lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#Cc1ccc(N2CCN(c3nc(NC4CC4)n4ncc(/C=C5\NC(=O)NC5=O)c4n3)CC2)nc1
InChIKey: LJXAYGXSYVBUEY-SXGWCWSVSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 471.49, ALogP 0.47, HBA 11, HBD 3, PSA 156.47, QED 0.35
Compound (SMILES): CC(C)OC(=O)/C=C\Cn1cnc(-c2cccc(Cl)c2)n1
InChIKey: SSCIPPJBPOZHKP-DAXSKMNVSA-N
Target: Exportin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14980
Assay: Inhibition of Crm1 in human U2OS cells expressing GFP-fused HIV1 Rev assessed as decrease in nuclear export of GFP-fused HIV1 Rev after 1 hr by Hoechst 33258 staining-based assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 305.77, ALogP 3.11, HBA 5, HBD 0, PSA 57.01, QED 0.63
Compound (SMILES): CC(=O)N[C@@H](C)c1ccc(CN2CCc3cc(OCCC(F)F)ccc3C2)cc1
InChIKey: HKGQZHLGAALOHX-INIZCTEOSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.Each assay microtiter plate contains wells with vehicle instead of compound as controls for the non-inhibited enzyme (100% CTL; HIGH) and wells without acetyl-CoA as controls for non-specific NADH degradation (0% CTL; LOW).
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 402.49, ALogP 4.48, HBA 3, HBD 1, PSA 41.57, QED 0.71
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(NC(=O)Cc2ccc(OCc3ccccc3)cc2)ccc1Oc1ccc(F)cc1F
InChIKey: IWFYZFJJVKDHND-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method B: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.47 mM to 7.8 nM. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 2320 nM (pChEMBL 5.630000114440918)
Descriptors: MW 596.63, ALogP 7.28, HBA 6, HBD 1, PSA 78.79, QED 0.17
Compound (SMILES): CCCN(CCC)C(=O)C1=Cc2ccc(C(=O)Nc3cc(C)cc(CN)c3)cc2N=C(N)C1
InChIKey: SBTHMGWKVZDJNK-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Pharmacological Assay: For TLR8 and TLR7 activity testing, HEK-Blue human TLR8 or TLR7 cells (Invivogen, San Diego, Calif., USA) are used, respectively. These cells are designed for studying the stimulation of human TLR8 or TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-κB and AP-1-binding sites. Therefore the reporter expression is regulated by the NF-κB promoter upon stimulation of human TLR8 or TLR7 for 20 hours. The cell culture supernatant SEAP reporter activity was determined using Quanti Blue kit (Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple/blue in the presence of alkaline phosphatase.
Measurement: EC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 447.58, ALogP 4.13, HBA 5, HBD 3, PSA 113.81, QED 0.56
Compound (SMILES): CC(C)C1CN(Cc2ccc3c(-c4ccc(F)cc4)cc(C(N)=O)nc3c2)CCN1
InChIKey: SEWPIHHWIJFHPE-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 116 nM (pChEMBL 6.940000057220459)
Descriptors: MW 406.51, ALogP 3.57, HBA 4, HBD 2, PSA 71.25, QED 0.68
Compound (SMILES): CC[C@H](C)[C@@H]1NCCOc2ccccc2CCCNC(=O)[C@@H](Cc2ccc(F)cc2)NC(=O)[C@@H](C)N(C)C1=O
InChIKey: OPCSZBKNRPRCRX-QIDJRYDTSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki = 0.1 nM (pChEMBL 10)
Descriptors: MW 540.68, ALogP 2.85, HBA 5, HBD 3, PSA 99.77, QED 0.55
Compound (SMILES): c1ccc2c(c1)C[C@H](N1CCCC1)[C@H]2Oc1ccc(-c2nccs2)cc1
InChIKey: VZPLWXCOSBOCJY-SFTDATJTSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human NHE3 expressed in LAP1 cell assessed as intracellular pH recovery measured for 2 mins by CECF-AM dye based FLIPR assay
Measurement: IC50 = 1710 nM (pChEMBL 5.769999980926514)
