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Small-Molecule Inhibitors of the β-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction The β-catenin/BCL9 protein-protein interaction (PPI) has emerged as a potential therapeutic target to suppress the hyperactive Wnt/β-catenin signaling that is vigorously involved in tumor initiation and development. Herein, we report the medicinal chemistry optimization on a screening hit to yield a series of novel small-molecule inhibitors of β-catenin/BCL9 PPI. The best compound 30 was found to disrupt the β-catenin/BCL9 PPI with a Ki of 3.6 μM in AlphaScreen competitive inhibition assays. Cell-based studies revealed that 30 selectively disrupted the β-catenin/BCL9 interaction while leaving the β-catenin/E-cadherin complex unaffected, dose-dependently suppressed transactivation of Wnt/β-catenin signaling, downregulated expression of Wnt target genes, and selectively inhibited viability of cancer cells with hyperactive β-catenin signaling. This compound could serve as a lead compound for further optimization of inhibitors of β-catenin/BCL9 PPI.
cAMP Assays Activation of GLP-1 receptor is known to stimulate cyclic AMP (cAMP) production in cells which indicates primary coupling to the G as subunit of the G protein heterotrimeric complex. Evidence suggests signaling through G as induced cAMP stimulation elicits the desired pharmacological response regarding insulin release from pancreatic β-cells.
5-HT1BR cAMP Secondary Messenger Agonist Assay The 5-HT1BR cAMP secondary messenger agonist assay used a panel of CHO-K1 cell lines stably expressing non-tagged GPCRs that endogenously signal through cAMP. Hit Hunter® cAMP assays monitored the activation of a GPCR via Gi and Gs secondary messenger signaling in a homogenous, non-imaging assay format using DiscoverX Enzyme Fragment Complementation (EFC) with β-galactosidase as the functional endpoint.
Measurement of beta-arrestin Recruitment to Human Dopamine D2L Receptors PathHunter CHO-K1 cells expressing tagged human D2L receptors and tagged β-arrestin-2 (Eurofins DiscoverX, Fremont, CA, USA) were seeded into 96-well white-walled clear bottom tissue culture plates in 90 μL/cell AssayComplete™ Cell Plating 2 (CP2) reagent (Eurofins DiscoverX) at a density of 20,000 cells/well. The plates were incubated overnight in a humidified atmosphere with 5% CO2 at 37° C. Twenty to twenty-four hours later, 20 μL of test compound or vehicle in CP2 reagent and containing 2.2% DMSO was added to the cells, and they were incubated for 90 minutes at 37° C. Then, 55 μL PathHunter® Detection Reagent (Eurofins DiscoverX) was added per well and plates were incubated for 60 minutes at 25° C., followed by luminescence detection using a PHERAstar® FS multimode plate reader (BMG Labtech, Ortenberg, Germany). Raw data were converted to percent stimulation above basal values. Values were further converted to percent of maximal stimulation of β-arrestin recruitment by 30 μM dopamine. EC50 values were calculated from concentration-response curves of at least six concentrations run in duplicates by sigmoidal fitting using Origin® 7.5 software (OriginLab Corporation, Northampton, MA, USA) and were defined as the concentration of the agonist with half-maximal stimulation. The pEC50 values were calculated as the negative logarithm of the EC50 value expressed in mol/liter and is shown in Table 6. The results indicate that the examples of the present disclosure are potent agonists of the G-protein-independent signaling pathway of human recombinant D2L receptors.
17beta-HSD13 Biochemical Assay More specifically, recombinant 17β-HSD13 protein was assayed in a buffer containing 200 mM Tris pH 7.5, 0.01% Triton X-100, and 0.02% BSA into a 384-well assay plate. Compounds were incubated with 17β-HSD13 (final 50 nM) and NAD+ (final 10 mM) at room temperature for 1 h prior to substrate addition. The assay reaction was then initiated by addition of β-estradiol (final 20 μM), and the reaction mixture was incubated for 2 hours at room temperature. Product formation was detected with chemiluminescence by adding equal volume of NAD+/NADH Glo reagent (Promega, #G9062) and read on a PHERAstar microplate reader (BMG LABTECH). IC50 values were determined with GraphPad Prism®, where log-transformed concentration values and the inhibition data were fitted to a four-parameter logistic equation. Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log IC50−X)*HillSlope)).
