51 was immobilized on a streptavidin chip (Cytiva Series S SA) using a Biacore 8K or 8 k+ (Cytiva). To achieve an immobilization level of 200 RU, 0.6 g/ml biotinylated avi-FKBP51 were injected for 150 sec at a flow rate of 10 l/min. Serial dilution of FRB was prepared (12 concentrations, 3-fold dilutions, 0.00011-20 M) and supplemented with 100 nM of rapalog. A-B-A injection mode was used to ensure saturation immobilized FKBP12 with respective rapalog. 100 nM solution of the respective rapalog was injected before FRB injection for 120 sec and during dissociation for 420 sec. The FRB dilutions were then injected 120 seconds contact time with increasing concentrations. Rapamycin was used as reference sample. 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20, 1% DMSO was used as running buffer at a flow rate of 30 l/min. The multi-cycle kinetics data were fit to a 1:1 binding model to measure the association rate ka (1/Ms), the dissociation rate kd (1/s) and the affinity Kd (M). In case of fast association and dissociation, steady state affinity analysis following the law of mass action was used to determine the affinity Kd (M).
- Surface Plasmon Resonance (SPR) SOS1 Binding Assay Binding to SOS1 was measured using a SPR assay with purified recombinant human SOS1 substrate (res. 564-1049 with N-terminal Avi tag; purified and biotinylated based on Hillig, et al., Proc Natl Acad Sci USA (2019); 116(7):2551-2560). SPR measurements were performed on a Biacore 8K SPR instrument (GE Healthcare, Sweden). Assays were performed at 25° C. using Series S SA sensor chips pre-coated with streptavidin (GE Healthcare, Cat. BR100531). Biotinylated SOS1 diluted in sample buffer (20 mM Tris HCl, 150 mM NaCl, 1 mM DTT, 0.05% TWEEN 20, 1 mM MgCl2, pH 8.0) was captured to one flow cell of the chip to about 3,000 resonance units (RU) using sample buffer supplemented with 5% DMSO as a running buffer. Serial dilutions of the assayed compounds in the running buffer at 100, 50 or 0.5 μM were injected for 60 s at a flow rate of 30 μL/min and association phases were recorded. Dissociation of the samples was monitored for 600 s. Data processing was performed using Biacore Insight Software (Biacore, GE Healthcare). Sensorgrams recorded on a SA flow cell without captured protein were subtracted from sensorgrams recorded on the SOS1 surface. Blank injections of running buffer were used for double referencing and solvent correction was applied to all sample sensorgrams to correct for buffer mismatches.
- TrkA Activity Reagents and consumables were purchased from Sigma Aldrich, Carna Biosciences, or Caliper Life Sciences. All assay reaction conditions for IC50 determinations were within the linear range with respect to time and enzyme concentration. In a 384 well polypropylene plate, TrkA (0.4 nM, Carna 08-186) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 μM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 200 pM TrkA, 1.5 μM peptide substrate and 55 μM ATP (ATP Km). The reaction was incubated at room temperature for 180 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi.
- Cbl-b and c-Cbl SPR Assay Affinity of binding to Cbl-b and c-Cbl for compounds described herein can be assessed by surface plasmon resonance (SPR) according to the following protocol. All experiments were recorded on a Biacore™ 8K or Biacore™ 8K+(Cytiva) with both surface preparation and experimental measurements performed at 20° C. in an assay buffer consisting of 50 mM HEPES, pH 7.5, 0.15 M NaCl, 0.001% (v/v) Tween® 20, 0.2 mM tris(2-carboxyethyl)phosphine, 0.025% (w/v) carboxymethylated dextran (average MW 10 kDa), 0.2% (w/v) PEG 3350, and 2% DMSO.Human Cbl-b (residues 40-426) or c-Cbl (residues 47-435) were irreversibly captured to a Series S sensor chip SA (Cytiva 29104992) via an N-terminal avi-tag, biotinylated by co-expression in E. coli with BirA. A surface capture range of 1300-1500 RU of protein was used for both isoforms.For SPR measurements, 6 concentrations with 2 fold serial dilution were measured with blanks flanking each series for double referencing. Initial concentrations between 20 and 0.5 μM were used depending on the anticipated affinity of the tested compound. SPR sensorgrams were recorded in multi-cycle kinetics format, with a contact time of 60 seconds and a flow rate of 40 μl/min, the dissociation time was varied between 120-1200 seconds aiming for 4-5 half-lives of the measured interaction.
