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- Insulin reductase assay Insulin reductase assay.
- ChEBML_90257 Inhibition of insulin receptor
- ChEBML_90271 Inhibition of Insulin receptor
- ChEBML_156464 Inhibition of canine cardiac Phosphodiesterase 3
- ChEMBL_2293166 Inhibition of hERG cardiac ion channel
- ChEMBL_759533 (CHEMBL1811059) Inhibition of insulin-induced autophosphorylation of human insulin receptor expressed in CHO cells
- ChEBML_90277 Inhibition of insulin receptor autophosphorylation
- ChEMBL_334490 (CHEMBL862525) Inhibition of insulin receptor
- ChEMBL_377345 (CHEMBL865408) Inhibition of Insulin receptor
- ChEMBL_506205 (CHEMBL941409) Inhibition of insulin receptor
- ChEMBL_581990 (CHEMBL1058970) Inhibition of insulin receptor
- ChEMBL_635332 (CHEMBL1119645) Inhibition of insulin receptor
- ChEMBL_698757 (CHEMBL1648482) Inhibition of insulin receptor
- ChEMBL_759532 (CHEMBL1811058) Inhibition of insulin receptor
- ChEMBL_794804 (CHEMBL1935987) Inhibition of insulin receptor
- ChEMBL_825876 (CHEMBL2044753) Inhibition of insulin receptor
- ChEMBL_90267 (CHEMBL699132) Inhibition of Insulin receptor
- ChEMBL_90276 (CHEMBL699141) Inhibition of Insulin receptor
- ChEBML_156450 Inhibition of bovine cardiac phosphodiesterase fraction III
- ChEMBL_156464 (CHEMBL764842) Inhibition of canine cardiac Phosphodiesterase 3
- ChEMBL_2223202 (CHEMBL5136536) Inhibition of hERG cardiac ion channel
- ChEMBL_154393 (CHEMBL759155) Inhibition of cardiac PDE 3 (phosphodiesterase III)
- ChEMBL_154394 (CHEMBL759156) Inhibition of cardiac PDE 3 (phosphodiesterase III)
- ChEBML_90279 Inhibition of Insulin receptor kinase-beta
- ChEMBL_2544064 Inhibition of insulin receptor (unknown origin)
- ChEMBL_563807 (CHEMBL994319) Inhibition of human insulin receptor
- ChEMBL_655044 (CHEMBL1244088) Inhibition of human insulin receptor
- ChEMBL_741336 (CHEMBL1764805) Inhibition of insulin receptor kinase
- ChEMBL_155704 (CHEMBL760583) Inhibition of guinea pig cardiac ventricle PDE 4
- ChEMBL_156315 (CHEMBL762548) Inhibition of guinea pig cardiac ventricle Phosphodiesterase 2
- ChEMBL_156484 (CHEMBL761729) Inhibition of guinea pig cardiac ventricle Phosphodiesterase 3
- ChEMBL_196055 (CHEMBL803054) Binding to ryanodine receptor in canine cardiac membranes.
- ChEMBL_217178 (CHEMBL821502) Inhibition of canine cardiac cAMP phosphodiesterase (cAMP-PDE)
- ChEMBL_1363800 (CHEMBL3294217) Inhibition of insulin receptor (unknown origin)
- ChEMBL_215892 (CHEMBL820824) Inhibition of beta-insulin receptor kinase
- ChEMBL_2251074 (CHEMBL5165284) Inhibition of Insulin receptor (unknown origin)
- ATPase assay Experiment on Inhibitory Effect of Cardiac Myosin ATPase Activity.
- ChEMBL_156148 (CHEMBL760754) Inhibition of Phosphodiesterase 1 of guinea pig cardiac ventricle
- ChEMBL_156462 (CHEMBL764840) Inhibition of cardiac phosphodiesterase-3 isolated from canine heart.
- ChEMBL_46319 (CHEMBL660720) Tested against neonatal rat cardiac myocyte carnitine palmitoyltransferase 1
- ChEMBL_46322 (CHEMBL660890) Tested against neonatal rat cardiac myocyte carnitine palmitoyltransferase 1
- ChEMBL_46324 (CHEMBL660892) Tested against neonatal rat cardiac myocyte carnitine palmitoyltransferase 2
- ChEMBL_46325 (CHEMBL660893) Tested against neonatal rat cardiac myocyte carnitine palmitoyltransferase 2
- ChEBML_90259 Inhibition of insulin beta-R in HepG2 cells
- ChEBML_90397 Inhibition of Insulin-like growth factor I receptor
- ChEMBL_1460545 (CHEMBL3395577) Inhibition of recombinant insulin receptor (unknown origin)
- ChEMBL_2267608 Inhibition of Insulin receptor tyrosine kinase (unknown origin)
- ChEMBL_2297251 Binding affinity to insulin regulated aminopeptidase (unknown origin)
- ChEMBL_614233 (CHEMBL1106061) Inhibition of insulin receptor by HTRF assay
- ChEMBL_699678 (CHEMBL1648321) Inhibition of insulin receptor after 1 hr
- ChEMBL_885942 (CHEMBL2215704) Inhibition of insulin receptor by AlphaScreen analysis
- ChEMBL_90266 (CHEMBL699131) Inhibition of insulin receptor (InsR) tyrosine kinase
- ChEMBL_2199955 (CHEMBL5112471) Inhibition of hERG cardiac ion channel by patch clamp assay
- ChEBML_90282 Inhibition of human insulin-like growth factor I receptor
- ChEMBL_305383 (CHEMBL832897) Inhibition of Insulin receptor kinase in P19 cells
- ChEMBL_566615 (CHEMBL960141) Inhibition of insulin receptor by virtual HTS assay
- ChEMBL_581979 (CHEMBL1058959) Inhibition of insulin receptor by cell based assay
- ChEMBL_88875 (CHEMBL698212) Activation of human insulin receptor tyrosine kinase (IRTK)
- ChEMBL_90285 (CHEMBL697504) Inhibition of Insulin-like growth factor I receptor
- ChEMBL_90396 (CHEMBL698351) Inhibition of Insulin-like growth factor I receptor
- ChEMBL_941897 (CHEMBL2329963) Inhibition of human recombinant IDE-mediated insulin degradation
- ChEBML_90404 Ability to displace Insulin-Like Growth Factor (IGF-I) from its binding to human insulin-like growth factor binding protein 3 (hIGFBP-3)
- ChEMBL_2527721 Inhibition of Insulin receptor expressed in HEK293 cells assessed as reduction insulin-stimulated INSR phosphorylation incubated for 1 hr by sandwich ELISA analysis
- ChEBML_90403 Ability of compound to displace Insulin-Like Growth Factor (IGF-I) from its binding to Insulin-like growth factor binding protein 3 (IGFBP-3)
- ChEBML_90543 Ability of compound to displace Insulin-Like Growth Factor (IGF-I) from its binding to Insulin-like growth factor binding protein 5 (IGFBP-5)
- ChEBML_42776 Inhibition of [3H]nitrendipine binding to calcium channels in Rabbit cardiac muscle.
- ChEBML_56503 Tested for beta1-receptor selectivity in canine cardiac tissue in anesthetized dogs
- ChEMBL_155193 (CHEMBL763666) Inhibitory activity against phosphodiesterase 5 (PDE V), isolated from cardiac ventricle
- ChEMBL_156018 (CHEMBL764110) Inhibitory activity against phosphodiesterase 1 (PDE I), isolated from cardiac ventricle
- ChEMBL_156458 (CHEMBL764836) Inhibitory activity against phosphodiesterase 3 (PDE 3) isolated from cardiac ventricle
- ChEMBL_609925 (CHEMBL1074787) Displacement of [3H]nitrendipine from dihydropyridine receptor in rat cardiac membrane
- ChEBML_88868 Inhibition of insulin receptor kinase (Inactive at 1 mM ATP)
- ChEMBL_1500622 (CHEMBL3587752) Inhibition of HSP27 (unknown origin) by insulin aggregation assay
- ChEMBL_2377430 Covalent inhibition of PDI (unknown origin) by insulin aggregation assay
- ChEMBL_2525422 Inhibition of insulin-R (unknown origin) in presence of ATP
- ChEMBL_305115 (CHEMBL831583) Inhibition of human insulin-like growth factor I receptor
- ChEMBL_306046 (CHEMBL874552) Inhibition of human Insulin receptor expressed in CHO cells
- ChEMBL_519660 (CHEMBL947811) Binding affinity to insulin receptor by liquid scintillation counting
- ChEMBL_90265 (CHEMBL699130) Inhibition of insulin receptor mediated mitogenesis of NIH3T3 cells
- ChEMBL_971459 (CHEMBL2406337) Inhibition of insulin receptor (unknown origin) after 60 mins
- ChEMBL_1869997 (CHEMBL4371164) Inhibition of human placental insulin receptor expressed in CHO cells assessed as decrease in insulin-stimulated [3H]-2-deoxyglucose uptake preincubated for 1 hr
- ChEMBL_950881 (CHEMBL2349705) Inhibition of Akt phosphorylation in human insulin-stimulated A549 cells incubated for 2 hrs prior to insulin-induction measured after 30 mins by ELISA
- ChEMBL_1767895 (CHEMBL4220007) Displacement of [TyrA14-125I]-human insulin from human insulin receptor isoform A expressed in baby hamster kidney cells after 2 days by gamma counting method
- ChEMBL_538724 (CHEMBL1035035) Activation of insulin receptor tyrosine kinase in mouse 3T3-L1 cells assessed as increase in 2-deoxy-D-[14C]glucose transport in presence of insulin
- ChEMBL_156472 (CHEMBL761549) In vitro inhibition of Phosphodiesterase 3 activity from guinea pig cardiac ventricle
- ChEMBL_1739758 (CHEMBL4155508) Inhibition of pig beta cardiac myosin 2 by NADH-coupled spectrophotometric analysis
- ChEMBL_214788 (CHEMBL815525) Inhibition of [3H]nitrendipine binding to membrane homogenates of rat cardiac muscle.
- ChEMBL_2199956 (CHEMBL5112472) Inhibition of NaV1.5 (unknown origin) cardiac ion channel by patch clamp assay
- ChEMBL_2199957 (CHEMBL5112473) Inhibition of NaV1.8 (unknown origin) cardiac ion channel by patch clamp assay
- ChEMBL_2199958 (CHEMBL5112474) Inhibition of Cav1.2 (unknown origin) cardiac ion channel by patch clamp assay
- ChEMBL_42772 (CHEMBL660036) Inhibition of [3H]nitrendipine binding to calcium channels in rabbit cardiac muscle
- ChEMBL_42776 (CHEMBL660040) Inhibition of [3H]nitrendipine binding to calcium channels in Rabbit cardiac muscle.
- ChEMBL_56503 (CHEMBL667338) Tested for beta1-receptor selectivity in canine cardiac tissue in anesthetized dogs
- ChEMBL_210560 (CHEMBL816512) Inhibition of thioredoxin reductase in the presence of thioredoxinand insulin
- ChEMBL_2377431 Non-covalent inhibition of PDI (unknown origin) by insulin aggregation assay
- ChEMBL_471122 (CHEMBL921275) Inhibition of human purified insulin receptor expressed in HepG2 cells
- ChEMBL_807032 (CHEMBL1959424) Inhibition of insulin receptor using fluorescent substrate by IMAP assay
- ChEMBL_90405 (CHEMBL698360) Inhibitory activity against Insulin-like growth factor binding protein 3
- ChEMBL_1706543 (CHEMBL4057776) Inhibition of insulin stimulated INSR phosphorylation in human HeLa cells preincubated for 1 hr followed by insulin addition measured after 5 mins by Western blot analysis
- ChEBML_46176 Inhibition of sodium channel blockade in CHO cells expressing human cardiac (hHI) sodium channel
- ChEMBL_202569 (CHEMBL805186) Blockade of Na+ current in frog oocytes expressing human cardiac sodium channel (HH1)
- ChEMBL_214787 (CHEMBL815524) Inhibition of [3H]nitrendipine binding to membrane homogenates of of rat cardiac muscle.