Measurement of beta-arrestin Recruitment to Human Dopamine D3 Receptors PathHunter CHO-K1 cells expressing tagged human D3 receptors and tagged β-arrestin-2 (Eurofins DiscoverX, Fremont, CA, USA) were seeded into 96-well white-walled clear bottom tissue culture plates in 90 μL AssayComplete™ Cell Plating 2 (CP2) reagent (Eurofins DiscoverX) at a density of 25,000 cells/well and incubated overnight in humidified atmosphere with 5% CO2 at 37° C. Twenty to twenty-four hours later, 20 μL of test compounds or vehicle in CP2 reagent containing 2.2% DMSO was added to the cells and the cells were incubated for 90 minutes at 37° C. Following incubation, 55 μL PathHunter® Detection Reagent (Eurofins DiscoverX) was added per well and the plates were incubated for 60 minutes at 25° C. followed by luminescence detection using a PHERAstar FS multimode plate reader (BMG Labtech, Ortenberg, Germany).
WecA Assay DP-Glucosamine-C6-FITC (2 mM stock solution, 0.56 μL), MgCl2 (0.5 M, 4 μL), β-mercaptoethanol (50 mM, 5 μL), CHAPS (5%, 11.25 μL), Tris buffer (pH 8.0, 50 mM), undecaprenyl phosphate (4 mM, 1.4 μL), and inhibitor molecule (0-100 μg/mL in Tris buffer) were place in a 500 μL Eppendorf tube. To a stirred reaction mixture, P-60 (10 μL) was added (total volume of reaction mixture: 50 μL adjust with Tris buffer). The reaction mixture was incubated for 4 h at 37° C. and quenched with n-butanol (150 μL). Two phases were mixed via vortex and centrifuged at 10,000 xg for 3 min. The upper organic phase was assayed via reverse-phase HPLC. The organic phase (30 μL) was injected into HPLC (solvent: gradient elution of 85:15 to 95:5 MeOH/0.05 M aq. NH4HCO3; UV: 485 nm; flow rate: 0.5 ml/min; column: Kinetex 5 μm C8, 100 Å, 150×4.60 mm), and the area of the peak for C55-P—P-glucosamine-C6-FITC was quantified to obtain the IC50 value.
ALPK1 In Vitro Kinase Assay In brief, dose-response studies were performed in 384-well assay plates. Each well contained 0.1 mg TIFA, ALPK1 (2 nM final concentration in reaction mixture) and kinase buffer (100 mM of HEPES pH 7.4, 4 mM DTT, 40 mM MgCl2, 20 mM of β-Glycerol phosphate disodium salt, 0.4 mM of Na3 VO4, 0.16 mg/mL). Titrations of the test compounds were prepared in dimethylsulphoxide (DMSO). The reaction was initiated by addition of ATP and ADP-Heptose.For HTRF, samples were incubated with a Tb cryptate-labeled anti-HA antibody for capturing HA-tagged proteins according to the manufacturer's instructions (PerkinElmer™, CisBio™) and the fluorescence signal was quantified (Tecan Infinite F NANO+). HTRF signals were calculated as the HTRF ratio (ratio of fluorescence measured at 665 nm and 620 nm)×104 (thereby using the signal at 620 nm as an internal standard).All compounds exhibited a dose-dependent decrease in TIFA phosphorylation in this assay. IC50 values were determined using 3- or 4-parameter logistic equation using GraphPad Prism version 6.00. The reference compound, A027, was used as a positive control for each plate.