Descriptors: MW 362.5, ALogP 4.95, HBA 4, HBD 0, PSA 25.36, QED 0.66
Compound (SMILES): CN1/C(=N\C(=O)OC(C)(C)C)N[C@](C)(c2sc(-c3ncc(-c4ccccn4)[nH]3)cc2Cl)CS1(=O)=O
InChIKey: WISROSNSFRIXNX-QFIPXVFZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 470 nM (pChEMBL 6.329999923706055)
Descriptors: MW 537.07, ALogP 4.22, HBA 7, HBD 2, PSA 129.64, QED 0.51
Compound (SMILES): O=[N+]([O-])c1cccc(C2NCCc3c2oc2ccccc32)c1
InChIKey: WYKURZVILQMQNZ-UHFFFAOYSA-N
Target: Sodium/iodide cotransporter (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q63008
Assay: Inhibition of sodium iodide symporter in rat FRTL5 cells assessed as decrease in [125I]-sodium iodide uptake after 60 mins by scintillation counting method
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 294.31, ALogP 3.58, HBA 4, HBD 1, PSA 68.31, QED 0.58
Compound (SMILES): Cc1cnc(C(=O)Nc2cccc(CN3CCC(C(=O)NC4CCCCC4)CC3)c2)cn1
InChIKey: XMXFSOOGAKPINO-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 435.57, ALogP 3.7, HBA 5, HBD 2, PSA 87.22, QED 0.72
Compound (SMILES): O=C1NCC2(CCCCC2)n2c1cc1cnc(Nc3ccc(C(=O)N4CCN(C5CCC5)CC4)cn3)nc12
InChIKey: ZFFPZCADMXKXTH-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: Caliper Assay: Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK6/CycD3 CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Phosphoacceptor substrate peptide concentration used was 1 μM for all assays and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below:CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM; Incubation time: 3 hr.CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM; Incubation time: 1 hr.CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 10 hr.CDK6/CyclinD3: Enzyme concentration: 1 nM; ATP concentration: 300 μM; Incubation time: 3 hr.
Measurement: IC50 = 202 nM (pChEMBL 6.699999809265137)
Descriptors: MW 514.63, ALogP 3.28, HBA 8, HBD 2, PSA 108.28, QED 0.55
Compound (SMILES): Cc1c(N(Cc2cccc(OC(F)(F)F)c2)S(=O)(=O)CCCn2ccnc2)sc2ccccc12
InChIKey: IQICZLMCQIIVAP-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 106.9 nM (pChEMBL 6.96999979019165)
Descriptors: MW 509.58, ALogP 5.73, HBA 6, HBD 0, PSA 64.43, QED 0.29
Compound (SMILES): CCC(CC)(COc1ccc(-c2ccc(-c3ncc(C(F)(F)F)[nH]3)cn2)cn1)C(=O)O
InChIKey: WZRZMFPDMQQWEW-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: DGAT1 Inhibitory Assay: As a buffer to be used in the enzymatic reaction of DGAT1, 100 mM Tris-HCl (pH 7.4), 200 mM Sucrose, 20 mM MgCl2, 0.125% Bovine Serum Albumin (BSA) were used. To the buffer were added Test compound with a predetermined concentration as well as 15 μM dioleoylglycerol, 5 μM [14C]-palmitoyl-CoA, 100 μg protein/nth DGAT1 highly expressed-expresSF+® microsome, 0.75% acetone, and 1% dimethylsulfoxide, and triglyceride (TG) synthetic reaction was carried out at 30 °C. for 20 minutes with a volume of 100 μL. 90 μL of the reaction solution was added to 810 μL of methanol to stop the reaction. The reaction solution was added to Oasie® μElution plate (available from Waters Corporation), and eluted with 150 μL of a mixed solution of acetonitrile: isopropanol (=2:3). To elute was added 150 μL of MicroScinti™-40 (available from PerkinElmer Inc.), and after thoroughly stirring the mixture, a [14C]-TG amount formed by the reaction was quantitated by measuring the same using TopCount™-NXT (available from PerkinElmer Inc.).