- ChEMBL_214942 (CHEMBL819393) Displacement of [3H](+)-PN200-110 binding from calcium channels of rat cardiac membranes.
- ChEMBL_46175 (CHEMBL660921) Blockade of Na+ current in frog oocytes expressing human cardiac sodium channel (HH1)
- ChEMBL_887445 (CHEMBL2217054) Inhibition of CPT1 in rat cardiac mitochondria using [3H]carnitine by radiometric method
- ChEMBL_92785 (CHEMBL704172) Competitive binding of 3[H]+isradipine to calcium channels of rat cardiac membranes
- ChEMBL_1570092 (CHEMBL3789694) Inhibition of insulin receptor (unknown origin) in presence of [gamma33P]ATP
- ChEMBL_519651 (CHEMBL946778) Inhibition of human insulin receptor autophosphorylation in transfected mouse NIH3T3 cells
- ChEMBL_591804 (CHEMBL1041544) Inhibition of insulin receptor by [gamma-33-P]ATP based assay
- ChEMBL_654586 (CHEMBL1243630) Inhibition of human insulin receptor-mediated Poly Glu4Tyr phosphorylation by ELISA
- ChEMBL_873671 (CHEMBL2188787) Agonist activity at rat P2Y1R assessed as glucose-dependent insulin secretion
- Reductase Activity Assay Reductase activity was assayed by measuring the PDI-catalyzed reduction of insulin in the presence of DTT, thus measuring the aggregation of reduced insulin chains at 650nm.
- ChEMBL_139918 (CHEMBL748586) Inhibition of [3H]- N-methyl-scopolamine ([3H]NMS) dissociation from porcine cardiac M2-receptors
- ChEMBL_42762 (CHEMBL653732) Displacement of [3H](+)-PN200-110 binding to calcium channels in guinea pig cardiac membranes.
- ChEMBL_71494 (CHEMBL680747) Selectivity for gardos channel; (Gardos channel vs 2000 nM for the cardiac IKs channel).)
- ChEMBL_2567995 Inhibition of INSR phosphorylation in insulin-stimulated human H4-II-E cells preincubated with compound for 30 mins followed by insulin stimulation and measured after 10 mins by immunoblot analysis
- ChEMBL_1513260 (CHEMBL3610968) Inhibition of insulin receptor (unknown origin) by homogeneous time resolved fluorescence assay
- ChEMBL_745322 (CHEMBL1775397) Activation of FFA1 in mouse islets assessed as glucose-dependent insulin secretion
- ChEMBL_796984 (CHEMBL1944059) Inhibition of insulin receptor after 20 mins by time-resolved fluorescence assay
- ChEMBL_162259 (CHEMBL772438) Inhibitory activity against Protein-tyrosine phosphatase 1B-mediated dephosphorylation of phosphorylated insulin receptor using (CHO) cell line transfected with an expression plasmid encoding the normal human insulin receptor [CHO/HIRc]
- ChEMBL_2542048 Inhibition of human IR autophosphorylation in insulin- stimulated mouse NIH-3T3-hIR cells pre incubated for 6 hr followed by insulin stimulation and measured after 15 mins by Western blot analysis
- ChEBML_139909 Allosteric inhibition of [3H]NMS (N-methyl-scopolamine) dissociation from porcine cardiac Muscarinic acetylcholine receptor M2
- ChEMBL_512033 (CHEMBL970836) Displacement of [3H]diltiazem from L-type calcium channel in Sprague-Dawley rat cardiac myocytes
- ChEMBL_162252 (CHEMBL770119) Inhibition of human Protein-tyrosine phosphatase 1B (PTP1B) dephosphorylation of insulin receptor peptide
- ChEMBL_521715 (CHEMBL1001626) Agonist activity at rat pancreas P2Y1 receptor assessed as enhancement of insulin secretion
- ChEMBL_801839 (CHEMBL1947451) Displacement of thioflavin T from insulin receptor by thioflavin-T fluorescent dye assay
- ChEMBL_90284 (CHEMBL697349) In vitro inhibition of Insulin-like growth factor I receptor expressed in baculovirus
- ChEMBL_154395 (CHEMBL759157) Inhibition of canine cardiac fraction III cAMP phosphodiesterase (PDE 3) at 0.875 mg/kg intravenous dose
- ChEMBL_154396 (CHEMBL759158) Inhibition of canine cardiac fraction III cAMP phosphodiesterase (PDE 3) at 1.875 mg/kg intravenous dose
- ChEMBL_1279808 (CHEMBL3095509) Activation of glucokinase in rat INS-1 cells assessed as glucose-stimulated insulin secretion
- ChEMBL_1672669 (CHEMBL4022698) Inhibition of human insulin receptor tyrosine kinase after 1 hr by scintillation counting method
- ChEMBL_2266110 Inhibition of Escherichia coil TrxR incubated for 2 hrs in presence of DTT and insulin
- ChEMBL_519642 (CHEMBL946769) Inhibition of GST-tagged insulin receptor expressed in baculovirus by time-resolved fluorescence assay
- ChEMBL_884453 (CHEMBL2212215) Inhibition of human recombinant TRX-2 up to 60 mins by insulin reduction assay
- ChEMBL_884454 (CHEMBL2212216) Inhibition of human recombinant TRX-1 up to 60 mins by insulin reduction assay
- ChEMBL_1909270 (CHEMBL4411716) Activation of insulin-stimulated glycogen synthase in human HepG2 cells using [U-14C]UDP glucose as substrate preincubated for 18 hrs followed by insulin stimulation and measured after 15 mins by beta-counting method
- ChEBML_140037 Binding affinity to the rat cardiac muscarinic acetylcholine receptor M2 using 0.3 nM [3H]N-methylscopolamine as radioligand
- ChEBML_53174 Inhibition of [3H]nitrendipine binding to L-type calcium channel dihydropyridine site of porcine cardiac sarcolemma membrane vesicles
- ChEMBL_1456351 (CHEMBL3368360) Inhibition of human cardiac slowly activating delayed rectifier K+ channel expressed in CHO cells by electrophysiology assay
- ChEMBL_46128 (CHEMBL660002) Binding affinity was determined by displacement of [3H]P1075 from its binding sites in canine cardiac membranes
- ChEMBL_762092 (CHEMBL1816131) Inhibition of human cardiac sodium channel Nav1.5 expressed in human HEK293 cells by patch-clamp electrophysiological assay
- Determination of the Effect of a SERCA Agonist on the Reduction of CSR Stress Assay is for cardiac SR.
- ChEMBL_2545794 Inhibition of PI3Kbeta in human adipocytes assessed as reduction in insulin-induced 2-deoxy-[U-14C]-glucose uptake preincubated for 3 hrs followed by insulin addition and measured after 30 mins by microbeta scintillation counting analysis
- ChEMBL_800636 (CHEMBL1947668) Activation of SUR1 in rat pancreatic islets assessed as inhibition of glucose-induced insulin secretion
- ChEMBL_90401 (CHEMBL698356) The compound was tested for binding affinity against insulin-like growth factor binding protein 1
- ChEMBL_90402 (CHEMBL698357) The compound was tested for binding affinity against insulin-like growth factor binding protein 2
- ChEMBL_90406 (CHEMBL698361) The compound was tested for binding affinity against Insulin-like growth factor binding protein 4
- ChEMBL_90407 (CHEMBL698362) The compound was tested for binding affinity against Insulin-like growth factor binding protein 4
- ChEMBL_90544 (CHEMBL700614) The compound was tested for binding affinity against Insulin-like growth factor binding protein 5
- ChEMBL_90545 (CHEMBL700615) The compound was tested for binding affinity against Insulin-like growth factor binding protein 6
- ChEMBL_959585 (CHEMBL2382530) Agonist activity at GPR40 in rat INS-1 cells assessed as glucose-stimulated insulin secretion
- ChEMBL_776271 (CHEMBL1914068) Inhibition of PIK3CA-mediated cell signaling in PTEN-deficient human U87MG cells assessed as inhibition of insulin-induced pAkt/PKB phosphorylation at Thr308 treated for 15 mins before insulin challenge measured after 5 mins by immunoblotting
- ChEMBL_776272 (CHEMBL1914069) Inhibition of PIK3CA-mediated cell signaling in PTEN-deficient human U87MG cells assessed as inhibition of insulin-induced pAkt/PKB phosphorylation at Ser473 treated for 15 mins before insulin challenge measured after 5 mins by immunoblotting
- ChEMBL_139910 (CHEMBL746846) Allosteric potency against the dissociation of radioligand [3H]N-methylscopolamine from the porcine cardiac Muscarinic acetylcholine receptor M2
- ChEMBL_140037 (CHEMBL744017) Binding affinity to the rat cardiac muscarinic acetylcholine receptor M2 using 0.3 nM [3H]N-methylscopolamine as radioligand
- ChEMBL_154397 (CHEMBL759159) Inhibitory activity against canine cardiac fraction III cyclic AMP phosphodiesterase (PDE III) at 8.75 mg/kg intravenous dose
- ChEMBL_1719739 (CHEMBL4134739) Inhibition of TrxR1 in human HL60 cells incubated for 24 hrs by endpoint insulin reduction assay
- ChEMBL_858381 (CHEMBL2168596) Activation of glucokinase in rat INS-1 cells assessed as stimulation of glucose-induced insulin secretion
- ChEMBL_90408 (CHEMBL698363) Dissociation constant binding to Insulin-like growth factor binding protein 5 at the residue L-81
- ChEMBL_90424 (CHEMBL698181) Dissociation constant in binding to Insulin-like growth factor binding protein 5 at the residue L73
- ChEMBL_90425 (CHEMBL698182) Dissociation constant in binding to Insulin-like growth factor binding protein 5 at the residue L81
- ChEMBL_90426 (CHEMBL697415) Dissociation constant in binding to Insulin-like growth factor binding protein 5 at the residue R87
- ChEMBL_90427 (CHEMBL697416) Dissociation constant in binding to Insulin-like growth factor binding protein 5 at the residue S85
- ChEMBL_90428 (CHEMBL697417) Dissociation constant in binding to Insulin-like growth factor binding protein 5 at the residue Y50
- ChEMBL_90429 (CHEMBL697418) Dissociation constant in binding to Insulin-like growth factor binding protein 5 at the residue Y86
- ChEMBL_2429862 Binding affinity to recombinant human Cardiac troponin C (1 to 89 residues) expressed in Escherichia coli assessed as dissociation constant
- ChEMBL_563140 (CHEMBL1009068) Displacement of [3H]diltiazem from L-type calcium channel in Sprague-Dawley rat cardiac myocytes by liquid scintillation counting
- ChEMBL_65811 (CHEMBL872385) Tested for the competitive binding versus [125I]- ET-1 determined in porcine cardiac ventricular membrane for Endothelin A receptor
- ChEBML_39162 In vitro Beta-1 adrenergic receptor affinity in partially purified membrane fractions from canine cardiac tissue using [SH]dihydroalprenolol (4.5 nM)
- ChEMBL_140036 (CHEMBL744016) Binding affinity of compound for cardiac Muscarinic acetylcholine receptor M2 in rat using 0.3 nM [3H]N-methylscopolamine as radioligand
- ChEMBL_1570094 (CHEMBL3789696) Inhibition of full length insulin receptor (unknown origin) transfected in Ba/F3 cells assessed as cell proliferation
- ChEMBL_1667905 (CHEMBL4017793) Inhibition of insulin receptor kinase (unknown origin) using TK as substrate after 30 mins by HTRF assay
- ChEMBL_1897038 (CHEMBL4399073) Inhibition of human recombinant IDE expressed in CHO cells in presence of [125I]-insulin by HTRF assay
- ChEMBL_581975 (CHEMBL1058955) Inhibition of insulin receptor-mediated proliferation of mouse NIH/3T3 cells after 48 hrs by MTT assay
- ChEMBL_90281 (CHEMBL697346) Concentration required to achieve 50% inhibition of tyrosine phosphorylation on human Insulin-like growth factor I receptor
- ChEMBL_90409 (CHEMBL698364) Dissociation constant in BS binding to Insulin-like growth factor binding protein 5 at the residue K91
- ChEMBL_90410 (CHEMBL698365) Dissociation constant in DMSO binding to Insulin-like growth factor binding protein 5 at the residue K91
- ChEMBL_90411 (CHEMBL698366) Dissociation constant in DMSO binding to Insulin-like growth factor binding protein 5 at the residue L73
- ChEMBL_90412 (CHEMBL698367) Dissociation constant in DMSO binding to Insulin-like growth factor binding protein 5 at the residue L81
- ChEMBL_90413 (CHEMBL698368) Dissociation constant in DMSO binding to Insulin-like growth factor binding protein 5 at the residue R87
- ChEMBL_90414 (CHEMBL698369) Dissociation constant in DMSO binding to Insulin-like growth factor binding protein 5 at the residue S85
- ChEMBL_90415 (CHEMBL698218) Dissociation constant in DMSO binding to Insulin-like growth factor binding protein 5 at the residue Y50
- ChEMBL_90416 (CHEMBL698219) Dissociation constant in DMSO binding to Insulin-like growth factor binding protein 5 at the residue Y86
- ChEMBL_90417 (CHEMBL698220) Dissociation constant in PBS binding to Insulin-like growth factor binding protein 5 at the residue K91
- ChEMBL_90418 (CHEMBL698221) Dissociation constant in PBS binding to Insulin-like growth factor binding protein 5 at the residue L73
- ChEMBL_90419 (CHEMBL698222) Dissociation constant in PBS binding to Insulin-like growth factor binding protein 5 at the residue L81
- ChEMBL_90420 (CHEMBL698223) Dissociation constant in PBS binding to Insulin-like growth factor binding protein 5 at the residue R87
- ChEMBL_90421 (CHEMBL698838) Dissociation constant in PBS binding to Insulin-like growth factor binding protein 5 at the residue S85
- ChEMBL_90422 (CHEMBL698839) Dissociation constant in PBS binding to Insulin-like growth factor binding protein 5 at the residue Y50
- ChEMBL_90423 (CHEMBL698180) Dissociation constant in PBS binding to Insulin-like growth factor binding protein 5 at the residue Y86
- ChEMBL_631420 (CHEMBL1111957) Inhibition of insulin receptor assessed as [33P]gamma-ATP incorporation into substrate after 60 mins by gamma counting
- ChEMBL_858884 (CHEMBL2167930) Displacement of photoactivatable [125I]-Tyr-Bpa-Ala-Hexarelin from CD36 in Sprague-Dawley rat cardiac membranes incubated for 60 mins by densitometry
- Cardiac Myofibril ATPase Assay Following example 15, the counter screen was done using frozen myofibril pellets obtained from cardiac tissue. The assay was done in the same manner as above, with the following notable exceptions: the final well concentration of myofibrils was 1.0 mg/mL and KCl was omitted from the recipe.