Fluorescence Polarization (FP) Assay The FP experiments were performed in 96-well Microfluor 2 black plates (Waltham, MA), and the sample signals were read by a Synergy 2 plate reader (Biotek, Winooski, VT). The polarization was measured at room temperature with an excitation wavelength at 485 nm and an emission wavelength at 535 nm. All of the FP experiments were performed in an assay buffer of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 100 pug/mL of bovine γ-globulin, and 0.010% Triton X-100. The final reaction volume was set to 100 μL. In the FP saturation binding experiments, the concentration of human BCL9 fluorescent tracer was fixed at 5 nM. The concentrations of β-catenin were ranged from 0 to 10 μM in the assay buffer giving a final volume of 100 μL. After the addition, each assay plate was covered black and gently mixed on an orbital shaker for 3 h before the polarization signals were recorded. The data were analyzed by nonlinear least-square analyses using GraphPad Prism 5.0 to derive the apparent Kd value. Each experiment was repeated three times, and the results were expressed as mean±standard deviation.
CAMP Assays Activation of GLP-1 receptor is known to stimulate cyclic AMP (cAMP) production in cells which indicates primary coupling to the Gαs subunit of the G protein heterotrimeric complex. Evidence suggests signaling through Gαs induced CAMP stimulation elicits the desired pharmacological response regarding insulin release from pancreatic β-cells. To optimize functional activity directed toward Gαs coupling, a HEK293/CRELuc cell line developed by HDB stably expressing the GLP-1 Receptor was used. 200× concentration of compound working solutions were prepared (Agilent Technologies Bravo) with 1/2 log serial dilution in 384-well Echo LDV plate (Labcyte, Cat #LP-0200). 50 nL/well 200× concentration of compound working solutions were moved to 384-well white low volume plate (Greiner, Cat #784075) using Labcyte ECHO550. 1×105 cells/mL HEK293/GLP1R/CRE-LUC (HD Biosciences) cell suspensions prepared with assay buffer [DPBS containing 0.5 mM IBMX (Sigma,Cat #I5879) and 0.1% BSA (GENVIEW, Cat #FA016-100 g)], 10 uL cell suspensions were added to each well of previous generated assay plate which already contains 50 nl compound at 200× concentration using ThermoFisher Multidrop Combi (1000cells/well). Seal the plate and incubate at 37° C. with 5% CO2 for 30 min.
HDAC Inhibition Assays The HDAC 1, 2 assays employed buffer A, which contained 20 mM HEPES, pH 8.0, 1 mM MgCl2, 137 mM NaCl, 2.7 mM KCl, 0.05% BSA. The HDAC3/SMRT assay employed buffer B, consisting of 20 mM HEPES, pH 8.0, 1 mM MgCl2, 50 mM NaCl, 2.7 mM KCl, 0.05% BSA, 0.005% Tween 20, and 10 μM IP4. The HDAC6 assay employed buffer C, consisting of 20 mM HEPES, pH 8.0, 1 mM MgCl2, 137 mM NaCl, 2.7 mM KCl, 0.5 mM TCEP (Calbiochem) and 0.05% BSA. The HDAC8 assay employed buffer D, consisting of 20 mM HEPES, pH 8.0, 1 mM MgCl2, 100 mM NaCl, 20 mM KCl, 0.1% n-octyl-β-D-glucoside (Anatrace) and 0.05% BSA. All steps were performed at room temperature (23° C.). The assay was performed by pre-incubating serial dilutions of test compounds with the target HDAC prior to initiation with substrate. Each compound was titrated in a 10-point dose response, using a 1:3 fold dilution scheme, with 0.15 ul of solution added by ECH0555 to the plate, followed by the addition of 20 μl of the appropriate HDAC isoform diluted in appropriate assay buffer. The incubation was allowed to proceed for 3 hours, then the appropriate substrate diluted in assay buffer (final substrate concentration ˜Km) was added and the reaction allowed to proceed for 60 min.