- Enzymatic HDAC Activity Assay Biochemical assays of HDAC activity were carried out by Nanosyn in a reaction volume of 10 ul in 384-well microplates. A standard enzymatic reaction contained 5 ul of 2x HDAC inhibitor, 4 ul of 2.5x enzyme, and 1 ul of 10x substrate in assay buffer (100 mM HEPES, pH 7.5, 25 mM KCl, 0.1% BSA, 0.01% Triton X-100, 1% DMSO). Final concentration of all HDACs in the enzymatic assays was between 0.5 and 5 nM. A final substrate concentration of 1 uM FAM-RHKK(Ac)-NH2 or FAM-RHKK(trifluoroacetyl)-NH2 was used in all assays and found to be below the determined Km,app for each enzyme. All inhibitors were serially diluted in DMSO prior to cross-dilution in assay buffer and were incubated with enzyme for 15 min prior to initiating the reaction by the addition of substrate. After incubation for 3 h, the reaction was terminated by the addition of EDTA and SDS to final concentrations of 24 mM and 0.04%, respectively. The product and substrate in each reaction were separated using a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, MA) run on a Caliper LC3000 (Caliper Life Sciences). The separation conditions used a downstream voltage of -800V, an upstream voltage of -3000 V, and a screening pressure of -1.4 p.s.i. The product and substrate fluorescence was excited at 488 nm and detected at 530 nm.
- JAK Caliper Enzyme Assay Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
- JAK Caliper Enzyme Assay Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 4 μM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
- JAK Caliper Enzyme Assay at 1 mM ATP Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration. Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 M peptide substrate. The JAK3 assays contained 1 M of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
- JAK Caliper Enzyme Assay at 4 uM ATP Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration. Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 4 uM ATP and 1 M peptide substrate. The JAK3 assays contained 1 M of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
- Surface Plasmon Resonance (SPR) Assay Recombinant full-length hnRNPA1 (aa 1-320) and truncated versions of hnRNPA1, including the N-terminal RNA binding domain (aa 1-196), the middle region (aa 182-268), and the C-terminal region (aa 268-320), were individually covalently coupled to a dextran matrix (CM5 chip) using a Biacore T200 system (GE Healthcare), following the manufacturer's protocols. Immobilized proteins were activated with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to form a carbodiamide linkage between carboxyl moieties on the dextran and primary amide groups on the protein. hnRNPA1 variants were dissolved in 10 mM acetate buffer (full-length, pH 4; N-terminal RNA binding domain and the C-terminal region, pH 4.5; middle region, pH 5) to a concentration of 20 μg/ml and flowed over the activated surface for 5 min with PBS. Unreacted groups were blocked with ethanolamine. PBS containing 0.05% (v/v) P20 (surfactant) and 5% DMSO was used for priming and conditioning the surface of the chips. Quercetin was serially diluted (from 200 to 3 μM) in PBS buffer and maintained in DMSO (5% (v/v)) (final concentrations from 10 to 0.15 μM). The baseline response was determined for 120 s (30 μl/min) before perfusing the immobilized proteins with the compounds to allow association to occur. Dissociation was then monitored over a period of an additional 120 s. The immobilized proteins were regenerated with 50 mM NaOH.
- SyncroPatch Recording Assay At the beginning of each assay, 20 μl of cell suspension was dispensed into each well of a multi-hole 384-well SyncroPatch chip by the onboard pipettor. Cell sealing was initiated, and seal enhancer solution was added to facilitate seal formation. Upon completion of sealing, cells were washed 3 times with extracellular recording solution and the assay voltage protocol was started. Human Kv7.2, Kv7.4 or Kv7.5/7.3 channels were evaluated using a voltage protocol in which cells were voltage-clamped at a holding potential of −60 mV. Potassium currents were continuously activated with a series of three voltage steps to −30 mV for 3 seconds, 40 mV for 1 second and −90 mV for 4 seconds with 12 seconds between successive voltage sweeps. Potassium currents were measured from the −90 mV repolarizing step. Baseline current was assessed for 3.5 minutes prior to the addition of 5.6 μM zinc pyrithione (1 μM for Kv7.4). Kv7.2, Kv7.4 or Kv7.5/7.3 current in the presence of zinc pyrithione was acquired over five minutes to allow channels to reach steady state activity prior to addition of test agents. Channel activity was monitored for three minutes preceding the addition of 30 μM ML-213 (3 minutes) to achieve maximum activation. 150 mM TEA with 10 μM XE-991 was applied for 2 minutes to measure the leak current during maximum inhibition of Kv7.2, Kv7.4 or Kv7.5/7.3 channels.