- ChEMBL_1339905 (CHEMBL3243742) Induction of human recombinant IDE-mediated insulin hydrolysis preincubated for 10 mins measured after 30 mins by spectrophotometer analysis
- ChEMBL_2524990 Inhibition of GST-tagged human Insulin receptor expressed in baculovirus infected Sf9 cells using retinoblastoma protein in presence of ATP
- ChEMBL_675743 (CHEMBL1272377) Inhibition of Pin1 in serum starved human PC3 cells assessed as reduction in insulin-induced p7)S6 kinase phosphorylation
- ChEMBL_1869996 (CHEMBL4371163) Non-competitive inhibition of human placental insulin receptor expressed in CHO cell membrane assessed as decrease in insulin-stimulated A2 phosphorylation preincubated for 15 mins followed by substrate addition and measured after 15 mins in presence of [gamma-32P]ATP by Dixon plot analysis
- ChEMBL_140185 (CHEMBL744902) Compound was evaluated for its binding affinity for Muscarinic acetylcholine receptor M2 by measuring displacement of [3H]- NMS ligand from rat cardiac cells.
- ChEMBL_1455264 (CHEMBL3362270) Agonist activity at mouse FFA4 receptor in mouse MIN6 cells assessed as induction of glucose-induced insulin secretion by AlphaLISA
- ChEMBL_1565351 (CHEMBL3782745) Inhibition of human recombinant PTP-1B using IR5 insulin receptor residues as substrate after 30 mins by malachite green assay
- ChEMBL_1623252 (CHEMBL3865604) Agonist activity at GPR40 in rat RINm cells assessed as increase glucose-stimulated insulin secretion after 1 hr by ELISA
- ChEMBL_1850470 (CHEMBL4351094) Inhibition of PI3Kdelta (unknown origin) preincubated for 30 mins followed by insulin stimulation for 5 mins by Western blot analysis
- ChEMBL_1922001 (CHEMBL4424846) Inhibition of human N-terminal His6-tagged insulin receptor (1005 to 1310 residues) expressed in baculovirus infected Sf21 insect cells
- ChEMBL_2335698 Displacement of N-terminal FAM labeled cTNI switch peptide probe from human full length cardiac troponin C in presence of CaCl2 by fluorescence polarization assay
- ChEMBL_1775688 (CHEMBL4232680) Inhibition of human IDE using insulin as substrate preincubated for 10 mis followed by substrate addition and measured after 30 mins
- ChEMBL_1883833 (CHEMBL4385332) Agonist activity at GPR40 in human islets assessed as induction of glucose-stimulated insulin secretion after 1 hr by HTRF assay
- ChEMBL_2028620 (CHEMBL4682778) Inhibition of PDIA3 (unknown origin) expressed in Escherichia coli BL21 (DE3) incubated for 1 hr and measured by insulin reduction assay
- ChEMBL_2028621 (CHEMBL4682779) Inhibition of PDIA4 (unknown origin) expressed in Escherichia coli BL21 (DE3) incubated for 1 hr and measured by insulin reduction assay
- ChEMBL_2028623 (CHEMBL4682781) Inhibition of PDIA6 (unknown origin) expressed in Escherichia coli BL21 (DE3) incubated for 1 hr and measured by insulin reduction assay
- ChEMBL_1655135 (CHEMBL4004501) Inhibition of human KCNQ1/KCNE1 expressed in HEK293 cells assessed as reduction in slowly activating delayed rectifier cardiac potassium current by patch clamp electrophysiology method
- ChEBML_1645791 Agonist activity at GLP1R in rat INS-1 cells assessed as increase in glucose-stimulated insulin secretion after 1 hr by HTRF assay
- ChEMBL_1897041 (CHEMBL4399076) Inhibition of recombinant IDE exosite (unknown origin) expressed in Escherichia coli using insulin as substrate incubated for 4 hrs by AlphaLisa assay
- ChEMBL_1655138 (CHEMBL4004504) Inhibition of human ERG expressed in CHOK1 cells assessed as reduction in rapidly activating delayed rectifier cardiac potassium current by whole-cell voltage-patch clamp assay
- ChEMBL_1296283 (CHEMBL3132611) Inhibition of Insulin receptor (unknown origin) using biotinylated poly-Glu-Tyr (4:1) as substrate preincubated for 5 mins measured after 12 hrs
- ChEMBL_1645791 (CHEMBL3994847) Agonist activity at GLP1R in rat INS-1 cells assessed as increase in glucose-stimulated insulin secretion after 1 hr by HTRF assay
- ChEMBL_1861481 (CHEMBL4362337) Inhibition of TrxR1 (unknown origin) using DTNB as substrate preincubated for 30 mins in presence of insulin followed by substrate addition by colorimetry
- ChEMBL_2069558 (CHEMBL4724811) Agonist activity at GPR119 in glucose-induced golden hamster HIT-T15 cells assessed as increase in insulin secretion after 2 hrs by A1phaLISA
- ChEMBL_971485 (CHEMBL2406363) Inhibition of TEL-fused insulin receptor (unknown origin) expressed in mouse BAF3 cells after 2 to 3 days by luciferase reporter gene assay
- ChEMBL_1784836 (CHEMBL4256353) Inhibition of NADPH-reduced recombinant rat TrxR1 expressed in Escherichia coli harboring gor mutant assessed as suppression of insulin reduction using Trx as substrate pretreated for 30 mins followed by Trx addition for 30 mins and subsequent eosin-labelled bovine insulin addition measured every 5 mins for 1 hr by fluorescence assay
- ChEMBL_2335697 Binding affinity to human BADAN labeled cardiac troponin C expressed in Escherichia coli BL21 (DE3) assessed as dissociation constant in presence of Ca2+ by fluorescence based spectra analysis
- ChEMBL_2104460 (CHEMBL4812963) Agonist activity at rat GPR119 in golden hamster HIT-T15 cells assessed as induction of glucose-induced insulin secretion after 2 hrs by A1phaLISA
- ChEMBL_1661563 (CHEMBL4011175) Induction of ERalpha degradation in human MCF7 cells assessed as inhibition of insulin-mediated cell proliferation after 6 days by Hoechst 33258 dye-based assay
- ChEMBL_1888984 (CHEMBL4390738) Inhibition of recombinant human N-terminal His-tagged PDI expressed in Escherichia coli BL21 (DE3) pLysS assessed as reduction in enzyme-mediated bovine insulin aggregation
- ChEMBL_863868 (CHEMBL2176258) Inhibition of INSR expressed in CHO cells assessed as inhibition of receptor phosphorylation pre-incubated before insulin stimulation by Meso-Scale Discovery pY-IR assay
- ChEMBL_218251 (CHEMBL819219) In vitro beta-adrenergic activity against beta-1 adrenergic receptor by the inhibition of insulin stimulated [14C]- glucose incorporation into glycogen in isolated rat soleus muscle
- ChEMBL_1933011 (CHEMBL4478663) Inhibition of AKT1 phosphorylation at Ser473 in EGF/insulin-stimulated human AN3CA cells incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- ChEMBL_1933012 (CHEMBL4478664) Inhibition of AKT1 phosphorylation at Ser473 in EGF/insulin-stimulated human A2780 cells incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- ChEMBL_1933013 (CHEMBL4478665) Inhibition of AKT1 phosphorylation at Thr308 in EGF/insulin-stimulated human AN3CA cells incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- ChEMBL_1933014 (CHEMBL4478666) Inhibition of AKT1 phosphorylation at Thr308 in EGF/insulin-stimulated human A2780 cells incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- Fluorescence-based biochemical high throughput dose response assay for activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF) Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC) Affiliation: The Scripps Research Institute, TSRI Assay Provider: James D. Potter, University of Miami School of Medicine Network: Molecular Library Probe Production Centers Network (MLPCN) Grant Proposal Number: 1 R21 NS064821-01 Grant Proposal PI: James D. Potter, University of Miami School of Medicine External Assay ID: RTF_ACT_FLINT_1536_3XEC50 DRUN SENS Name: Fluorescence-based biochemical high throughput dose response assay for activators of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF). Description: Cardiomyopathies are myocardial diseases that often lead to cardiac remodeling to compensate for deficiencies in cardiac output (1). Cardiomyopathies are characterized as having systolic dysfunctions (i.e. reduced ejection fraction) in dilated cardiomyopathy or diastolic dysfunctions (i.e. impaired relaxation) in hypertrophic and restrictive cardiomyopathies (2). The
- Fluorescence-based biochemical high throughput dose response assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF) Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC) Affiliation: The Scripps Research Institute, TSRI Assay Provider: James D. Potter, University of Miami School of Medicine Network: Molecular Library Probe Production Centers Network (MLPCN) Grant Proposal Number 1 R21 NS064821-01 Grant Proposal PI: James D. Potter, University of Miami School of Medicine External Assay ID: RTF_INH_FLINT_1536_3XIC50 DRUN DESENS Name: Fluorescence-based biochemical high throughput dose response assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF). Description: Cardiomyopathies are myocardial diseases that often lead to cardiac remodeling to compensate for deficiencies in cardiac output (1). Cardiomyopathies are characterized as having systolic dysfunctions (i.e. reduced ejection fraction) in dilated cardiomyopathy or diastolic dysfunctions (i.e. impaired relaxation) in hypertrophic and restrictive cardiomyopathies (2). Th
- ChEMBL_2335696 Binding affinity to human recombinant full length N-terminal Alexa647-NHS labeled cardiac troponin C expressed in Escherichia coli BL21 (DE3) assessed as dissociation constant incubated for 20 mins by microscale thermophoresis analysis
- ChEMBL_1897052 (CHEMBL4399087) Inhibition of wild type human IDE catalytic site using insulin as substrate preincubated for 10 mins followed by substrate addition and measured after 10 mins by Luminex kit method
- ChEMBL_1988385 (CHEMBL4621932) Inhibition of PI3Kalpha in human BT474 cells expressing P3KCA mutant assessed as reduction in AKT phosphorylation at residue S473 incubated for 2 hrs followed by insulin addition by ELISA
- Metabolic Measurements Glucose in tail blood was measured using a glucometer (One-Touch Basic; Lifescan, CA). For glucose tolerance tests (GTTs), mice were fasted for 10 hours and then injected with 20% D-glucose (2 mg/g body weight) and the blood glucose was monitored immediately before and at 15, 30, 60 and 120 mins following the injection. For insulin tolerance tests (ITTs), 4-h fasted animals were given insulin (0.75 mU/g) and blood glucose was measured immediately before and at 30, 60 and 120 minutes postinjection. Serum insulin, cholesterol, triglycerides (Stanbio Labs, TX), BDNF (Abnova), IGF1 and IGFBPs (R&D Systems) were determined by enzyme-linked immunosorbent assay.