KRAS-BRAF with CYPA (50 nM) Interaction Assay In this example, TR-FRET was also used to measure the compound or compound-CYPA dependent disruption of the KRAS G12C-BRAF complex. This protocol was also used to measure disruption of KRAS G12D or KRAS G12V binding to BRAF by a compound of the invention, respectively. In assay buffer containing 25 mM HEPES PH=7.4 (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Thermo, 15630080), 0.002% Tween20, 0.1% BSA, 100 mM NaCl, 5 mM MgCl2, 10 μM GMPPNP (Guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate, Sigma, G0635), tagless CYPA, GMPPNP loaded 6His-KRAS proteins, and GST-BRAFRBD were mixed in a well of a 384-well assay plate at final concentrations of 50 nM, 6.25 nM and 1 nM, respectively. Compound was present in plate wells as a 16-point 3-fold dilution series starting at a final concentration of 10 μM and incubated for 3 hours. A mixture of MAb Anti-6His-XL665 (Cisbio, 61HISXLB) and Mab anti-GST-TB cryptate (Cisbio, 61GSTTLB) was then added at a final concentration of 6.67 nM and 0.21 nM, respectively, and the plate was incubated for an additional 1.5 hours. TR-FRET signal was read on a PHERstar FSX microplate reader (Ex320 nm, Em 665/615 nm). Compounds that facilitate disruption of the KRAS-BRAF complex were identified as those eliciting a decrease in the TR-FRET ratio relative to DMSO control wells.
KRAS-BRAF with CYPA (500 nM) Interaction Assay In this example, TR-FRET was also used to measure the compound or compound-CYPA dependent disruption of the KRAS G12C-BRAF complex. This protocol was also used to measure disruption of KRAS G12D or KRAS G12V binding to BRAF by a compound of the invention, respectively. In assay buffer containing 25 mM HEPES PH=7.4 (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Thermo, 15630080), 0.002% Tween20, 0.1% BSA, 100 mM NaCl, 5 mM MgCl2, 10 μM GMPPNP (Guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate, Sigma, G0635), tagless CYPA, GMPPNP loaded 6His-KRAS proteins, and GST-BRAFRBD were mixed in a well of a 384-well assay plate at final concentrations of 50 nM, 6.25 nM and 1 nM, respectively. Compound was present in plate wells as a 16-point 3-fold dilution series starting at a final concentration of 10 μM and incubated for 3 hours. A mixture of MAb Anti-6His-XL665 (Cisbio, 61HISXLB) and Mab anti-GST-TB cryptate (Cisbio, 61GSTTLB) was then added at a final concentration of 6.67 nM and 0.21 nM, respectively, and the plate was incubated for an additional 1.5 hours. TR-FRET signal was read on a PHERstar FSX microplate reader (Ex320 nm, Em 665/615 nm). Compounds that facilitate disruption of the KRAS-BRAF complex were identified as those eliciting a decrease in the TR-FRET ratio relative to DMSO control wells.
Menin/MLL Homogenous Time-Resolved Fluorescence (HTRF) Assay To an untreated, white 384-well microtiter plate was added 40 nL 200× test compound in DMSO and 4 μL 2× terbium chelate-labeled menin (vide infra for preparation) in assay buffer (40 mM Tris·HCl, pH 7.5, 50 mM NaCl, 1 mM DTT (dithiothreitol) and 0.05% Pluronic F-127). After incubation of test compound and terbium chelate-labeled menin for 30 min at ambient temperature, 4 μL 2×FITC-MBM1 peptide (FITC-β-alanine-SARWRFPARPGT-NH2) (“FITC” means fluorescein isothiocyanate) in assay buffer was added, the microtiter plate centrifuged at 1000 rpm for 1 min and the assay mixtures incubated for 15 min at ambient temperature. The relative amount of menin-FITC-MBM1 complex present in an assay mixture is determined by measuring the homogenous time-resolved fluorescence (HTRF) of the terbium/FITC donor/acceptor fluorphore pair using an EnVision microplate reader (ex. 337 nm/terbium em. 490 nm/FITC em. 520 nm) at ambient temperature. The degree of fluorescence resonance energy transfer (the HTRF value) is expressed as the ratio of the fluorescence emission intensities of the FITC and terbium fluorophores (Fem 520 nm/Fem 490 nm). The final concentrations of reagents in the binding assay are 200 pM terbium chelate-labeled menin, 75 nM FITC-MBM1 peptide and 0.5% DMSO in assay buffer. Dose-response titrations of test compounds are conducted using an 11 point, four-fold serial dilution scheme, starting typically at 10 μM.