Measurement: IC50 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 434.42, ALogP 4.82, HBA 5, HBD 2, PSA 100.99, QED 0.53
Compound (SMILES): COc1ccccc1-c1cc(Nc2ccc(F)c(NS(C)(=O)=O)c2)ncn1
InChIKey: QTORRJJDONDBFZ-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: Binding affinity to full length human CDK2 (1 to 298 residues) expressed in bacterial expression system by KINOMEscan competition assay
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 388.42, ALogP 3.41, HBA 6, HBD 2, PSA 93.21, QED 0.67
Compound (SMILES): Cc1ccc(C(=O)N2CCOC[C@H]2Cc2cccc(-c3ncon3)c2)c(-n2nccn2)c1
InChIKey: DCMSTVVYTKCBDX-LJQANCHMSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: OX Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 ug/mL G418, 100 U/mL penicillin, 100 ug/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37 C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 430.47, ALogP 2.71, HBA 8, HBD 0, PSA 99.17, QED 0.48
Compound (SMILES): O=C(Nc1ccc(C(F)(F)F)cn1)N1CC=C(c2ncc([C@@H](O)CO)cc2Cl)CC1
InChIKey: BMWVHVOIXZOJTJ-HNNXBMFYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Capsaicin-Based Assay: Two days prior to performing this assay, cells are seeded in poly-D-lysine-coated 96-well clear-bottom black plates (50,000 cells/well) in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4, and cells are loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final). After one hour, the cells are washed twice with 0.2 mL of wash buffer and resuspended in 0.1 mL of wash buffer. The plates are transferred to a FLIPR for assay. 50 μL of test compound diluted with assay buffer (1× Hank's Balanced Salt Solution containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4) are added to the cell plates and incubated for 2 min. The final concentration of the compound is adjusted to range from about 50 picoM to about 3 μM. Human TRPV1 is activated by the addition of 50 μL of capsaicin (400 nM), and the plates are incubated for an additional 3 min.
Measurement: IC50 = 161.1 nM (pChEMBL 6.789999961853027)
Descriptors: MW 442.83, ALogP 3.5, HBA 5, HBD 3, PSA 98.58, QED 0.67
Compound (SMILES): CO[C@H]1COC(c2c(NC(=O)c3nc(-c4c(F)cccc4F)sc3N)cnn2C)CC[C@@H]1N
InChIKey: ZUQMFIJENVDQFJ-JDLVMGNASA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Assay: LC3K assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO. PIM reactions were carried out in a final volume of 10 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000® (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of −500V, an upstream voltage of −2150V, and a screening pressure of −1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm.
Measurement: Ki = 0.476 nM (pChEMBL 9.319999694824219)
Descriptors: MW 478.53, ALogP 2.85, HBA 9, HBD 3, PSA 130.31, QED 0.51
Compound (SMILES): Nc1ncnc(N2CCC(c3nc(-c4ccc(F)c(C(F)(F)F)c4)cn3CCN3CCCC3)CC2)c1F
InChIKey: OUIQTZMFLWJZDO-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 521.54, ALogP 4.7, HBA 7, HBD 1, PSA 76.1, QED 0.47
Compound (SMILES): C=C(c1ccc(C(=O)NS(=O)(=O)c2ccc(NCCSc3ccccc3)c([N+](=O)[O-])c2)cc1)c1cc2c(cc1C)C(C)(C)CCC2(C)C
InChIKey: UQOYPUQPZRJYPK-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Inhibition of 5-FAM-Bid peptide binding to Bcl-2 (unknown origin) by fluorescence polarization assay
Measurement: Ki = 4200 nM (pChEMBL 5.380000114440918)
Descriptors: MW 683.9, ALogP 8.64, HBA 7, HBD 2, PSA 118.41, QED 0.07
Compound (SMILES): NCCC[C@@H]1N[C@H](CNC(=O)/C=C/c2ccc(F)cc2F)CCN(CC(c2ccccc2)c2ccccc2)C1=O