- Testing the Affinity of DB Compounds to PSMA Protein Using Biacore Table 5: The affinity of DB compounds to PSMA protein was tested using the Biacore 8K (Cytiva) instrument to detect ligand binding to PSMA protein (Sino Biological). PSMA protein was captured on an SA chip. Before fixing the ligand ( flow paths 1 and 2, flow rate of 10 μL/min), PSMA protein (10 μg/mL, flow rate of 5 μL/min, injection time of 600 s) was fixed on flow path 2 using a flow buffer. The sensor surface was adjusted by continuously injecting 1 M NaCl three times in 50 mM NaOH. After each ligand injection, additional cleaning was performed using isopropanol in 1 M NaCl and 50 mM NaOH (flow paths 1 and 2, flow rate of 10 μL/min, injection time of 60 s).[0170]All compounds were dissolved in 100% DMSO and diluted to 10 mM, then further diluted to the appropriate highest concentration in the analysis buffer (PBS, PH 7.4, 1 mM TCEP (tris-(2-hydroxyethyl) phosphine), 0.05% P20, 2% DMSO). The analysis was conducted under the following conditions: 15° C. analysis temperature; analysis steps=all set to LMW kinetics; cycle type=single cycle (90 s contact time, 1800 s dissociation time, 30 μL/min flow rate, flow paths 1 and 2); detection in flow=path 2-1. Data evaluation was performed using Biacore Insight Evaluation Software, fitting the data to a 1:1 binding model.
- Binding Assay hERG (human ether go go-related gene) potassium channels are essential for normal electrical activity in the heart. Arrhythmia can be induced by a blockage of hERG channels by a diverse group of drugs. This side effect is a common reason for drug failure in preclinical safety trials and therefore minimisation of hERG channel blocking activity may be a desirable property for drug candidates.The purpose of the hERG binding assay is to evaluate the effects of test compounds on the voltage-dependent potassium channel encoded by the human ether go go-related gene (hERG) using a constitutively expressing CHO cell line on the Nanion Syncropatch 384PE automated patch clamp system.The assay was conducted as follows with all reagents used at room temperature unless otherwise stated.Reagent Preparations Include:1. Internal IC700 solution used to perfuse the underside of chip (in mM), KF 130, KCl 20, MgCl2 1, EGTA 10 and HEPES 10, (all Sigma-Aldrich; pH 7.2-7.3 using 10 M KOH, 320 mOsm) and supplemented with 25 □M escin.2. External and cell buffer (in mM), NaCl 137, KCl 4, HEPES 10, D-glucose 10, CaCl2 2, MgCl2 1 (pH7.4, NaOH)3. NMDG reference buffer used to establish a stable baseline prior to the addition of test compounds, NaCl 80, KCl 4, CaCl2 2, MgCl2 1, NMDG Cl 60, D-Glucose monohydrate 5, HEPES 10 (pH7.4 NaOH 298 mOsm)4. Seal enhancer used to improve seal quality of cells, NaCl 80, KCl 3, CaCl2 10, HEPES 10, MgCl2 1 (pH7.4 NaOH)
- Caliper Enzyme Assay (4 mM ATP) JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 4 μM or 1 mM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
- Caliper Enzyme Assay (4 uM ATP) JAK Caliper Enzyme Assay at 4 μM: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 4 μM or 1 mM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
- In Vitro Biochemical Kinase Assay of FGFR 1-3 1. The kinase reaction in the present invention was carried out in 384-well plates, the kinase(Carna) at a certain concentration and ATP at a certain concentration and 1 μM of peptide FAM-P22 (GL Biochem, Cat. No. 112393)) was incubated to react for a certain time at 28° C. in a reaction system consisting of 50 mM HEPES, pH47.5, 0.0015% Brij-35 and basic kinase buffer; for FGFR1, the enzyme concentration was 0.25 nM and ATP concentration was 382 μM and the reaction time was 20 minutes; for FGFR2, the enzyme concentration was 2.5 nM, ATP concentration was 1 μM, and the reaction time was 40 minutes; for FGFR3, the enzyme concentration was 8 nM, ATP concentration was 4.7 μM, and the reaction time was 30 minutes:2. The reaction was terminated with a stop solution (100 mM HEPES, pH 7.5, 0.2% Caliper coating, reagent, 50 mM EDTA and 0.015% Brij35);3. The plate with the terminated kinase reaction was transferred to the Caliper workstation to read the data;4. the phosphorylated and unphosphorylated peptides were separated by the Caliper microfluid migration shift technique, and the analyte was transferred by a constant buffer flow through the chip, the migration of the substrate peptide was monitored by the labeled fluorescent signal, and the kinase activity was calculated by the amount of the phosphate-based peptide formed.5. IC50 was determined by non-linear regression analysis of percent inhibition at different concentration level of the compound.