- Myofibril Assays To evaluate the effect of compounds on the ATPase activity of full-length cardiac myosin in the context of the native sarcomere, skinned myofibril assays were performed. Bovine cardiac myofibrils were obtained by homogenizing bovine cardiac left ventricular tissue in the presence of a detergent such as triton X-100. Such treatment removes membranes and a majority of the soluble cytoplasmic proteins but leaves intact the cardiac sarcomeric acto-myosin apparatus. Myofibril preparations retain the ability to hydrolyze ATP in an Ca2+ regulated manner. ATPase activities of such myofibril preparations in the presence and absence of compounds were assayed at Ca2+ concentrations activating to a defined fraction of the maximal rate (i.e., 25%, 75%). Small molecule agents were assessed for their ability to inhibit the steady-state ATPase activity of bovine cardiac myofibrils using pyruvate kinase and lactate dehydrogenase (PK/LDH)-coupled enzyme system. This assay regenerates myosin-produced ADP into ATP by oxidizing NADH, producing an absorbance change at 340 nm. Prior to testing small molecule agents, the bovine cardiac myofibrils were assessed for their calcium responsiveness and the calcium concentration that achieves either a 50% (pCa50) or 75% (pCa75) activation of the myofibril system was chosen as the final condition for assessing the inhibitory activity of the small molecule agents. All enzymatic activity was measured in a buffered solution containing 12 mM PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid), 2 mM magnesium chloride at pH 6.8 (PM 12 buffer). Final assay conditions were 1 mg/mL of bovine cardiac myofibrils, 4 U/mL pyruvate kinase, 6 U/mL lactate dehydrogenase, 50 μM ATP, 0.1 mg/mL BSA (bovine serum albumin), 10 ppm antifoam, 1 mM DTT, 0.5 mM NADH, 1.5 mM PEP, 0.6 mM EGTA, and an amount of CaCl2) sufficient to achieve either 50% or 75% activation of the myofibril ATPase activity.
- ChEMBL_1570093 (CHEMBL3789695) Inhibition of full length insulin receptor (unknown origin) autophosphorylation transfected in HEK293 cells pretreated for 60 mins followed by IGF-1 stimulation measured after 10 mins by quantitative Western blot analysis
- ChEMBL_1933015 (CHEMBL4478667) Inhibition of AKT1 in EGF/insulin-stimulated human AN3CA cells assessed as reduction in phosphorylation at Thr246 incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- ChEMBL_1933016 (CHEMBL4478668) Inhibition of AKT1 in EGF/insulin-stimulated human A2780 cells assessed as reduction in phosphorylation at Thr246 incubated for 1 hr followed by stimulation for 15 mins by Western blot analysis
- ChEMBL_2074276 (CHEMBL4729810) Inhibition of recombinant human wild-type N-terminal His-tagged PDIA1 expressed in Escherichia coli strain BL21(DE3) using bovine insulin as substrate preincubated for 1 hr followed by substrate addition
- ChEMBL_2516987 Inhibition of Ins-R in A14 [Human melanoma] cells assessed as inhibition of autophosphorylation of Ins-R incubated for 90 mins followed by insulin stimulation measured after 10 mins by ELISA analysis
- ChEMBL_901085 (CHEMBL3062690) Agonist activity at Homo sapiens (human) PPARgamma expressed in mouse 3T3-L1 cells incubated for 2 days followed by compound wash out measured after 4 days by insulin receptor binding assay
- ChEMBL_1767894 (CHEMBL4220006) Agonist activity at B6D2F1 mouse insulin receptor assessed as increase in glucose incorporation into lipid phase after 2 hrs in presence of D-[3-3H]glucose by TopCount microplate scintillation counting method
- ChEMBL_1897053 (CHEMBL4399088) Inhibition of human recombinant IDE expressed in Escherichia coli BL21 (DE3) cells using insulin as substrate preincubated for 10 mins followed by substrate addition and measured after 15 mins by fluorescence based assay
- ChEMBL_2264503 Inhibition of GST-tagged recombinant human IR ( 919 to 1343 residues) autophosphorylation expressed in mouse NIH-3T3-A14 cells preincubated for 90 mins followed by insulin stimulation and measured after 10 mins by ELISA
- ChEMBL_2248960 (CHEMBL5163170) Positive allosteric modulator activity at GLP-1R in rat INS1 beta-cells assessed potentiation of GLP-1(7-36)NH2 induced insulin secretion incubated for 30 mins in presence of high glucose condition by ELISA
- ChEMBL_1717891 (CHEMBL4132891) Antagonist activity at GLUT4 in rat adipocytes assessed as reduction in insulin-stimulated [3H]2-deoxyglucose uptake preincubated for 1 min followed by [3H]2-deoxyglucose addition measured after 1 min by liquid scintillation counting analysis
- ChEMBL_2248961 (CHEMBL5163171) Positive allosteric modulator activity at GLP-1R in rat INS1 beta-cells assessed potentiation of GLP-1(7-36)NH2 induced insulin secretion incubated for 30 mins in presence of 0.2 nM of GLP-1 by ELISA
- ChEMBL_2248962 (CHEMBL5163172) Positive allosteric modulator activity at GLP-1R in rat INS1 beta-cells assessed potentiation of GLP-1(7-36)NH2 induced insulin secretion at 0.1 nM incubated for 30 mins in presence of exendin(9-39)NH2 by ELISA
- Assaying Skeletal Muscle Myosin II The skeletal muscle myosin II actin-activated ATPase assay is performed the same as for cardiac muscle myosin II with the following exceptions: Rabbit skeletal myosin is obtained from Cytoskeleton and the final assay conditions contain 20 nM skeletal muscle myosin II.
- ChEMBL_1661323 (CHEMBL4010935) Agonist activity at TGR5 in STC1 cells derived from double transgenic mouse expressing rat insulin promoters linked to SV40 large T antigen and to polyomavirus small T antigen assessed as increase in cAMP level after 40 mins by mass spectrometric method
- ChEMBL_662256 (CHEMBL1252348) Inhibition of PKB/Akt activation in human overnight starved MCF7 cells assessed as phosphorylated Akt at Ser473 level at 50 ug/ml after 1 hr followed by treated with 1 ug/mL of insulin for 15 mins by Western blotting
- 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.
- Potassium Channel Assay Drugs belonging to different classes have been shown to be associated with QT prolongation and in some cases serious ventricular arrhythmias. The most common mechanism for these adverse events is the inhibition of one or more cardiac potassium channels, in particular hERG. This current is important for cardiac myocyte repolarization and is a common target for drugs that prolong the QT interval. Test articles in this study were therefore characterized to determine their ability to inhibit the hERG channel. Ion channel activity was measured using a stably transfected Chinese Hamster Ovary (CHO) cell line expressing the hERG mRNA. The pharmacology of this cloned channel expressed in the CHO cell line is very similar to that observed in native tissue.
- Inhibition of ALK IR Anaplastic Lymphoma Kinase Activity To measure the activity of the N2-(2-methoxyphenyl)pyrimidine derivative represented by formula 1 of the present invention to inhibit anaplastic lymphoma kinase (ALK) activity at enzyme level, the following experiment was performed by the same manner as described in experimental example 1 except that IR (Insulin Receptor) protein was used instead of ALK WT protein.
- In vitro Assay The method of determining thioredoxin activity is based on the reduction of insulin by thioredoxin. Thioredoxin is reconstituted is by thioredoxin reductase, with NADPH. The resulting free thiol -SH groups react with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). The termination of the reaction results in the formation of a red colour. The intensity of colouration is determined spectrophotometrically at 412 nm and this corresponds to the number of reduced sulfahydryl groups. The reaction was performed at 37° C. over 30 min. in a buffer containing 50 mM Tris-HCl and 20 mM EDTA at a pH of 7.6. The substrate concentrations used were: 0.25 μM human recombinant thioredoxin and 0.325 μM rat recombinant thioredoxin reductase (IMCO Corporation Ltd AB, Sweden) and 316 μM of insulin, 0.8 μM NADPH, 8 mM DTNB (Sigma Aldrich).
- Nav1.5 Assay To assess the potential cardiac liability of compounds at an early stage in the drug discovery process, a Nav1.5 sodium channel screening assay was be performed on Molecular Device's PatchXpress™ automated electrophysiology platform. Under voltage-clamp conditions, Nav1.5 currents were recorded from HEK cells expressing the human Nav1.5 channel in the absence and presence of increasing concentrations of the test compound to obtain an IC50 value. The external recording solution contained (in mM): 90 TEACl, 50 NaCl, 1.8 CaCl, 1 MgCl2, 10 HEPES, 10 glucose, adjusted to pH 7.4 with TEA-OH and to 300 mOsm with sucrose (if necessary), while the internal patch pipette solution contained (in mM): 129 CsF, 2 MgCl2, 11 EGTA, 10 HEPES, 3 Na2ATP adjusted to pH 7.2 with CsOH and to 290 mOsm with sucrose (if necessary). Nav1.5 channel currents were evoked using a cardiac action potential waveform at 1 Hz, digitized at 31.25 kHz and low-pass filtered at 12 kHz.