InChIKey: HSMIYJGCJMTPNG-FMDWORLUSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 546.66, ALogP 4.22, HBA 4, HBD 3, PSA 87.46, QED 0.31
Compound (SMILES): O=C(Nc1cc2cccnc2cc1N1CCCCC1)c1cnn2ccccc12
InChIKey: ZDXFUOXCTQMSTQ-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: In vitro IRAK-1 and IRAK-4 Assay: Purified recombinant IRAK-4 protein was incubated with 250 uM synthetic peptide(KKARFSRFAGSSPSQSSMVAR) in 30 ul of kinase buffer including (20 mM MOPS pH7.2, 25 mM beta glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 50 uM ATP, 20 mM MgCl2, 10 uCi gamma-33P, 0.1% BSA) for the indicated time. For purified recombinant IRAK-1 protein kinase assay, 50 uM ATP was used. A 25 ul aliquot of the reaction mixture was transferred on to p81 phosphocellulose squares (Upstate Biotechnology, Lake Placid, N.Y.). The assay squares were washed three times with 0.75% phosphoric acid and once with acetone. Enzyme activity was measured by determining the bound radioactivity by liquid scintillation counting.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 371.44, ALogP 4.13, HBA 5, HBD 1, PSA 62.53, QED 0.59
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3ccc(CP(=O)(OCC)OCC)cc3)ncc2F)c1
InChIKey: VISDEYWCEAZMCJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of human recombinant full-length N-terminal His-tagged BTK expressed in baculovirus infected Sf9 insect cells measured after 60 mins by ADP-Glo kinase assay
Measurement: IC50 = 32.1 nM (pChEMBL 7.489999771118164)
Descriptors: MW 499.48, ALogP 5.99, HBA 8, HBD 3, PSA 114.47, QED 0.21
Compound (SMILES): NS(=O)(=O)OC[C@H]1C[C@@H](NC(=O)c2ccncc2)[C@H](O)[C@@H]1O
InChIKey: IINUZRFVTDDCRA-DBIOUOCHSA-N
Target: NEDD8 activating enzyme (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13564
Assay: Inhibition of recombinant human NAEalpha/N-terminal His-tagged NAEbeta expressed in baculovirus infected insect Sf9 cells using N-terminal FLAG tagged Nedd8 as substrate incubated for 105 mins in presence of N-terminal GST-tagged Ubc12 by FRET assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 331.35, ALogP -1.86, HBA 7, HBD 4, PSA 151.84, QED 0.49
Compound (SMILES): Cc1cccc(C(O)(c2cc(-c3c(C)noc3C)cc3[nH]c(C4CC4)nc23)C2CCCO2)n1
InChIKey: GBTIYPSXKAVJCF-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Ligand KI Assay: The labeled ligand specifically binds BRD4-1 and BRD4-2 and can be displaced by a small molecule inhibitor that shares a similar or overlapping binding site. BRD4-I and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6, tagged proteins. A Eu-cryptate labeled anti-His antibody (Perkin Elmer) was used to specifically bind BRD4. Binding of BRD4 to the labeled probe/ligand resulted in an increase in FRET signal whereas displacement of this labeled ligand from BRD4 with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 10 nM labeled ligand at the following concentrations for each BRD4 isoform: 2 nM BRD4-1 and 0.5 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM Eu-cryptate labeled anti-His antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 us delay and 200 us read window). Data were normalized based on a positive (2 uM I-BET) and negative (DMSO) controls and IC50 values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations. The IC50 values were converted to Ki values (dissociation constant for BRD4-inhibitor complex) using the Cheng and Prusoff equation for a competitive inhibitor mode of action.