- Surface Plasmon Resonance (SPR) SOS1 Binding AssaySurface Plasmon Resonance (SPR) SOS1 Binding Assay Binding to SOS1 was measured using a SPR assay with purified recombinant human SOS1 substrate (res. 564-1049 with N-terminal Avi tag; purified and biotinylated based on Hillig, et al., Proc Natl Acad Sci USA (2019); 116(7):2551-2560). SPR measurements were performed on a Biacore 8K SPR instrument (GE Healthcare, Sweden). Assays were performed at 25° C. using Series S SA sensor chips pre-coated with streptavidin (GE Healthcare, Cat. BR100531). Biotinylated SOS1 diluted in sample buffer (20 mM Tris HCI, 150 mM NaCl, 1 mM DTT, 0.05% TWEEN 20, 1 mM MgCh, pH 8.0) was captured to one flow cell of the chip to about 3,000 resonance units (RU) using sample buffer supplemented with 5% DMSO as a running buffer. Serial dilutions of the assayed compounds in the running buffer at 100, 50 or 0.5 µM were injected for 60 s at a flow rate of 30 µL/min and association phases were recorded. Dissociation of the samples was monitored for 600 s. Data processing was performed using Biacore Insight Software (Biacore, GE Healthcare). Sensorgrams recorded on a SA flow cell without captured protein were subtracted from sensorgrams recorded on the SOS1 surface. Blank injections of running buffer were used for double referencing and solvent correction was applied to all sample sensorgrams to correct for buffer mismatches. KDS were estimated using a kinetic or steady state, where applicable, fitting model describing a reversible equilibrium with 1:1 binding between SOS1 and the compound.
- Chip Based Microfluidic Mobility Shift Assay All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, as well as four 8-point serial dilutions of staurosporine as reference compound, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on a Thermo CatX workstation equipped with a Innovadyne Nanodrop Express. Between pipetting steps, tips were cleaned in wash cycles using wash buffer. The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 ul per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 1 mM MgCl2, 3 mM MnCl2, 4 uM ATP, 4 uM peptide (5-Fluo-Ahx-GAPDYENLQELNKK-Amid) (purchased from Biosyntan, Berlin, Germany) and 4.5 ul per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 1 mM MgCl2, 3 mM MnCl2, 4 nM Syk (Syk(2-635) (UniProtKB/Swiss-Prot: KSYK_HUMAN, P43405), produced in-house from insect cells). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 ul per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology.
- Kinase Inhibition Assay Measurement of the kinase inhibitory activity of each compound produced in Examples was conducted using the Off-chip Mobility Shift Assay. For this test, a human recombinant VEGF receptor 2 was prepared in a baculovirus expression system. A recombinant protein was expressed as a GST fusion protein by using 790-1356 amino acids of a cytosolic domain in the VEGF receptor 2 (NP 002244.1) and binding a glutathione-S-transferase (GST) to N-terminal thereof. The expressed GST-VEGF receptor 2 fusion protein was purified using glutathione-sepharose chromatography. In addition, the test substance was dissolved in dimethylsulfoxide to prepare a solution at a concentration about 100 times higher than the test concentration. Furthermore, the solution was diluted with an assay buffer (20 mM HEPES, 0.01% Triton X-100 and 2 mM DTT, pH7.5) by 25 times to prepare a 4-time concentrated test substance solution. In the kinase inhibition assay, CSKtide was used as a substrate. In the kinase reaction, 10 mL of 2-time concentrated VEGF receptor 2 kinase solution, 5 mL of 4-time concentrated test substance solution prepared with the assay buffer, and 5 mL of 4-time concentrated substrate/ATP/metal solution were mixed in wells of a polypropylene 384-well plate, and reacted at room temperature for 1 hour (substrate concentration: CSKtide 1000 nM, ATP concentration: 75 μM, Magnesium: 5 mM). One hour after, 60 mL of Termination Buffer (QuickScout Screening Assist MSA) was added so as to terminate the reaction. After that, the substrate peptide and the phosphorylated peptide in the reaction solution were separated by LabChip3000 system (Caliper Life Science), and the both peptides were quantified.
- SPR Binding Assay Table A2: In vivo biotinylated PRMT5-MEP50 was diluted to 4.5 μM in 25 mM Bicine pH 7.6, 100 mM NaCl, 1 mM TCEP, and 0.05% Tween-20 and injected at 5 μl/min flow rate into flow cell 2 (FC2) of a Series S Sensor Chip SA (Cytiva) in a Biacore T200 or in a Biacore 8K plus (Cytiva). SPR screening was performed in MTA running buffer (25 mM Bicine pH 7.6, 100 mM NaCl, 1 mM TCEP, 20 μM MTA, 0.05% Tween-20 and 2% DMSO). The biotinylated PRMT5-MEP50 surface was equilibrated with MTA running buffer for 12 hours prior to the start. The test compound affinity was determined using multi-cycle injection of each fragment from 0.001 to 500 μM over the PRMT5•MTA at a flow rate of 30 μl/min and with association and dissociation times of 20 and 60 seconds respectively. PRMT5•MTA surface activity was confirmed at the initiation, and the end of the run by titration of EPZ015666 (KD=11 and 13 μM respectively). Subsequently, compound titration was repeated in SAM-running buffer (25 mM Bicine pH 7.6, 100 mM NaCl, 1 mM TCEP, 20 μM SAM, 0.05% Tween-20, and 2% DMSO). The PRMT5•SAM surface was equilibrated for at least 5 hours prior to compound titration and the PRMT5•SAM surface activity was confirmed at the end of the fragment titration run by titration of EPZ015666 (KD<1 nM). After double referencing, the steady-state response was extracted for each fragment concentration and was fit to the Langmuir isotherm equation to determine the equilibrium dissociation constant (KD).