Inhibition Assays of p110α/p85α, p110β/p85α, p110δ/p85α, and p110γ The commercially available kits or systems can be used to screen for inhibitors and/or agonists of PI3-Ks including but not limited to PI3-Kinase α, β, δ, and γ. An exemplary system is PI3-Kinase (human) HTRF™ Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader. Data is analyzed and IC50s are generated using GraphPad Prism 5.
Inhibition of KRASG12C and PI3Ka Binding The AlphaScreen technology was used to determine IC50s for compound inhibition of KRAS G1 2C (present as the Cys-light (C51S, C80L and C118S), truncated version comprising amino acids 1-169) and P13Ka interaction. Compounds were diluted in 100% DMSO and each compound concentration was spotted at 200 nl/well onto low volume, white 384 well plates. The KRAS G12C contained a biotin-AviTag and the P13Ka, as Ras-binding domain (amino acids 157-300, RBD), was His-tagged. KRAS G12C was preloaded with the GTP analogue Guanosine 5′-[β, γ-imido]triphosphate (GMPPNP). The KRAS GT2C was diluted in 25 mM Hepes, pH 7.4, 150 mM NaCl, 5 mMv MgCl2, 0.0100 TritonX-100 and 10 μM GMPPNP and added at 10 ul/well to compound-spotted plates resulting in a DMR concentration of 2%. Plates were incubated for 2 hours. A mixture of RBD and the AlphaScreen streptavidin donor and nickel chelate acceptor beads diluted in 25 mMv Hepes, pH 7.4, 150 mMv NaCl, 5 mM MgCl2, 0.0100 TritonX-100 and 2% DMSO was then added at 10 ul/well and incubated for 60-90 minutes before the samples were read for emission at 570 nm after excitation of the donor beads at 680 nm. All incubations were performed at room temperature. The final top compound concentration was 50 μM with 1:3 titrations for 10-point dose response curves. Final assay conditions were 1.5 nM KRAS GT2C, 100 nM RBD, 1.25 ug/ml of AlphaScreen donor beads and 10 μg/ml AlphaLISA acceptor beads. IC50S were determined using nonlinear regression fit of [inhibitor]vs. response (4 parameters).
Inhibition of KRASG12C and cRAF Binding The AlphaScreen technology was used to determine IC50s for compound inhibition of KRAS G12C (present as the Cys-light (C51S, C80L and C118S), truncated version comprising amino acids 1-169) and cRAF interaction. Compounds were diluted in 100% DMSO and each compound concentration was spotted at 200 nl/well onto low volume, white 384 well plates. The KRAS G12C contained a biotin-AviTag and the cRaf, as Ras-binding domain (amino acids 50-131, RBD), was GST-tagged. KRAS G12C was preloaded with the GTP analogue Guanosine 5′-[β, γ-imido]triphosphate (GMPPNP). The KRAS G12C was diluted in 25 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.01% TritonX-100 and 10 μM GMPPNP and added at 10 ul/well to compound-spotted plates resulting in a DMSO concentration of 2%. Plates were incubated for 19-20 hours. A mixture of RBD and the AlphaScreen streptavidin donor and glutathione acceptor beads diluted in 25 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.01% TritonX-100 and 2% DMSO was then added at 10 ul/well and incubated for 60-90 minutes before the samples were read for emission at 570 nm after excitation of the donor beads at 680 nm. All incubations were performed at room temperature. The final top compound concentration was 50 μM with 1:3 titrations for 10-point dose response curves. Final assay conditions were 0.5 nM KRAS G12C, 0.75 nM RBD and 5 μg/ml each of AlphaScreen donor and acceptor beads. IC50s were determined using nonlinear regression fit of [inhibitor]vs. response (4 parameters).