- Assaying Cardiac Muscle Myosin II The cardiac muscle myosin II actin-activated ATPase assay is a biochemical assay. Specifically, it is an NADH (nicotinamide adenine dinucleotide)-coupled ATPase assay that relies on NADH fluorescence as a readout. Cardiac myosin is a mechanochemical energy transducer that hydrolyzes ATP to generate force in the presence of its activator, F-actin. The resulting ADP is regenerated to ATP by pyruvate kinase (PK) that transforms one molecule of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH) that, in turn, oxidizes one molecule of NADH to NAD. Therefore, the decrease in NADH concentration as a function of time equals the ATP hydrolysis rate. Bovine cardiac myosin is obtained from a commercial source, Cytoskeleton. PK, LDH, ATP, PEP, and NADH are obtained from Sigma. F-actin is prepared in house from Rabbit Muscle Acetone Powder. The assay is run at 25° C. in 384 well black-wall polystyrene microplates with a total volume of 20 μl per well. NADH fluorescence is monitored for 30 minutes with an EnVision Multimode Plate Reader. The slope of the fluorescence response, which is proportional to the reaction rate, is determined by simple linear regression. Final assay conditions are 300 nM cardiac myosin, 10 μM actin, 40 U/ml LDH, 200 U/ml PK, 220 μM NADH, 1 mM PEP, 1 mM ATP in a buffer containing 10 mM 3-(N-morpholino)propanesulfonic acid (pH=7.0), 2 mM MgCl2, 0.15 mM ethylene glycol-bis(p-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 0.1 mg/mL bovine serum albumin, 0.5% (V/V) dimethyl sulfoxide (DMSO) and 1 mM dithiothreitol. Prior to testing the inhibitory activity of the compounds, a two-fold dilution series starting at 10 mM compound concentration is prepared in DMSO. Subsequently, 100 nl is transferred to each well of the measuring plate containing a mixture of myosin, LDH and PK. The enzymatic reaction is started with the addition of a mixture containing ATP, PEP, NADH and actin. The highest final compound concentration is 50 uM. 20 μM para-aminoblebbistatin in 0.5% DMSO serves as the positive control and 0.5% DMSO alone is the negative control. Reaction rates are determined by using the fluorescence responses of a dilution series of NADH included in all plates and plotted as a function of inhibitor concentration. All measurements are carried out in triplicate. Inhibitory constants are determined by fitting the 16 point dose-response data to a quadratic equation corresponding to a simple one-to-one binding model. Small molecules showing no observable inhibition at or below their solubility are reported as inactive.
- In Vitro Assay The in vitro effects of the selected compounds on the hERG (human ether- -go-go-related gene) potassium channel current (a surrogate for IKr, the rapidly activating, delayed rectifier cardiac potassium current) expressed in mammalian cells were evaluated at room temperature using the PatchXpress 7000A (Molecular Devices), an automatic parallel patch clamp system. Each compound was evaluated at varying concentrations in duplicate up to 10 μM, and the duration of exposure to each test article concentration was 5 minutes.
- Dose Response: Fluorescence polarization-based cell-based high throughput dose response assay for inhibitors of insulin-degrading enzyme (IDE) Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC) Center Affiliation: The Scripps Research Institute (TSRI) Assay Provider: Malcolm Leissring, Mayo Clinic College of Medicine Network: Molecular Libraries Probe Production Centers Network (MLPCN) Grant Proposal Number: 1 R03 DA024888-01 Grant Proposal PI: Malcolm Leissring, Mayo Clinic College of Medicine External Assay ID: IDE_INH_FP_1536_3XIC50 DRUN Name: Dose Response: Fluorescence polarization-based cell-based high throughput dose response assay for inhibitors of insulin-degrading enzyme (IDE). Description: Alzheimer's disease (AD) is characterized by accumulation of amyloid beta-protein (A-beta; Abeta) in brain regions involved in memory and cognition (1). The steady-state levels of AB reflect a balance between its production via beta- and gamma-secretases and its catabolism by proteolytic degradation (2-4). Because Abeta cleavage products are less neurotoxic than intact Abeta, e
- Assay of Inhibitory Effect on Cardiac yosin ATPase acAtivity a) 250 μL of ice-cold PM12 buffer was added to 250 μg of S1 myosin to a protein concentration of 1 mg/mL.b) The reagents were successively added in the following order and mixed to give a reaction mixture:400 μL of PM12,400 μL of 5× MSEG (from the ATPase assay biochem kit),1200 μL of actin/cardiac tropomyosin/troponin complex,40 μL of myosin S1,40 μL of 100× PNP (from the ATPase assay biochem kit),10.4 μL of 100 mM ATP.c) 10 μL of 440 μM CaCl2 solution was added to the 96-well plate, and the plate was placed in a 37° C. incubator to pre-warm.d) 100 μL of the reaction mixture was added to the 96-well plate, and the plate was centrifuged at 1000 rpm for 10 seconds.e) The plate was read continuously for 10 min with a 30 seconds interval on a SpectraMax 340PC, with an instrument temperature of 37° C. and a wavelength of 360 nm.
- PDI Inhibition Assay The assay was carried out in 384-well plates according to literature procedures [46]. Each well contained 100mM sodium phosphate and 0.2mM EDTA pH 7.0. 10ng PDI was pre-incubated with various concentration of probes (4% DMSO) in 20uL buffer for 30 min at 37 degrees Celsius, followed by the addition of insulin (0.16mM final concentration) and DDT (1mM final concentration). The enzyme reaction was monitored at 650nm on a Bioteck microplate reader.
- Biochemical Assay In our experiments, bovine skinned cardiac myofibrils were isolated from the frozen bovine left ventricle as myosin's source in the ATPase assay. The calcium concentration that achieves a 50% (pCa50 or pCa=6.25) activation of the myofibril system was chosen as the final condition for assessing the activation activity according to the literature (DOI: 10.1074/jbc.M117.776815). Myofibrils ATPase activity was measured in a buffered solution containing 12 mM PIPES (piperazine-N, N′-bis(2-ethane sulfonic acid) and 2 mM magnesium chloride at pH 6.8 (PM12 buffer). Final assay conditions were 1 mg/mL of bovine cardiac myofibrils, 1:20 of stock PK/LDH (Sigma-Aldrich, Cat No. P0294-5X5ML), 50 μM ATP, 1 mM DTT (dithiothreitol), 0.75 mM NADH, 1.5 mM PEP at pCa50 (pCa=6.25). Compounds were dissolved in DMSO (dimethyl sulfoxide). Serial dilution of compounds was created such that the final desired concentration of compound would be achieved in a volume of 150 μL with a fixed DMSO concentration of 2% (v/v). 75 μL of a solution containing bovine cardiac myofibrils, PK/LDH, and calcium were added to a 96 well plate for a 7 point dose-response. In some circumstances, 10 point-response was used to repeat the ATPase assays on compounds of interest. Compounds were added to the myofibrils solution and incubated for 5 minutes. The enzymatic reaction was started with the addition of 75 μL of a solution containing ATP, PEP, NADH, compounds, and calcium. The ATPase activity was measured by reading absorbance at 340 nm in a PerkinElmer Victor Nivo plate reader at 25° C. in kinetic mode for 15 minutes using clear bottom plates. The slopes of the absorbance changes as a function of time for the first 10 minutes were normalized to slopes on the control wells containing all reagents, including DMSO, but without compounds.
- In Vitro Kinase Assay Inhibition of kinase activity against a variety of recombinant kinases [FLT3, FLT3 D835Y, anaplastic lymphoma kinase (ALK), insulin receptor, and epidermal growthfactor receptor (EGFR)] was measured using homogeneous time-resolved fluorescence (HTRF) assays [Choi et al., Bioorg. Med. Chem. Lett., 20:2033-2037]. Assays consist of enzymes mixed with serially diluted compounds and peptide substrates in a kinase reaction buffer (250 mM HEPES (pH 7.0), 0.5 mM orthovanadate, 0.05% bovine serum albumin, 0.1% NaN3). Following the addition of reagents for detection, the TR-FRET signal was measured using an EnVision multi-label reader (Perkin Elmer, Waltham, MA).
- In vitro degradation assay In vitro degradation assay protocol for compounds 3-33: Panc02.13 cells were purchased from ATCC and cultured in RPMI-1640 (Gibco), supplemented with 15% FBS (ATCC) and 10 Units/mL human recombinant insulin (Gibco). PROTAC treatments were carried out in 12-well plates for 16 h. TLR3 agonist Poly I:C (Invivogen; tlr1-pic) was added for the final 3 h. Cells were harvested, and lysed in RIPA buffer (50 mM Tris pH8, 150 mM NaCl, 1% Tx-100, 0.1% SDS, 0.5% Sodium Deoxycholate) supplemented with protease and phosphatase inhibitors. Lysates were clarified at 16,000 g for 10 minutes, and supernatants were separated by SDS-PAGE. Immunoblotting was performed using standard protocols. The antibodies used were TBK1 (Cell Signaling#3504), pIRF3 (abcam#ab76493), and GAPDH (Cell Signaling#5174).