- Caliper Assay CHK1 kinase activity was measured in a microfluidic assay that monitors the separation of a phosphorylated product from its substrate. The assay was run on an EZ Reader II (Caliper Life Sciences Ltd, Runcorn, UK) using separation buffer (#760367 Caliper LS) containing CR-8 (500 nM, #760278, Caliper LS). An ECHO 550 (Labcyte Inc) acoustic dispenser was used to generate duplicate 8 pt dilution curves directly into 384 polypropylene assay plates (Greiner Bio-One, Gloucestershire, UK). For each test compound a 50 μM stock concentration in 100% DMSO was used. The total amount of DMSO dispensed per well was 250 nL to give a final assay concentration of 2.5% DMSO and test compound concentrations in the range 0.5-1000 nM. To this assay plate, 6 μL CHK1 (2 nM final concentration, in-house protein preparation), 2 μL peptide 10 (5-FAM-KKKVSRSGLYRSPSMPENLNRPR COOH, 1.5 μM final concentration, #760354 Caliper LS) and 2 μL ATP (90 μM final concentration) all diluted in kinase buffer (HEPES 50 mM, NaN3 0.02%, BSA 0.01%, sodium orthovanadate 0.1 mM, DTT 1 mM, MgCl2 2 mM, Tween 20 0.1%) were added. The plate was sealed and centrifuged (1 minute, 1000 rpm) before incubation for one hour at room temperature. The reaction was stopped by the addition of separation buffer (90 μL). The plate was read on an EZ Reader II, using a 12-sipper chip (760137-0372R, Caliper LS) with instrument settings of 1.5 psi and 1750 ΔV. The percentage conversion of product from substrate was generated automatically and the percentage inhibition was calculated relative to blank wells (containing no enzyme and 2.5% DMSO) and total wells (containing all reagents and 2.5% DMSO).
- In Vitro c-FMS Activity In a 384 well polypropylene plate, c-FMS (0.14 nM, Carna 08-155) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 uM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 70 pM c-FMS, 1.5 μM peptide substrate and 500 μM ATP (ATP Km). The reaction was incubated at room temperature for 120 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi. The LabChip 3000 separates and measures the fluorescent signal of fluorescein labeled peptide substrate and fluorescein labeled peptide product present in each well. Results are expressed as percent conversion by measuring peak height for both the substrate and product and dividing the product peak height by the sum of peak heights for both substrate and product. On every plate 100% inhibition (with a saturating concentration of staurosporine) and 0% inhibition (substrate with enzyme and DMSO) controls were used to calculate percent inhibition of tested compounds and a Z′ prime value.
- In Vitro TrkA Activity In a 384 well polypropylene plate, TrkA (0.4 nM, Carna 08-186) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 μM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 200 pM TrkA, 1.5 μM peptide substrate and 55 μM ATP (ATP Km). The reaction was incubated at room temperature for 180 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi. The LabChip 3000 separates and measures the fluorescent signal of fluorescein labeled peptide substrate and fluorescein labeled peptide product present in each well. Results are expressed as percent conversion by measuring peak height for both the substrate and product and dividing the product peak height by the sum of peak heights for both substrate and product. On every plate 100% inhibition (with a saturating concentration of staurosporine) and 0% inhibition (substrate with enzyme and DMSO) controls were used to calculate percent inhibition of tested compounds and a Z′ prime value.
- In Vitro TrkB Activity In a 384 well polypropylene plate, TrkB (0.6 nM, Carna 08-187) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 uM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 300 pM TrkB, 1.5 μM peptide substrate and 70 μM ATP (ATP Km). The reaction was incubated at room temperature for 180 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi. The LabChip 3000 separates and measures the fluorescent signal of fluorescein labeled peptide substrate and fluorescein labeled peptide product present in each well. Results are expressed as percent conversion by measuring peak height for both the substrate and product and dividing the product peak height by the sum of peak heights for both substrate and product. On every plate 100% inhibition (with a saturating concentration of staurosporine) and 0% inhibition (substrate with enzyme and DMSO) controls were used to calculate percent inhibition of tested compounds and a Z′ prime value.