15-PGDH Enzyme Activity Assay a. A solution of pH 7.5 containing 50 mM Tris-HCl, 0.01% Tween 20 was prepared with ultrapure water as a reaction buffer;b. A 10 mM mother liquor of the compound to be tested was prepared with DMSO, and then the reaction buffer was used to dilute the mother liquor of the compound to be tested to obtain solution 1 of the compound to be tested at a concentration of 40,000 nM, and then the solution 1 of the compound to be tested was serially diluted into solutions 2-9 (or 2-12) of the compound to be tested at 9 (or 11) concentrations with a gradient difference of three-fold. 5 μL of each concentration of solutions of the compound to be tested was respectively taken and added into a 384-well plate as test wells;c. Then 5 μL of the reaction buffer was added to the blank wells of the 384-well plate as positive control and blank control wells, respectively;d. The reaction buffer was used to prepare a 15-PGDH protein solution at a concentration of 5 ng/μL, 5 μL of the 15-PGDH protein solution was taken and added to the test wells and positive control wells, and meanwhile another 5 μL of the reaction buffer was added to the blank control wells, then the plate was centrifuged at 2000 rpm for 30 seconds;e. The reaction buffer was used to prepare 5 mM β-NAD and 2 mM PGF2α, respectively, which were mixed at 1:1 by volume to obtain a substrate mixture, 10 μL of the substrate mixture was taken and added to the test wells, positive control wells and blank control wells to start the reaction;f. The fluorescence signal value (Ex/Em=340/450) of each well was detected continuously by using a multifunctional microplate reader.
Detection of Activity of 15-PGDH Kinase a. A solution of pH 7.5 containing 50 mM Tris-HCl, 0.01% Tween 20 was prepared with ultrapure water as a reaction buffer;b. A 10 mM mother liquor of the compound to be tested was prepared with DMSO, and then the reaction buffer was used to dilute the mother liquor of the compound to be tested to obtain solution 1 of the compound to be tested at a concentration of 40,000 nM, and then the solution 1 of the compound to be tested was serially diluted into solutions 2-9 (or 2-12) of the compound to be tested at 9 (or 11) concentrations with a gradient difference of three-fold. 5 μL of each concentration of solutions of the compound to be tested was respectively taken and added into a 384-well plate as test wells;c. 5 μL of the reaction buffer was added to the blank wells of the 384-well plate as positive control and blank control wells, respectively;d. The reaction buffer was used to prepare a 15-PGDH protein solution at a concentration of 5 ng/μL, 5 μL of the 15-PGDH protein solution was taken and added to the test wells and positive control wells, and meanwhile 5 μL of the reaction buffer was added to the blank control wells, then the plate was centrifuged at 2000 rpm for 30 seconds;e. The reaction buffer was used to prepare 5 mM β-NAD and 2 mM PGF2α, respectively, which were mixed at 1:1 by volume to obtain a substrate mixture, 10 μL of the substrate mixture was taken and added to the test wells, positive control wells and blank control wells to start the reaction;f. The fluorescence signal value (Ex/Em=340/450) of each well was detected continuously by using a multifunctional microplate reader.
Inhibition of KRASG12C and PI3Ka Binding Assay The AlphaScreen technology was used to determine IC50S for compound inhibition of KRAS G12C (present as the Cys-light (C51S, C80L and C118S), truncated version comprising amino acids 1-169) and PI3Ka interaction. Compounds were diluted in 100% DMSO and each compound concentration was spotted at 200 nl/well onto low volume, white 384 well plates. The KRAS G12C contained a biotin-AviTag and the PI3Ka, as Ras-binding domain (amino acids 157-300, RBD), was His-tagged. KRAS G12C was preloaded with the GTP analogue Guanosine 5′-[β,γ-imido]triphosphate (GMPPNP). The KRAS G12C was diluted in 25 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.01% TritonX-100 and 10 μM GMPPNP and added at 10 ul/well to compound-spotted plates resulting in a DMSO concentration of 2%. Plates were incubated for 2 hours. A mixture of RBD and the AlphaScreen streptavidin donor and nickel chelate acceptor beads diluted in 25 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.01% TritonX-100 and 2% DMSO was then added at 10 ul/well and incubated for 60-90 minutes before the samples were read for emission at 570 nm after excitation of the donor beads at 680 nm. All incubations were performed at room temperature. The final top compound concentration was 50 μM with 1:3 titrations for 10-point dose response curves. Final assay conditions were 1.5 nM KRAS G12C, 100 nM RBD, 1.25 ug/ml of AlphaScreen donor beads and 10 μg/ml AlphaLISA acceptor beads. IC50s were determined using nonlinear regression fit of [inhibitor] vs. response (4 parameters).A counter assay was also set up to rule out inhibitors of the AlphaScreen technology itself. Compound plates were incubated for 19-20 hours as above with buffer only. The AlphaScreen beads were added as above except an unrelated biotinylated His-tagged peptide was substituted for the RBD.