- AKT counterscreen of confirmed PKD inhibitors-57K library The PKD HTS assay was developed and run at the University of Pittsburgh Molecular Screening Center (PMLSC) as part of the Molecular Library Screening Center Network (MLSCN)(1R03DA24898-01). Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by diacylglycerol. It regulates many fundamental cell functions including cell proliferation, survival, differentiation and protein trafficking, and plays important roles in pathological conditions such as cardiac hypertrophy and cancer in multiple organ systems. However, the mechanisms underlying these effects of PKD are not clearly understood, and the role of PKD in cancer and other diseases has not been fully defined. This is partly due to the lack of effective pharmacological tools that specifically target PKD in normal cellular processes and in pathological conditions. The immediate goal of this proposal is to demonstrate the feasibility of an IMAP-based HTS fluorescent polarization (FP) assay and its use to identi
- CDK7 counterscreen of confirmed PKD inhibitors-57K library addition The PKD HTS assay was developed and run at the University of Pittsburgh Molecular Screening Center (PMLSC) as part of the Molecular Library Screening Center Network (MLSCN)(1R03DA24898-01). Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by diacylglycerol. It regulates many fundamental cell functions including cell proliferation, survival, differentiation and protein trafficking, and plays important roles in pathological conditions such as cardiac hypertrophy and cancer in multiple organ systems. However, the mechanisms underlying these effects of PKD are not clearly understood, and the role of PKD in cancer and other diseases has not been fully defined. This is partly due to the lack of effective pharmacological tools that specifically target PKD in normal cellular processes and in pathological conditions. The immediate goal of this proposal is to demonstrate the feasibility of an IMAP-based HTS fluorescent polarization (FP) assay and its use to identi
- Fluorescence polarization assay for PKD inhibitiors-interference assay-57K HTS campaign The PKD HTS assay was developed and run at the University of Pittsburgh Molecular Screening Center (PMLSC) as part of the Molecular Library Screening Center Network (MLSCN)(1R03DA24898-01). Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by diacylglycerol. It regulates many fundamental cell functions including cell proliferation, survival, differentiation and protein trafficking, and plays important roles in pathological conditions such as cardiac hypertrophy and cancer in multiple organ systems. However, the mechanisms underlying these effects of PKD are not clearly understood, and the role of PKD in cancer and other diseases has not been fully defined. This is partly due to the lack of effective pharmacological tools that specifically target PKD in normal cellular processes and in pathological conditions. The immediate goal of this proposal is to demonstrate the feasibility of an IMAP-based fluorescent polarization (FP) assay for high throughput scree
- PLK1 counterscreening assay for identified PKD inhibitors The PKD HTS assay was developed and run at the University of Pittsburgh Molecular Screening Center (PMLSC) as part of the Molecular Library Screening Center Network (MLSCN)(1R03DA24898-01). Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by diacylglycerol. It regulates many fundamental cell functions including cell proliferation, survival, differentiation and protein trafficking, and plays important roles in pathological conditions such as cardiac hypertrophy and cancer in multiple organ systems. However, the mechanisms underlying these effects of PKD are not clearly understood, and the role of PKD in cancer and other diseases has not been fully defined. This is partly due to the lack of effective pharmacological tools that specifically target PKD in normal cellular processes and in pathological conditions. The immediate goal of this proposal is to demonstrate the feasibility of an IMAP-based HTS fluorescent polarization (FP) assay and its use to identi
- SAR analysis for the identification of translation initiation inhibitors (PABP) Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG) Source Affiliation: Sanford-Burnham Medical Research Institute(SBMRI, San Diego, CA) Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN) Grant Number: 1R03MH084835-01 Assay Provider: Jerry Pelletier, Ph.D, McGill University, Montreal, Canada Translation is an essential cellular process whose deregulation is associated with alterations in cell growth, cell cycle progression, and cell death responses. The initiation phase of translation is a key target for regulation when cells are exposed to various environmental cues (e.g. insulin, amino acid starvation, mitogenic stimulation, hypoxia, etc). As well, translation initiation control is usurped upon viral infection and is deregulated in many human cancers. Over-expression of certain translation factors can lead to malignant transformation and many of the components of the translational apparatus are over-expressed in human cancers. Several tumor sup
- SAR analysis for the identification of translation initiation inhibitors (eIF4H) Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG) Source Affiliation: Sanford-Burnham Medical Research Institute (SBIMR, San Diego, CA) Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN) Grant Number: 1R03MH084835-01 Assay Provider: Jerry Pelletier, Ph.D, McGill University, Montreal, Canada Translation is an essential cellular process whose deregulation is associated with alterations in cell growth, cell cycle progression, and cell death responses. The initiation phase of translation is a key target for regulation when cells are exposed to various environmental cues (e.g. insulin, amino acid starvation, mitogenic stimulation, hypoxia, etc). As well, translation initiation control is usurped upon viral infection and is deregulated in many human cancers. Over-expression of certain translation factors can lead to malignant transformation and many of the components of the translational apparatus are over-expressed in human cancers. Several tumor su
- uHTS fluorescence polarization assay for the identification of translation initiation inhibitors (PABP) Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG) Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA) Network: NIH Molecular Libraries Production Centers Network (MLPCN) Grant Number: 1R03MH084835-01 Assay Provider: Jerry Pelletier, Ph.D, McGill University, Montreal, Canada Translation is an essential cellular process whose deregulation is associated with alterations in cell growth, cell cycle progression, and cell death responses. The initiation phase of translation is a key target for regulation when cells are exposed to various environmental cues (e.g. insulin, amino acid starvation, mitogenic stimulation, hypoxia, etc). As well, translation initiation control is usurped upon viral infection and is deregulated in many human cancers. Over-expression of certain translation factors can lead to malignant transformation and many of the components of the translational apparatus are over-expressed in human cancers. Several tumor suppresso
- uHTS fluorescence polarization assay for the identification of translation initiation inhibitors (eIF4H) Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG) Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA) Network: NIH Molecular Libraries Production Centers Network (MLPCN) Grant Number: 1R03MH084835-01 Assay Provider: Jerry Pelletier, Ph.D, McGill University, Montreal, Canada Translation is an essential cellular process whose deregulation is associated with alterations in cell growth, cell cycle progression, and cell death responses. The initiation phase of translation is a key target for regulation when cells are exposed to various environmental cues (e.g. insulin, amino acid starvation, mitogenic stimulation, hypoxia, etc). As well, translation initiation control is usurped upon viral infection and is deregulated in many human cancers. Over-expression of certain translation factors can lead to malignant transformation and many of the components of the translational apparatus are over-expressed in human cancers. Several tumor suppresso
- In Vitro Inhibition Activity To screen for isoform selectivity of the digoxin derivatives we compared inhibition of Na,K-ATPase activity of purified detergent-soluble human isoform complexes α1β1FXYD1, α2β1FXYD1, α2β2FXYD1 and α2β3FXYD1. Although all the preparations and assays were conducted with FXYD1 in order to stabilize the complexes, the FXYD1 suffix is omitted in naming of isoform complexes for simplicity.Na,K-ATPase activity of α/βFXYD1 complexes was measured over one hour at 37° C. in a medium containing 130 mM NaCl, 5 mM KCl, 3 mM MgCl2, 1 mM EGTA, 25 mM Histidine, pH 7.4 and 1 mM ATP using the PiColor Lock gold malachite green assay (Inova Biosciences).The Na,K-ATPase activities were α1β1, 21.5±5.3 moles/min/mg; α2β1, 18.7±1.8 moles/min/mg, and α2β3, 10.7±1.9 moles/min/mg protein. As discussed below, an important kinetic property in relation to inhibition by cardiac glycosides is K0.5 for activation by K: α1β1 1.25±0.05 mM, α2β1 2.7±0.14 mM and α2β3 6.4±0.50 mM, respectively.
- Selective Inhibition Assays of Isolated Na,K-ATPase To screen for isoform selectivity of the digoxin derivatives we compared inhibition of Na,K-ATPase activity of purified detergent-soluble human isoform complexes α1β1FXYD1, α2β1FXYD1, α2β2FXTD1 and α2β3FXYD1. Although all the preparations and assays were conducted with FXYD1 in order to stabilize the complexes, the FXYD1 suffix is omitted in naming of isoform complexes for simplicity.Na,K-ATPase activity of α/βPFXYD1 complexes was measured over one hour at 37° C. in a medium containing 130 mM NaCl, 5 mM KCl, 3 mM MgCl2, 1 mM EGTA, 25 mM Histidine, pH 7.4 and 1 mM ATP using the PiColor Lock gold malachite green assay (Inova Biosciences).The Na,K-ATPase activities were α1β1, 21.5±5.3 μmoles/min/mg; α2β1, 18.7±1.8 μmoles/min/mg, and α2β3, 10.7±1.9 μmoles/min/mg protein. As discussed below, an important kinetic property in relation to inhibition by cardiac glycosides is K0.5 for activation by K: α1β1-1.25±0.05 mM, α2β1-2.7±0.14 mM and α2β3 6.4±0.50 mM, respectively.Selectivity of the compounds for various isolated isoforms of human Na,K-ATPase was determined essentially as described before [Katz, A. et al., J Biol Chem., 2010, 285(25), pp. 19582-19592].ATPase activity assays as well as titrations with NaCl, KCl and vanadate were performed as described in Lifshitz-2007 and Loayza-1998 using PiColorLock malachite green assay (Inova Bioscience). Inhibitor assays were performed as described in Katz-2010. [3H]ouabain binding and K+-[3H]digoxin displacement assays were performed as described in Katz-2010.The percent inhibition VCG/V0 was calculated and Ki values were obtained by fitting the data to the function VCG/V0=Ki/([CG]+Ki)+c (CG stands for cardiac glycoside). Inhibition was estimated in 3-5 separate experiments and average Ki values±standard error of the mean (SEM) were calculated. The ratios Ki α1β1/α2β1, α1β1/α2β2 and α1β1/α2β3 was calculated for each compound.
- hERG Inhibition Assay The human Ether-a-go-go Related Gene (hERG) encodes the inward rectifying voltage gated potassium channel in the heart (IKr) which is involved in cardiac repolarisation. IKr current inhibition has been shown to elongate the cardiac action potential, a phenomenon associated with increased risk of arrhythmia. IKr current inhibition accounts for the vast majority of known cases of drug-induced QT-prolongation. A number of drugs have been withdrawn from late stage clinical trials due to these cardiotoxic effects, therefore it is important to identify inhibitors early in drug discovery.The hERG inhibition study aims at quantifying the in vitro effects of compounds of the invention on the potassium-selective IKr current generated in normoxic conditions in stably transfected HEK 293 cells with the human ether-a-go-go-related gene (hERG).Whole-cell currents (acquisition by manual patch-clamp) elicited during a voltage pulse were recorded in baseline conditions and following application of tested compounds (5 minutes of exposure). The concentrations of tested compounds (0.3 μM; 3 μM; 10 μM; 30 μM) reflect a range believed to exceed the concentrations at expected efficacy doses in preclinical models.The pulses protocol applied is described as follow: the holding potential (every 3 seconds) was stepped from −80 mV to a maximum value of +40 mV, starting with −40 mV, in eight increments of +10 mV, for a period of 1 second. The membrane potential was then returned to −55 mV, after each of these incremented steps, for 1 second and finally repolarized to −80 mV for 1 second.The current density recorded were normalized against the baseline conditions and corrected for solvent effect and time-dependent current run-down using experimental design in test compound free conditions.Inhibition curves were obtained for compounds and the concentrations which decreased 50% of the current density determined in the baseline conditions (IC50) were determined. All compounds for which the IC50 value is above 10 M are not considered to be potent inhibitors of the hERG channel whereas compounds with IC50 values below 1 μM are considered potent hERG channel inhibitors.
- hERG Inhibition Assay The human ether-a-go-go related gene (hERG) encodes the inward rectifying voltage gated potassium channel in the heart (IKr) which is involved in cardiac repolarisation. IKr current inhibition has been shown to elongate the cardiac action potential, a phenomenon associated with increased risk of arrhythmia. IKr current inhibition accounts for the vast majority of known cases of drug-induced QT-prolongation. A number of drugs have been withdrawn from late stage clinical trials due to these cardiotoxic effects, therefore it is important to identify inhibitors early in drug discovery.The hERG inhibition study aims at quantifying the in vitro effects of compounds of the invention on the potassium-selective IKr current generated in normoxic conditions in stably transfected HEK 293 cells with the human ether-a-go-go-related gene (hERG).Whole-cell currents (acquisition by manual patch-clamp) elicited during a voltage pulse were recorded in baseline conditions and following application of tested compounds (5 minutes of exposure). The concentrations of tested compounds (0.3 μM; 3 μM; 10 μM; 30 μM) reflect a range believed to exceed the concentrations at expected efficacy doses in preclinical models.The pulses protocol applied is described as follow: the holding potential (every 3 seconds) was stepped from −80 mV to a maximum value of +40 mV, starting with −40 mV, in eight increments of +10 mV, for a period of 1 second. The membrane potential was then returned to −55 mV, after each of these incremented steps, for 1 second and finally repolarized to −80 mV for 1 second.The current density recorded were normalized against the baseline conditions and corrected for solvent effect and time-dependent current run-down using experimental design in test compound free conditions.Inhibition curves were obtained for compounds and the concentrations which decreased 50% of the current density determined in the baseline conditions (IC50) were determined. All compounds for which the IC50 value is above 10 μM are not considered to be potent inhibitors of the hERG channel whereas compounds with IC50 values below 1 μM are considered potent hERG channel inhibitors.
- hERG Inhibition Assay The human ether-a-go-go related gene (hERG) encodes the inward rectifying voltage gated potassium channel in the heart (Igr) which is involved in cardiac repolarisation. IK r current inhibition has been shown to elongate the cardiac action potential, a phenomenon associated with increased risk of arrhythmia. IK r current inhibition accounts for the vast majority of known cases of drug-induced QT-prolongation. A number of drugs have been withdrawn from late stage clinical trials due to these cardiotoxic effects, therefore it is important to identify inhibitors early in drug discovery.The hERG inhibition study aims at quantifying the in vitro effects of compounds of the invention on the potassium-selective IK r current generated in normoxic conditions in stably transfected HEK 293 cells with the human ether-a-go-go-related gene (hERG).Whole-cell currents (acquisition by manual patch-clamp) elicited during a voltage pulse were recorded in baseline conditions and following application of tested compounds (5 minutes of exposure). The concentrations of tested compounds (0.3 μM; 3 μM; 10 μM; 30 μM) reflect a range believed to exceed the concentrations at expected efficacy doses in preclinical models.The pulses protocol applied is described as follow: the holding potential (every 3 seconds) was stepped from −80 mV to a maximum value of +40 mV, starting with −40 mV, in eight increments of +10 mV, for a period of 1 second. The membrane potential was then returned to −55 mV, after each of these incremented steps, for 1 second and finally repolarized to −80 mV for 1 second.The current density recorded were normalized against the baseline conditions and corrected for solvent effect and time-dependent current run-down using experimental design in test compound free conditions.Inhibition curves were obtained for compounds and the concentrations which decreased 50% of the current density determined in the baseline conditions (IC50) were determined. All compounds for which the IC50 value is above 10 μM are not considered to be potent inhibitors of the hERG channel whereas compounds with IC50 values below 1 μM are considered potent hERG channel inhibitors.