- JAK Caliper Enzyme Assay at 1 mM ATP Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
- KD Determination by SPR Affinity and Reversibility -The binding affinity and kinetics of binding were measured using Surface Plasmon Resonance based binding assay. These experiments were carried out on Bruker SPR MASS-1 and MASS-2 instruments. There was no significant difference in results obtained on both these instruments. Bap-tagged TTR protein was captured on a Streptavidin coated sensor chip to achieve about 2000 to 3000 RUs of surface density. All the samples were prepared in buffer consisting of 10 mM Sodium Phosphate, pH 7.6, 100 mM KCI, 0.005% Tween-20 and 2% DMSO. The same buffer was used as the running buffer during the experiments. Compound samples were injected at a flow rate of 30 pL/min for 90 seconds of association time followed by at least 240 seconds of dissociation period. The compounds were tested in a concentration series consisting of at least 6 samples (usually 10) made with 5-fold, 3-fold, or 2-fold dilution. The highest concentration was 10 mM or selected based on compound binding affinity observed in a previous experiment. Multiple blank injections were run before and after each compound series to allow double reference subtraction during data processing and analysis. Tafamidis or another compound with >10 replicates was tested in every experiment as a positive control to assess activity of the captured protein on the surface. A DMSO curve was run during each experiment to properly correct for excluded volume. The data were processed and analyzed using Bruker Analyzer and Scrubber to calculate binding affinities by fitting the data to 1:1 binding model. The binding parameters obtained for tafamidis binding to TTR (n=24) are listed below. Tafamidis binds to TTR in a reversible manner with calculated residence time of around 40 seconds.
- Pim Kinase Binding Activity PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 μM PIM1, 50 μM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of −500V, an upstream voltage of −2150V, and a screening pressure of −1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm.
- TrkB Activity Reagents and consumables were purchased from Sigma Aldrich, Carna Biosciences, or Caliper Life Sciences. All assay reaction conditions for IC50 determinations were within the linear range with respect to time and enzyme concentration. In a 384 well polypropylene plate, TrkB (0.6 nM, Carna 08-187) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 uM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 300 pM TrkB, 1.5 μM peptide substrate and 70 μM ATP (ATP Km). The reaction was incubated at room temperature for 180 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi. The LabChip 3000 separates and measures the fluorescent signal of fluorescein labeled peptide substrate and fluorescein labeled peptide product present in each well. Results are expressed as percent conversion by measuring peak height for both the substrate and product and dividing the product peak height by the sum of peak heights for both substrate and product. On every plate 100% inhibition (with a saturating concentration of staurosporine) and 0% inhibition (substrate with enzyme and DMSO) controls were used to calculate percent inhibition of tested compounds and a Z′ prime value.
- TrkB Activity Reagents and consumables were purchased from Sigma Aldrich, Carna Biosciences, or Caliper Life Sciences. All assay reaction conditions for IC50 determinations were within the linear range with respect to time and enzyme concentration. In a 384 well polypropylene plate, TrkB (0.6 nM, Carna 08-187) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 uM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 300 pM TrkB, 1.5 μM peptide substrate and 70 μM ATP (ATP Km). The reaction was incubated at room temperature for 180 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi. The LabChip 3000 separates and measures the fluorescent signal of fluorescein labeled peptide substrate and fluorescein labeled peptide product present in each well. Results are expressed as percent conversion by measuring peak height for both the substrate and product and dividing the product peak height by the sum of peak heights for both substrate and product. On every plate 100% inhibition (with a saturating concentration of staurosporine) and 0% inhibition (substrate with enzyme and DMSO) controls were used to calculate percent inhibition of tested compounds and a Z′prime value.
- Assays of Enzymatic Activity Unless otherwise indicated, assays of DOT1L enzymatic activity were performed under balanced conditions (all substrates present at concentrations equal to their respective KM values) using a radiometric assay of S-[methyl-3H] adenosyl-L-methionine transfer from SAM to chicken erythrocyte nucleosomes as previously described. Reactions were initiated by addition of S-[methyl-3H] adenosyl-L-methionine and allowed to run at room temperature for 120 minutes before being quenched by the addition of 800 μM cold SAM.Compound IC50 values were determined from assays of enzymatic activity in which compound was titrated into reaction mixtures by 3-fold serial dilution from DMSO stocks. For each titration, 10 concentrations of inhibitor were used along with 100% inhibition (2.5 μM SAH) and 0% inhibition (1 μL of neat DMSO per well) controls. Plots of residual enzyme velocity as a function of inhibitor concentration were fit to a standard