Measurement: IC50 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 444.54, ALogP 4.83, HBA 6, HBD 2, PSA 97.06, QED 0.46
Compound (SMILES): O=C(NC1CCN(Cc2ccccc2)CC1)c1cccc(-c2n[nH]c3ccc(-c4nc[nH]n4)cc23)c1
InChIKey: JDIRNAZNRMEUIW-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 477.57, ALogP 4.41, HBA 5, HBD 3, PSA 102.59, QED 0.34
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H](C(=O)N1c2ccccc2C[C@H]1C(=O)NCc1ccccc1)c1ccccc1
InChIKey: CQOHSROJVMHERP-LQGLAIQGSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 723 nM (pChEMBL 6.139999866485596)
Descriptors: MW 470.57, ALogP 2.73, HBA 4, HBD 3, PSA 90.54, QED 0.47
Compound (SMILES): Nc1c2c(nc3c1C(c1ccccc1)c1c(-c4ccco4)nn(-c4ccccc4)c1O3)CCCC2
InChIKey: UVCXEIZZXFEHCV-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P06276
Assay: Inhibition of human recombinant BChE using butyrylthiocholine iodide as substrate incubated for 5 mins followed by substrate addition measured for 1 min by Ellman's method
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 460.54, ALogP 6.27, HBA 6, HBD 1, PSA 79.1, QED 0.34
Compound (SMILES): Cc1cccc(F)c1S(=O)(=O)NCC1(C(=O)NC2C3CC4CC2CC(C(N)=O)(C4)C3)CC1
InChIKey: PCHPQIFQXAIEPO-UHFFFAOYSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: HTRF Assay : The inhibiting activity of 11β-HSD1 derived from microsomal fractions was measured using the HTRF assay (62CO2PEB, Cisbio). Different concentrations of compounds were added to 96-well plates, followed by the addition of TE buffer (20 mM Tris buffer and 5 mM EDTA, pH 6.0) containing 200 μM NADPH (N1630, Sigma) and 160 nM cortisone (C2755, Sigma), The reactions were initiated by the addition of human microsomal fractions (M0317, Sigma), and were allowed to incubate for 2 h at 37° C. Europium (Eu3+) cryptate and XL665-conjugated cortisol were then added to each well and incubated for an additional 2 h at room temperature.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 463.58, ALogP 1.99, HBA 4, HBD 3, PSA 118.36, QED 0.57
Compound (SMILES): COC(=O)[C@@H](NC(=O)Nc1cc2c(cn1)c(OC)nn2C(c1ccccc1)(c1ccccc1)c1ccccc1)c1ccccc1
InChIKey: VHHUICNAAGIEAU-YTTGMZPUSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 597.68, ALogP 6.32, HBA 7, HBD 2, PSA 107.37, QED 0.15
Compound (SMILES): O=C(Nc1cnc(Oc2ccc(-c3cnc4[nH]ncc4c3)cc2)nc1)Nc1cc(C(F)(F)F)ccc1-c1cccnc1
InChIKey: FBBVXRHXBBBOJS-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Activity Assay: On the day before the assay, CellSenser TrkA-NFAT-bla CHO-K1 cells were suspended in an assay medium (Opti-MEM1 Reduced Serum Medium (Invitrogen) containing 0.5% dialysed fetal bovine serum (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen) and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin (Invitrogen))) and plated at a density of 2.4x104 cells/40 uL/well in a 96-well clear bottom plate (Corning, Catalogue No.: 3882). In some wells were added only the assay medium at 40 uL/well (Cell-free). On the day of the assay, 10 mM of the present compound (DMSO solution) was distributed in a 96-well plate (Costar, Catalogue No.: 3363) and serially diluted with DMSO with the geometrical ratio of 3. The serial dilutions were diluted with the assay medium to 100-fold to prepare a solution of the present compound with a 10-fold concentration (DMSO concentration: 1%). To the plate where cells were plated was added the present compound at 5 L/well and the plate was incubated in a CO2 incubator with 5% CO2, 95% Air at 37° C. for 30 minutes. For a control and a blank, the assay medium containing 1% DMSO was added at 5 L/well in place of the solution of the present compound. Subsequently the assay medium containing NGF (Mouse 2.5s, Natural, Invitrogen) was added to the plate at 5 L/well (final concentration of NGF: 50 ng/ml) and the plate was incubated in a CO2 incubator with 5% CO2, 95% Air at 37° C. for 5 hours. For the blank group, the assay medium was added in place of NGF at 5 L/well. A reporter assay detection reagent (10 L/well) was added to the plate which was then incubated in the dark at room temperature for 120 minutes. The reporter assay detection reagent was prepared from LiveBLAzer -FRET B/G Loading Kit (Invitrogen). On the Analyst GT (Molecular Devices Japan, K.K.) the wells were irradiated with excitation light at 405 nm and the fluorescence intensities at 460 nm and 530 nm were measured.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 568.52, ALogP 6.93, HBA 7, HBD 3, PSA 130.6, QED 0.2
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3C(C(=O)O)O4)cc12
InChIKey: GXDOOCNCYZAKJD-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: ELISA-Based Assay: Measurement of inhibition by compounds was performed using the HCV replicon system. Several different replicons encoding different HCV genotypes or mutations were used. In addition, potency measurements were made using different formats of the replicon assay, including different ways of measurements and different plating formats. See Jan M. Vrolijk et al., A replicons-based bioassay for the measurement of interferons in patients with chronic hepatitis C, 110 J. VIROLOGICAL METHODS 201 (2003); Steven S. Carroll et al., Inhibition of Hepatitis C Virus RNA Replication by 2'-Modified Nucleoside Analogs, 278(14) J. BIOLOGICAL CHEMISTRY 11979 (2003). However, the underlying principles are common to all of these determinations, and are outlined below.Stable neomycin phosphotransferase encoding replicons-harboring cell lines were used, so all cell lines were maintained under G418 selection prior to the assay. Potency was determined using a cell ELISA assay with an antibody to the replicons encoded NS3/4a protease. See Caterina Trozzi et al., In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor, 77(6) J. Virol. 3669 (2003).