- Determination of a Possible Activity on hERG The hERG (human ether-A-go-go-related gene) channel corresponds to an important anti-target for potential new drugs since its inhibition may lead to sudden death. In order to establish whether the compounds of the present invention act on hERG, the following experiment was carried out.The in vitro effects of the compounds indicated in Table 5 on the hERG potassium channel current (a surrogate for Iκr, the rapidly activating, delayed rectifier cardiac potassium current) expressed in mammalian cells were evaluated at room temperature using the QPatch HT (Sophion Bioscience A/S, Denmark), an automatic parallel patch clamp system. Each compound indicated in Table 5 was evaluated at 0.1, 1, 3, 10 and 30 μM with each concentration tested in a minimum of two cells (n≥2). The duration of exposure to each compound concentration was 3 minutes. A summary of the results is shown in Table 5. The positive control (E4031) confirmed the sensitivity of the test system to hERG inhibition (98.6% of inhibition at 0.5 μM). Generally, compounds displaying an IC50> about 0.5 μM are regarded as not acting on hERG and thus as safe.
- MK-499 Filter Binding Assay Drug cardiac arrhythmia is an important safety concern for pharmaceutical development and health regulatory authorities. Blockade of heterologously-expressed human ether-a-go-go gene (hERG) channel prolongs the duration of the cardiac action potential leading to a long QT interval that can lead to sudden death (De Ponti, F.; et al Drug Safety 2002, 25, pp. 263-286). It is important to have compounds devoid of hERG channel activity as measured by an in vitro assay. Affinity of compounds for the hERG channel was evaluated in radioligand competition experiments using HEK293 cells that were stably transfected with the hERG channel and radiolabeled ligand, MK-499 a potent antiarrhythmic. This assay correlates well with QT prolongation in vivo (Jamieson, C.; et al., J. Med. Chem. 2006, 49, pp. 5029-5046). 25 μL Target membranes (in assay buffer: 10 mM HEPES/NaOH, pH 7.4, 70 mM NaCl, 60 mM KCl, 2 mM MgCl2, 1 mM CaCl2) purified from a HEK293 cell line expressing the human Ether- -go-go Related Gene (hERG) ion channel, 1 μL test compound in 10 mM DMSO and 25 μL (6,000 cpm/well; in assay buffer) [35S]MK-0499 radioligand (Merck/Perkin Elmer) were added to the assay plate (Axygen; 384 Deep well Diamond Plate , clear). After incubation of the binding reaction at room temperature (RT) for 90 min 50 μL of the assay were transferred to a Multiscreen HTS 384 FC filter plate (Millipore), which had been pre-wetted with 20 μL 0.01% PEI/0.01% Triton X-100 for at least 30 min at RT. Then, 30 μL wash buffer (10 mM HEPES/NaOH, pH 7.4, 130 mM NaCl, 2 mM MgCl2, 1 mM CaCl2) equilibrated to RT were added to each well of the assay plate and subsequently transferred to the filter plate. The assay mixture was aspirated through the filter plate using a Biotek ELx405 washer. The filter plate was washed twice with 100 μl cold wash buffer per wash and well and then dried in a drying oven for at least 75 min at 55° C. Afterwards, the bottom of each filter plate was heat sealed with a solid foil seal, then 10 μL of Microscint 0 (Perkin Elmer) were added to each well of the filter plate and finally, the top of each filter plate was sealed with a clear seal. The plates were stored for at least 20 min in a MicroBeta2 reader (Perkin Elmer) before they are counted (60 sec/well).
- 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.
- Fluorescence Intensity-Based Assay To test the synthesized compounds for their potential to inhibit SHP2 activity, a fluorescence intensity-based assay using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as the substrate was adapted for three recombinant SHP2 constructs: 1) the SHP2 catalytic domain (SHP2cat; residues 248-527), 2) the full-length SHP2-E76K oncogenic mutant, and 3) the full-length SHP2 wild-type (SHP2-WT). The recombinant SHP2 proteins were expressed and purified. A dually phosphorylated peptide derived from the insulin receptor substrate 1 (IRS-1) served as a surrogate binding protein and was used to activate SHP2-WT. The constitutively active E76K mutant did not require activation. Similarly, the SHP2cat construct, which lacks the SH2 domains, did not need to be activated. Michaelis-Menten experiments to determine the DiFMUP Michaelis-Menten constant (Kmn) for each SHP2 construct were performed and yielded the following values: SHP2-WT, Km=60 μM; SHP2-E76K, Km=20 μM; SHP2cat, Km=20 μM. Relative maximum rates (Vmax) of the DiFMUP reactions were as follows: SHP2-WT, Vmax=871 AFU/min; SHP2-E76K, Vmax, =2730 AFU/min; SHP2cat, Vmax=2912 AFU/min. IC50 values for each compound were determined from initial rates in 10-point dose-response assays using DiFMUP at a concentration corresponding to its Km value for the respective SHP2 construct.
- hERG Inhibition Assay The effect of compounds of the formula I on the cloned human cardiac hERG channel was evaluated in an in vitro model using a whole-cell patch-clamping technique. CHO (Chinese hamster ovary) cells stably expressing hERG, the potassium channel underlying IKr currents in human hearts, were grown in HAM's F-12 media supplemented with 10% fetal bovine serum, 1x penicillin/streptomycin and 500 ug/ml G418 (Invitrogen, Carlsbad, Calif., USA) in an atmosphere of 95% air and 5% carbon dioxide. Cells used for patch-clamping were seeded on glass or plastic coverslips 12 to 36 hours before use. hERG channel currents were recorded at room temperature using the whole-cell configuration of the patch clamp technique with an Axopatch 200B amplifier (Axon Instruments, Foster City, Calif., USA). Briefly, electrodes (3 to 6 MΩ resistance) were fashioned from TW150F glass capillary tubes (World Precision Instruments, Sarasota, Fla., USA) and filled with pipette solution (containing 120 mM potassium aspartate, 20 mM potassium chloride, 4 mM adenosine triphosphate disodium salt, 5 mM HEPES, 1 mM magnesium chloride; pH adjusted to 7.2 with potassium hydroxide). hERG currents were initiated by a positive voltage pulse (20 mV) followed by a negative pulse (-40 mV) and were recorded for off-line analyses. Once hERG current from a cell perfused with external solution (containing 130 mM sodium chloride, 5 mM potassium chloride, 2.8 mM sodium acetate, 1 mM magnesium chloride, 10 mM HEPES, 10 mM glucose, 1 mM calcium chloride; pH adjusted to 7.4 with sodium hydroxide) without the test compound, i.e. control solution, was stabilized, the cell was perfused with external solution containing the test compound at specific concentrations.
- HepaRG-CAR Cell-Based Assay for Quantitation of Glycolate Oxidase Inhibition A HepaRG human hepatic cell line was transfected for stable overexpression of the constitutive androstane receptor (i.e., HepaRG-CAR cells), as reported by van der Mark et al. (Drug Metab. Dispos., 2017, 45:56-67. Overexpression of CAR in these cells resulted in higher levels of glycolate oxidase (GOX) expression compared to the parental HepaRG cells. HepaRG-CAR cells were plated in a 12-wells plate and incubated for 4 weeks until fully differentiated.To measure cellular glycolate flux, the HepaRG-CAR cells were incubated in Williams medium supplemented with 10% fetal bovine serum (FBS), 5 μg/mL insulin, 50 μM hydrocortisone hemisuccinate, 2 mM glutamine, 5000 U/mL penicillin and 5 mg/mL streptomycin. Test compounds were added to the medium at 0, 0.3, 1, 3 or 10 μM and incubated for 30 minutes, after which 500 μM glycolate was also added. After incubation for 48 hours, 400 μL medium was taken from the culture plate and added to 60 μL 37% HCl.Internal standards (2,2-d2 glycolate, 1,2-13C2 oxalate and 13C2-glyoxylate) and hydroxylamine were added followed by another 30 minute incubation at 80° C. The acids were extracted using ethyl acetate with NaCl. The organic phase was dried under nitrogen and derivatized with N-tert-butyldimethylsilyl-N-methyl trifluoroacetamide (MTBSTFA) for 30 minutes at 80° C. The amounts of glycolate, glyoxylate and oxalate were determined by gas chromatography-mass spectrometry (GC-MS) analysis, using a 25 meter CP-Sil 5 CB low bleed column. A standard curve was used to calculate the concentrations of each acid in the culture medium.
- Mobility Shift Assay Compound activity was determined using in house His tagged full-length PTPN1 protein (SEQ ID NO: 3) in an in vitro enzymatic reaction. The enzymatic assay used to determine activity is a mobility shift assay using a LabChip EZ Reader by Caliper Life Sciences. The enzymatic reaction was carried out in assay buffer (50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM EDTA, 0.01% Tween 20, and 2 mM DTT). The compounds were dispensed on a white 384 well ProxiPlate™ (PerkinElmer Cat #6008289) plate using a Labcyte Echo liquid handler at varying concentrations (12 point, 1:3 dilution). The enzyme (at 0.5 nM) was incubated with compound for 10 minutes at room temperature. Thereafter, the substrate (phosphorylated insulin receptor probe sequence: ((OG488)-(NH-CH2-CH2-O-CH2-CH2-O-CH2-CO)-T-R-D-I-(PY)-E-T-D-Y-Y-R-K-K-NH2) (SEQ ID NO: 2) was added at 2 μM to the plates and incubated for another 10 minutes at room temperature. Finally, a quench solution (water and 4-bromo-3-(2-oxo-2-propoxyethoxy)-5-(3-{[1-(phenylmethanesulfonyl)piperidin-4-yl]amino}phenyl)thiophene-2-carboxylic acid) was added to the plates, which were then run on the EZ Reader (excitation 488 nm, emission 530 nm) to measure % conversion (the amount of phosphorylated substrate which was de-phosphorylated by PTPN1). Each plate had a 100% control (inhibitor: 4-bromo-3-(2-oxo-2-propoxyethoxy)-5-(3-{[1-(phenylmethanesulfonyl)piperidin-4-yl]amino}phenyl)thiophene-2-carboxylic acid) and 0% control (DMSO), which were used to calculate % inhibition. The % inhibition was then used to calculate the IC50 values.
- pAKT Protocol Inhibition of the PI3K-AKT-mTOR pathway was measured by quantifying the loss of (Ser-473) pAKT using AlphaScreen (Perkin Elmer). B103 (Rat Neuroblastoma) cells were seeded in serum containing medium (High Glucose DMEM (-Phenol Red)+10% FBS+2× Glutamax+1 mM Sodium Pyruvate+10 mM HEPES+1× Non-Essential Amino Acids+1× Pen/Strep) on a 96-well tissue culture treated plate and grown for 20 hours. Cells were then serum starved in serum free medium (High Glucose DMEM (-Phenol Red)+1× Glutamax+1 mM Sodium Pyruvate+1× Pen/Strep) for 6 hours prior to a 2-hour pretreatment with inhibitors of the pathway, including reference inhibitor LY294002. These inhibitors were prepared at a 200× final concentration as a 6-point, 1:3 serial dilution in DMSO series, with DMSO as the 7th point. The inhibitors were then diluted in experimental medium (High Glucose DMEM (-Phenol Red)+1× Glutamax+1 mM Sodium Pyruvate+1× Pen/Strep+25 mM HEPES+0.1% BSA) and combined with the cells at 1× final concentration in 0.5% DMSO. The cells were then stimulated for 20 minutes with (2.5 μg/mL) insulin, an activator of the PI3K-AKT-mTOR pathway and a demonstrated (Ser-473) pAKT agonist. Cells were promptly lysed using Perkin Elmer proprietary lysis buffer and the (Ser-473) pAKT and total AKT contained in the lysate was measured by AlphaScreen. In AlphaScreen, donor beads were coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads were coated with Protein A to immobilize the other antibody. In the presence of target protein, the two antibodies bring the donor and acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of target protein present in the sample. For each inhibitor tested: the ratio of measured (Ser-473) pAKT/totalAKT was plotted in GraphPad Prism as a 7-point, non-linear regression, 4-parameter curve with bottom constrained to reference control bottom and unconstrained top anchored to DMSO.