Langmuir isotherm equation (12) to derive estimates of the IC50 value of the compound. As described herein, the inhibition modality of key compounds within the aminonucleoside series were tested and always found to be competitive with SAM and noncompetitive with respect to nucleosome substrate. For most compounds, the Ki value was calculated from the IC50 value using the appropriate equation for competitive inhibition with respect to SAM.For selected compounds, the inhibition modality with respect to the two substrates (SAM and nucleosomes) were determined by dual titration of compound and varied substrate concentration while holding the other substrate fixed at its KM value. Plots of velocity as a function of varied substrate at multiple inhibitor concentrations were globally fit to a general equation for enzyme inhibition using Graphpad Prism. Selection of the modality for each data set was done by evaluating the value of a, a term related to the degree of cooperative or anticooperative interaction between substrate and inhibitor binding, as previously described. A value of α≥10 was taken as consistent with competitive inhibition, while a value of α≤0.1 was taken as consistent with uncompetitive inhibition. Values of α between 10 and 0.1 were considered to be consistent with noncompetitive inhibition.Compounds that displayed an IC50 value within 50-fold of the enzyme concentration used in initial assays ([E]=0.25 nM) were treated as tight binding inhibitors. In this case, the Ki value was determined by measuring the IC50 value of the compound (vide supra) at varying concentrations of enzyme from 5 nM to 0.25 nM. A plot of IC50 as a function of enzyme concentration was fit to a linear equation and the y-intercept value was equivalent to Ki(1+[S]/KM) where [S] and KM refer to SAM, the substrate with which these inhibitors compete. Knowing the values of [S] and KM used in the assay, the Ki value was then calculated from the y-intercept value.Determination of Ligand Association and Dissociation Rate ConstantsLigand association and dissociation rate constants were determined by surface plasmon resonance (SPR). DOT1L was stored in 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.8 and immobilized by direct amine coupling, diluting enzyme into coupling buffer containing 10 mM Hepes pH 7.4, 1 mM TCEP. Immobilization run buffer contained 10 mM Hepes pH 7.4, 150 mM NaCl, 500 μM TCEP, and approximately 10,000 RUs (response units) of DOT1L was captured. A reference channel of a surface that was activated in parallel and blocked was created in a second flow cell was also created. Data was captured on either a Biacore 4000 (chip CM5) or a Biorad ProteOn (chip GLM).Kd determinations were determined using run buffer containing 20 mM Tris pH 8.0, 10 mM NaCl, 100 mM KCl, 0.002% Tween-20, 500 μM TCEP, 2% DMSO, with the following injection parameters: 30 μl/min flow rate, with a 30 second association phase followed by monitoring dissociation for 30 seconds. Exp
- Kinase Binding Assay PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2 ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
- LATS1 Biochemical Caliper Assay The LATS1 biochemical Caliper assay was performed as following.Human LATS1 kinase domain protein was purchased from Carnabio (catalogue number 01-123; lot 15CBS-0098D). Human LATS1, catalytic domain [589-1130(end) amino acids of accession number NP_004681.1] was co-expressed as N-terminal GST-fusion protein (90 kDa) with human His-tagged MOBKL1A [1-216(end) amino acids of accession number NP_775739.1] using baculovirus expression system. GST-LATS1 was purified by using glutathione sepharose chromatography. The substrate (Fluo-SGKtide; Peptide for LATS1; lot BS-41067) has the following sequence: 5-Fluo-Nva-KKRNRRLSVA-amide (SEQ ID NO: 27) x TFA and was purchased from Biosyntan.The reaction is performed in reaction buffer containing 50 mM Hepes pH 7,5; 0.02% Tween20; 0.02% BSA; 1 mM DTT; 10 uM Na3VO4 and 10 mM beta-Glycerolphosphat and fresh added 1 mM MgCl2 and qsp H2O.The substrate solution (2× conc.) in Reaction Buffer contains 300 μM ATP and 4 μM Fluo-SGKtide.The kinase solution (2× conc.) in Reaction Buffer contains 20 nM LATS1 kinase. 4.5 μL of 2× conc. Kinase solution, 50 nL of 1.8 mM compounds and 4.5 μL of substrate solution were added into 384-well plates black small volume from Greiner and incubated at 32° C. for 1 hour. 15 μL of stop buffer containing 100 mM Hepes pH 7,5; 5% DMSO; 0.1% Coating reagent; 10 mM EDTA and 0.02% Brij35 and qsp H2O to each well to terminate the reaction.Substrates and products were electrophoretically separated using the microfluidic-based Caliper EZ Reader System (Caliper Life Sciences) using a 12 sipper chip (cat 760404). The separation takes place in Coating Buffer (idem Stop buffer) and containing 0.1% Coating reagent CR3 and 0.5% coating reagent CR8 (Perkin Elmer).Plates were read using a LED with an excitation at 488 nm and a detection at 520 nm to 5 quantify the fluorescence intensity. The IC50 is measured when the effect of the compound reduces the product fluorescence signal by 50%.
- PIM Kinase Binding Assay LC3K assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO. PIM reactions were carried out in a final volume of 10 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000® (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of −500V, an upstream voltage of −2150V, and a screening pressure of −1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm.