Inhibition of KRASG12C and cRAF Binding Assay The AlphaScreen technology was used to determine IC50S for compound inhibition of KRAS G12C (present as the Cys-light (C51S, C80L and C118S), truncated version comprising amino acids 1-169) and cRAF interaction. Compounds were diluted in 100% DMSO and each compound concentration was spotted at 200 nl/well onto low volume, white 384 well plates. The KRAS G12C contained a biotin-AviTag and the cRaf, as Ras-binding domain (amino acids 50-131, RBD), was GST-tagged. KRAS G12C was preloaded with the GTP analogue Guanosine 5′-[β,γ-imido]triphosphate (GMPPNP). The KRAS G12C was diluted in 25 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.01% TritonX-100 and 10 μM GMPPNP and added at 10 ul/well to compound-spotted plates resulting in a DMSO concentration of 2%. Plates were incubated for 2 hours. A mixture of RBD and the AlphaScreen streptavidin donor and glutathione acceptor beads diluted in 25 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.01% TritonX-100 and 2% DMSO was then added at 10 ul/well and incubated for 60-90 minutes before the samples were read for emission at 570 nm after excitation of the donor beads at 680 nm. All incubations were performed at room temperature. The final top compound concentration was 50 μM with 1:3 titrations for 10-point dose response curves. Final assay conditions were 0.5 nM KRAS G12C, 0.75 nM RBD and 5 μg/ml each of AlphaScreen donor and acceptor beads. IC50S were determined using nonlinear regression fit of [inhibitor] vs. response (4 parameters). A counter assay was also set up to rule out inhibitors of the AlphaScreen technology itself. Compound plates were incubated for 2 hours as above with buffer only. The AlphaScreen beads were added as above except biotin-AviTag-GST was substituted for the RBD.
FLIPR Calcium 4 Assay The antagonistic activity of human P2X3 receptor (hP2X3) antagonists against hP2X3 was evaluated using the FLIPR Calcium 4 Assay Kit (Molecular Devices, R8141) and FLIPR TETRA instrument (Molecular Devices, 0296) to detect calcium flux signals. 24 h before the experiment, human cells stably transfected with the hP2X3 receptor were seeded into a 384-well plate at a density of 2×105 cells/mL, with 50 μL of cell suspension per well. The cells were then incubated in a 5% CO2 incubator at 37° C. for 16-24 h. Each test compound was prepared in DMSO at 180 times the desired concentration (20-50 mM DMSO stock solution). 500 nL of the solution was then added to each well of the 384-well plate, followed by addition of 30 μL of FLIPR Assay buffer (lx HBSS containing 1.26 mM Ca2++2 mM CaCl2), 20 mM HEPES). The mixture was shaken for 20-40 min to ensure homogeneous mixing. An agonist (α,β-meATP) was prepared using FLIPR Assay buffer at 3 times the desired concentration (desired final concentration of 400 nM). 45 μL of the agonist was then added to each well of another 384-well plate. The cell culture plate prepared one day ago was taken, and the cell supernatant was aspirated and discarded. 30 μL of Dye (FLIPR® Calcium 4 Assay Kit, diluted in FLIPR buffer) was added to each well. The plate was then incubated for 1 h. 15 μL of the compound was added to the cells in each well (using the FLIPR instrument). After 15 min, 22.5 μL of the agonist was added to each well. The fluorescence signal was detected (with an excitation wavelength of 470-495 nm and an emission wavelength of 515-575 nm). Taking the difference between the peak and trough values of the signal as the base data, the data for the highest concentration of the positive drug as the 100% inhibition rate, and the DMSO data as the 0% inhibition rate, the inhibition effect curve of the compound was fitted on the software Graphpad Prism 6, and the IC50 value was calculated.