- PHH Natural Infection Assay Detailed procedures regarding primary human hepatocyte (PHH) HBV natural infection assay are described as below. One tube of frozen PHH (10 million cells) is thawed in 37 oC water bath and then transferred to 20 mL of PHH thawing medium (Sigma, InVitroGRO HT Medium, Cat. S03319) with gently mixing. The cells were then centrifuged at 80 g/min for 5 min, the supernatant was discarded and the tube was refilled with 25 mL of PHH plating medium (Sigma , InVitroGRO CP Medium, Cat. S03317). The tube was shaken very gently to re-suspend all cells, and then 50 µl of cells were transferred to each well 384-well collagen I coated plate withappropriate liquid handling equipment, e.g. Integra VI AFL0384 or Agilent Bravo. The cells were then cultured for 24 hours in a cell incubator. For HBV infection, after PHH attachment on the culture plate, the plating medium was removed and replenished with PHH culture medium containing HBV virus. The PHH culture medium was prepared with Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1 in volume ratio) containing 10% fetal bovine serum (Gibco, Cat.10099141), 5 ng/mL human epidermal growth factor (Gibco, Cat.PHG0311L), 20 ng/mL dexamethasone (Sigma, Cat.D4902-100mg), 250 ng/mL human recombinant insulin (Gibco, Cat.41400045) and 100 U/mL penicillin. HBV virus at 200 genome equivalent (GE) per cell with 4% PEG8000 (Sigma, Cat.P1458) containing culture medium were added to the PHH culture medium for infection. The cells were then cultured for 24 hours in cell incubator. Then the cell culture supernatant was removed. The HBV-infected PHH were cultured with sandwich culture method with PHH culture medium containing 1% DMSO and 0.25 mg/mL matrix gel for 72 hours. The supernatant was then refreshed with PHH culture medium containing different concentrations of testing compounds for two times with 72-hour interval. At the end of treatment, the supernatant was collected for viral markers measurements, including HBsAg, HBeAg, HBV DNA and cytotoxicity. HBsAg and HBeAg were detected using alphalisa method using their specific antibodies. For HBV DNA detection, HBV DNA Quantitative Fluorescence Diagnostic Kit (Sansure Biotech Inc.) was used following the manufacture s protocol.
- PHH Natural Infection Assay Detailed procedures regarding primary human hepatocyte (PHH) HBV natural infection assay are described as below. One tube of frozen PHH (10 million cells) is thawed in 37 oC water bath and then transferred to 20 mL of PHH thawing medium (Sigma , InVitroGRO HT Medium, Cat. S03319) with gently mixing. The cells were then centrifuged at 80 g/min for 5 min, the supernatant was discarded and the tube was refilled with 25 mL of PHH plating medium (Sigma , InVitroGRO CP Medium, Cat. S03317). The tube was shaken very gently to re-suspend all cells.50 μl of cells were transferred to each well 384-well collagen I coated plate with appropriate liquid handling equipment, e.g. Integra VIAFLO384 or Agilent Bravo. The cells were then cultured for 24 hours in a cell incubator. For HBV infection, after PHH attachment on the culture plate, the plating medium was removed and replenished with PHH culture medium containing HBV virus. The PHH culture medium was prepared with Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1 in volume ratio) containing 10% fetal bovine serum (Gibco, Cat.10099141), 5 ng/mL human epidermal growth factor (Gibco, Cat.PHG0311L), 20 ng/mL dexamethasone (Sigma, Cat.D4902-100mg), 250 ng/mL human recombinant insulin (Gibco, Cat.41400045) and 100 U/mL penicillin. HBV virus at 200 genome equivalent (GE) per cell with 4% PEG8000 (Sigma, Cat.P1458) containing culture medium were added to the PHH culture medium for infection. The cells were then cultured for 24 hours in cell incubator. Then the cell culture supernatant was removed. The HBV-infected PHH were cultured with sandwich culture method with PHH culture medium containing 1% DMSO and 0.25 mg/mL matrix gel for 72 hours. The supernatant was then refreshed with PHH culture medium containing different concentrations of testing compounds for two times with 72-hour interval. At the end of treatment, the supernatant was collected for viral markers measurements, including HBsAg, HBeAg, HBV DNA and cytotoxicity. HBsAg and HBeAg were detected using alphalisa method using their specific antibodies. For HBV DNA detection, HBV DNA Quantitative Fluorescence Diagnostic Kit (Sansure Biotech Inc.) was used following the manufactures protocol. Cytotoxicity was determined using Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies, Inc.).
- PHH Natural Infection Assay Detailed procedures regarding primary human hepatocyte (PHH) HBV natural infection assay are described as below. One tube of frozen PHH (10 million cells) is thawed in 37 oC water bath and then transferred to 20 mL of PHH thawing medium (Sigma , InVitroGRO HT Medium, Cat. S03319) with gently mixing. The cells were then centrifuged at 80 g/min for 5 min, the supernatant was discarded and the tube was refilled with 25 mL of PHH plating medium (Sigma , InVitroGRO CP Medium, Cat. S03317). The tube was shaken very gently to re-suspend all cells.50 μl of cells were transferred to each well 384-well collagen I coated plate with appropriate liquid handling equipment, e.g. Integra VIAFLO384 or Agilent Bravo. The cells were then cultured for 24 hours in a cell incubator. For HBV infection, after PHH attachment on the culture plate, the plating medium was removed and replenished with PHH culture medium containing HBV virus. The PHH culture medium was prepared with Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1 in volume ratio) containing 10% fetal bovine serum (Gibco, Cat.10099141), 5 ng/mL human epidermal growth factor (Gibco, Cat.PHG0311L), 20 ng/mL dexamethasone (Sigma, Cat.D4902-100mg), 250 ng/mL human recombinant insulin (Gibco, Cat.41400045) and 100 U/mL penicillin. HBV virus at 200 genome equivalent (GE) per cell with 4% PEG8000 (Sigma, Cat.P1458) containing culture medium were added to the PHH culture medium for infection. The cells were then cultured for 24 hours in cell incubator. Then the cell culture supernatant was removed. The HBV-infected PHH were cultured with sandwich culture method with PHH culture medium containing 1% DMSO and 0.25 mg/mL matrix gel for 72 hours. The supernatant was then refreshed with PHH culture medium containing different concentrations of testing compounds for two times with 72-hour interval. At the end of treatment, the supernatant was collected for viral markers measurements, including HBsAg, HBeAg, HBV DNA and cytotoxicity. HBsAg and HBeAg were detected using alphalisa method using their specific antibodies. For HBV DNA detection, HBV DNA Quantitative Fluorescence Diagnostic Kit (Sansure Biotech Inc.) was used following the manufacture s protocol. Cytotoxicity was determined using Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies, Inc.).
- PHH Natural Infection Assay Detailed procedures regarding primary human hepatocyte (PHH) HBV natural infection assay are described as below. One tube of frozen PHH (10 million cells) is thawed in 37 °C water bath and then transferred to 20 mL of PHH thawing medium (Sigma, InVitroGRO HT Medium, Cat. S03319) with gently mixing. The cells were then centrifuged at 80 g/min for 5 min, the supernatant was discarded and the tube was refilled with 25 mL of PHH plating medium (Sigma , InVitroGRO CP Medium, Cat. S03317). The tube was shaken very gently to re-suspend all cells, and then 50 mΐ of cells were transferred to each well 384-well collagen I coated plate with appropriate liquid handling equipment, e.g. Integra VIAFL0384 or Agilent Bravo. The cells were then cultured for 24 hours in a cell incubator. For HBV infection, after PHH attachment on the culture plate, the plating medium was removed and replenished with PHH culture medium containing HBV virus. The PHH culture medium was prepared with Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1 in volume ratio) containing 10% fetal bovine serum (Gibco, Cat.10099141), 5 ng/mL human epidermal growth factor (Gibco, Cat.PHG0311L), 20 ng/mL dexamethasone (Sigma, Cat.D4902- 1 OOmg), 250 ng/mL human recombinant insulin (Gibco, Cat.41400045) and 100 U/mL penicillin. HBV virus at 200 genome equivalent (GE) per cell with 4% PEG8000 (Sigma, Cat.P1458) containing culture medium were added to the PHH culture medium for infection. The cells were then cultured for 24 hours in cell incubator. Then the cell culture supernatant was removed. The HBV-infected PHH were cultured with sandwich culture method with PHH culture medium containing 1% DMSO and 0.25 mg/mL matrix gel for 72 hours. The supernatant was then refreshed with PHH culture medium containing different concentrations of testing compounds for two times with 72 -hour interval. At the end of treatment, the supernatant was collected for viral markers measurements, including HBsAg, HBeAg, HBV DNA and cytotoxicity. HBsAg and HBeAg were detected using alphalisa method using their specific antibodies. For HBV DNA detection, HBV DNA Quantitative Fluorescence Diagnostic Kit (Sansure Biotech Inc.) was used following the manufacture s protocol. Cytotoxicity was determined using Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies, Inc.).
- PHH Natural Infection Assay Detailed procedures regarding primary human hepatocyte (PHH) HBV natural infection assay are described as below. One tube of frozen PHH (10 million cells) is thawed in 37 °C water bath and then transferred to 20 mL of PHH thawing medium (Sigma, InVitroGRO HT Medium, Cat. S03319) with gently mixing. The cells were then centrifuged at 80 g/min for 5 min, the supernatant was discarded and the tube was refilled with 25 mL of PHH plating medium (Sigma, InVitroGRO CP Medium, Cat. S03317). The tube was shaken very gently to re-suspend all cells, and then 50 μL of cells were transferred to each well 384-well collagen I coated plate with appropriate liquid handling equipment, e.g. Integra VIAFL0384 or Agilent Bravo. The cells were then cultured for 24 hours in a cell incubator. For HBV infection, after PHH attachment on the culture plate, the plating medium was removed and replenished with PHH culture medium containing HBV virus. The PHH culture medium was prepared with Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1 in volume ratio) containing 10% fetal bovine serum (Gibco,Cat.10099141), 5 ng/mL human epidermal growth factor (Gibco, Cat.PHG0311L), 20 ng/mL dexamethasone (Sigma, Cat.D4902-100mg), 250 ng/mL human recombinant insulin (Gibco,Cat.41400045) and 100 U/mL penicillin. HBV virus at 200 genome equivalent (GE) per cell with 4% PEG8000 (Sigma, Cat.P1458) containing culture medium were added to the PHH culture medium for infection. The cells were then cultured for 24 hours in cell incubator. Then the cell culture supernatant was removed. The HBV-infected PHH were cultured with sandwichculture method with PHH culture medium containing 1% DMSO and 0.25 mg/mL matrix gel for 72 hours. The supernatant was then refreshed with PHH culture medium containing different concentrations of testing compounds for two times with 72-hour interval. At the end of treatment, the supernatant was collected for viral markers measurements, including HBsAg, HBeAg, HBV DNA and cytotoxicity. HBsAg and HBeAg were detected using alphalisa method using their specific antibodies. For HBV DNA detection, HBV DNA Quantitative Fluorescence Diagnostic Kit (Sansure Biotech Inc.) was used following the manufacture s protocol. Cytotoxicity was determined using Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies, Inc.).