- TrkA Activity Reagents and consumables were purchased from Sigma Aldrich, Carna Biosciences, or Caliper Life Sciences. All assay reaction conditions for IC50 determinations were within the linear range with respect to time and enzyme concentration. In a 384 well polypropylene plate, TrkA (0.4 nM, Carna 08-186) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 μM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 200 pM TrkA, 1.5 μM peptide substrate and 55 μM ATP (ATP Km). The reaction was incubated at room temperature for 180 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi. The LabChip 3000 separates and measures the fluorescent signal of fluorescein labeled peptide substrate and fluorescein labeled peptide product present in each well. Results are expressed as percent conversion by measuring peak height for both the substrate and product and dividing the product peak height by the sum of peak heights for both substrate and product. On every plate 100% inhibition (with a saturating concentration of staurosporine) and 0% inhibition (substrate with enzyme and DMSO) controls were used to calculate percent inhibition of tested compounds and a Z′ prime value.
- c-FMS Activity Reagents and consumables were purchased from Sigma Aldrich, Carna Biosciences, or Caliper Life Sciences. All assay reaction conditions for IC50 determinations were within the linear range with respect to time and enzyme concentration. In a 384 well polypropylene plate, c-FMS (0.14 nM, Carna 08-155) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 uM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 70 pM c-FMS, 1.5 μM peptide substrate and 500 μM ATP (ATP Km). The reaction was incubated at room temperature for 120 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi. The LabChip 3000 separates and measures the fluorescent signal of fluorescein labeled peptide substrate and fluorescein labeled peptide product present in each well. Results are expressed as percent conversion by measuring peak height for both the substrate and product and dividing the product peak height by the sum of peak heights for both substrate and product. On every plate 100% inhibition (with a saturating concentration of staurosporine) and 0% inhibition (substrate with enzyme and DMSO) controls were used to calculate percent inhibition of tested compounds and a Z′ prime value.
- c-FMS Activity Reagents and consumables were purchased from Sigma Aldrich, Carna Biosciences, or Caliper Life Sciences. All assay reaction conditions for IC50 determinations were within the linear range with respect to time and enzyme concentration. In a 384 well polypropylene plate, c-FMS (0.14 nM, Carna 08-155) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 uM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 70 pM c-FMS, 1.5 μM peptide substrate and 500 μM ATP (ATP Km). The reaction was incubated at room temperature for 120 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi. The LabChip 3000 separates and measures the fluorescent signal of fluorescein labeled peptide substrate and fluorescein labeled peptide product present in each well. Results are expressed as percent conversion by measuring peak height for both the substrate and product and dividing the product peak height by the sum of peak heights for both substrate and product. On every plate 100% inhibition (with a saturating concentration of staurosporine) and 0% inhibition (substrate with enzyme and DMSO) controls were used to calculate percent inhibition of tested compounds and a Z′prime value.
- In Vitro Assay for the Inhibition of BTK Kinase Activity 1: Test Principle:Mobility-Shift Assay, which is microfluidic chip technology, applies the basic concept of capillary electrophoresis to microfluidic environments. The substrate used for the experiment is a polypeptide with a fluorescent label. Under the action of enzyme in the reaction system, the substrate is transformed into a product, and the charge the substrate carries also changes accordingly. The use of the charge difference between the substrate and the product involved in Mobility-Shift Assay achieves the separating of the two, and they are tested respectively. The test results are expressed by conversion rates.2: Test Method:(1) preparation of samples to be tested: diluted with 100% DMSO to 50 times the final concentration of the reaction, i.e. 25 μmol/L;(2) dilution: 25 μmol/L is the initial concentration, then diluted with 4 times the concentration and diluted with 10 concentration gradients;(3) 100% DMSO was added to the positive control well and the negative control well, respectively;(4) the prepared compounds with 10 concentrations were diluted 10-fold with 1 xkinase buffer, respectively; wherein, the kinase buffer contained hydroxyethyl piperazine ethanesulfonic acid at a concentration of 50 mmol/L and a pH of 7.5, 0.01% dodecyl polyethylene glycol ether, 10 mmol/L magnesium chloride, 2 mmol/L dithiothreitol;(5) preparation of 2.5×enzyme solution: the kinase was added to 1×kinase buffer to form 2.5×enzyme solution;(6) preparation of 2.5×substrate solution: FAM-labeled polypeptide and ATP were added to the 1 xkinase buffer to form 2.5×substrate solution; (7) addition of the enzyme solution to the 384-well plate: 5 μl of 5×compound dissolved in 10% DMSO contained in the 384-well reaction plate, then 10 μl of 2.5×enzyme solution was added, the obtained system was incubated for 10 minutes at room temperature;(8) addition of the substrate solution to the 384-well plate: 10 μl of 2.5×substrate solution was added to the 384-well reaction plate;(9) kinase reaction and termination: after incubation at 28° C. for 1 hour, 25 μl stop solution was added to terminate the reaction; wherein, the stop solution contained hydroxyethyl piperazine ethanesulfonic acid at a concentration of 100 mmol/L and a pH of 7.5, 0.015% dodecyl polyethylene glycol ether, 0.2% surface reagent No. 3, 20 mmol/L ethylenediaminetetraacetic acid; (10) Caliper data reading: conversion rate data was read from Caliper.