CDK12/CycK High ATP Kinase Assay For the assay 50 nanoL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into either a black low volume 384 well microtiter plate or a black 1536 well microtiter plate (both Greiner Bio-One, Frickenhausen, Germany), 2 microL of a solution of CDK12/CycK in aqueous assay buffer [25 millimol/L HEPES pH 7.5, 20 millimol/L MgCl2, 5 millimol/L β-glycerophosphate, 2 millimol/L EGTA, 1.0 millimol/L dithiothreitol, 0.01% (v/v) Nonidet-P40 (Sigma), 0.01% (w/v) bovine serum albumin] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 microL of a solution ATP (3.33 millimol/L=>final conc. in the 5 microL assay volume is 2 millimol/L) and substrate (1.67 micromol/L=>final conc. in the 5 microL assay volume is 1 micromol/L) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of CDK12/CycK was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were about 0.75 nanomol/L. The reaction was stopped by the addition of 3 microL of a solution of TR-FRET detection reagents (125 nanomol/L streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 0.67 nanomol/L anti-Phospho-c-Myc (Ser 62) (E1J4K)-antibody from Cell Signalling [#13748] and 2 nanomol/L LANCE EU-W1024 labeled anti-rabbit IgG antibody [Perkin-Elmer, product no. 0083]) in an aqueous EDTA-solution (133 millimol/L EDTA, 0.27% (w/v) bovine serum albumin in 66.7 millimol/L HEPES pH 7.5).The resulting mixture was incubated 1 h at 22° C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a TR-FRET reader, e.g. a Pherastar FS (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer).
LOX Inhibition Assay Lysyl oxidase (LOX) is an extracellular copper dependent enzyme which oxidizes peptidyl lysine and hydroxylysine residues in collagen and lysine residues in elastin to produce peptidyl alpha-aminoadipic-delta-semialdehydes. This catalytic reaction can be irreversibly inhibited by β-aminopropionitrile (BAPN) that binds to the active site of LOX (Tang S. S., Trackman P C and Kagan H. M., Reaction of aortic lysyl oxidase with beta-aminoproprionitrile. J Biol Chem 1983; 258: 4331-4338). There are five LOX family members; these are LOX, LOXL1, LOXL2, LOXL3 and LOXL4. LOX and LOXL family members can be acquired as recombinant active proteins from commercial sources, or extracted from animal tissues like bovine aorta, tendons, pig skin; or prepared from cell cultures. The inhibitory effects of the compounds of the present invention were tested against the given LOX-LOXL preparation using a method based on the detection of hydrogen peroxide with an Amplex Red oxidation assay (Zhou et al. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem. 1997; 253, 162-168). The assay was developed using either 384 or 96 well format. Briefly, in a standard black, clear bottom 384 well plate assay 25 μL of a dilution of any of the isoenzymes and orthologues in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were added into each well in the presence of 1 μM mofegiline and 0.5 mM pargyline (to inhibit SSAO and MAO-B and MAO-A, respectively; not necessary if the enzyme is from a recombinant or purified form). Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 11 data points, typically in the micromolar or nanomolar range after incubation with the enzyme for 30 min at 37° C. Twenty five μL of a reaction mixture containing twice the KM concentration of putrescine (Sigma Aldrich, e.g. 20 mM for LOX, or 10 mM for LOXL2 and LOXL3), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were then added to the corresponding wells. The above volumes were doubled in the case of 96 wells plate. The fluorescence (RFU) was read every 2.5 min for 30 min at a range of temperatures from 37° C., excitation 565 nm and emission 590 (Optima; BMG labtech). The slope of the kinetics for each well was calculated using MARS data analysis software (BMG labtech) and this value was used to deduce the IC50 value (Dotmatics).

Query String: β-elemene