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 658.64, ALogP 5.79, HBA 8, HBD 2, PSA 143.97, QED 0.23
Compound (SMILES): Cn1cc(-c2ccc(N3CCC(n4ccc5ccc(Cl)cc54)CC3)nn2)cn1
InChIKey: PTMMQFPPPKXWPY-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Enzymatic Assay: The SCD1 enzymatic assay was done in a volume of 50 μL using 10 μg of RLM (prepared as described above) in a 96-well polypropylene plate (enzyme reaction buffer contains 0.1 M K-Phosphate Buffer, 10 mM ATP, 6 mM MgCl2. 1 mM CoA, 1 mM β-NADH, 1.6 mM L-glutathione, 20 μM Stearoyl-CoA). Stearoyl-[9,10-3H]-CoA (ARC-0390, 1 mCi/mL, 60 Ci/mmol,) was added at a final concentration of 2 μCi/mL. Test compound was then added to the reaction mixture at the selected concentration. After incubation at room temperature for 2 hours, 5 μL 1 N HCl was added to stop the reaction, followed by addition of 25 μL of 10% charcoal. The reaction mixture was then transferred to 96-well Multiscreen plate (Millipore, Cat#MSFCN6B50). [3H2O] was collected into Opti-plate (PE, Cat#6005290) by centrifuge, at 1,000 rpm for 2 minutes. 150 μL Microscint 40 (PE, cat #6013641) was then added to each well and counted on Topcount for [3H] counts per minute (cpm).
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 392.89, ALogP 4.33, HBA 6, HBD 0, PSA 51.77, QED 0.52
Compound (SMILES): CCOC(=O)N1CCC(S(=O)(=O)c2c(Cl)ccc(NC(=O)Nc3cccc(F)c3Cl)c2O)CC1
InChIKey: UNSQETVNQPAXKQ-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P25024
Assay: Displacement of [125I]IL8 from recombinant human CXCR1 expressed in CHO cell membranes measured after 1 hr by TopCount scintillation counting method
Measurement: IC50 < 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 534.39, ALogP 4.88, HBA 6, HBD 3, PSA 125.04, QED 0.47
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)nc(N[C@H]4c5ccccc5C[C@@H]4O)nc23)N1
InChIKey: IFVKGXXFFHIRNY-BKZJWICZSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 432.44, ALogP 0.95, HBA 9, HBD 5, PSA 145.57, QED 0.3
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1cnccn1)c1nc2cccc(C(F)F)c2c(=O)n1-c1cccc(C#N)c1
InChIKey: WRBCSBMSBYWGCN-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 535.52, ALogP 3.93, HBA 10, HBD 2, PSA 148.29, QED 0.32
Compound (SMILES): O=C(NCc1ccc(Cl)cc1Cl)N1CC2(C1)CN(c1ncccn1)C2
InChIKey: COEQVCNZOXVPHM-UHFFFAOYSA-N
Target: Bifunctional epoxide hydrolase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34913
Assay: Enzyme Assay: Test compounds (1.6 mM stock in DMSO) were diluted 3 fold in series in DMSO and 0.8 microliters per well were added into 384-well NBS microplates (Corning). Resorufin epoxide substrate (20 uM stock in DMSO) was diluted to 5 μM with Assay Buffer (25 mM bis-Tris-HCl, pH 7.0, 1 mM DTT and 0.2 mg/ml BSA) and 8 microliters per well were added to the microplates. Thirty two microliters per well of 3.6 nM soluble epoxide hydrolase in Assay Buffer was then added. The samples were incubated at room temperature and assay signals were monitored by reading excitation at 530 nm and emission fluorescence at 590 nm on a PlateVision (Zeiss) reader every 2 minutes for 8 times.
Measurement: IC50 = 2323 nM (pChEMBL 5.630000114440918)
Descriptors: MW 378.26, ALogP 2.82, HBA 4, HBD 1, PSA 61.36, QED 0.89
Compound (SMILES): C=CC(=O)N1CCCC(n2cc(-c3cc4c(-c5cccc(N6CCOc7cc(C8CC8)cc(F)c7C6=O)c5CO)ncnc4[nH]3)cn2)C1
InChIKey: SGCPAQMSTLOCLE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Recombinant Human BTK Inhibition Assay: The inhibition assays were performed using the generic tyrosine kinase assay kit from C is Bio (HTRF KinEASE-TK #62TK0PEB). The assay is based on the principle of fluorescence resonance energy transfer (FRET) between the Europium Cryptate labeled anti-phosphotyrosine antibody and streptavidin labeled fluorophore, XL-665. In an uninhibited kinase reaction the biotinylated peptide substrate (Tk-Biotin) gets phosphorylated at an intermediate tyrosine site which is captured by anti-phosphotyrosine antibody which eventually acts as a FRET donor. The streptavidin labeled XL-665 binds strongly to the Tk-Biotin substrate and act as a FRET acceptor. The resultant FRET signal is measured at 665 nm. A ratio of this specific fluorescence (665 nm) to the background fluorescence (620 nm), conferred by the free antibody, is directly proportional to the kinase activity. Inhibition of the kinase activity by a pharmacological intervention such as a Btk inhibitor of the present disclosure will result into decreased FRET signal.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 647.71, ALogP 5.39, HBA 8, HBD 2, PSA 129.47, QED 0.23
Compound (SMILES): Cc1cccc(C)c1N1CC(C(=O)Nc2cc(C(F)(F)F)cc(C(F)(F)F)c2)(C2CCN(C)CC2)CC1=O
InChIKey: MMOQGJYMOUFBCF-UHFFFAOYSA-N
Target: Chemerin-like receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99788
Assay: Displacement of [125I]-chemerin from ChemR23 (unknown origin) transfected in mouse BaF3 cells after 3 hrs by scintillation counting method
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 541.54, ALogP 6.04, HBA 3, HBD 1, PSA 52.65, QED 0.48
Compound (SMILES): Cc1ccc2c(NCc3ccc(NC(=O)c4ccc(Cl)cc4)cc3)nc(N(C)C)nc2c1
InChIKey: AOCHSTRFBNLJOW-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf33.13 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 227 nM (pChEMBL 6.639999866485596)
Descriptors: MW 445.95, ALogP 5.52, HBA 5, HBD 2, PSA 70.15, QED 0.41
Compound (SMILES): COc1ccccc1OCCN1CCC(CNS(=O)(=O)c2cccc(Cl)c2F)CC1
InChIKey: ZTNYEEHAUAMDPR-UHFFFAOYSA-N
Target: Alpha-1A adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35348
Assay: Intrinsic activity at alpha1A adrenergic receptor (unknown origin) assessed as inhibition of agonist induced effect preincubated for 30 mins followed by agonist addition measured after 5 hrs in presence of CCF4-AM by cell based beta lactamase reporter gene assay
Measurement: EC50 = 84.2 nM (pChEMBL 7.079999923706055)
Descriptors: MW 456.97, ALogP 3.56, HBA 5, HBD 1, PSA 67.87, QED 0.62
Compound (SMILES): COc1cc(N2CCN(C(C)=O)CC2)ccc1Nc1ncc(Cl)c(Nc2ccc(N3CCN(C(=O)CO)CC3)cc2OC)n1
InChIKey: JZMRVGGBILYPDP-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 625.13, ALogP 2.94, HBA 11, HBD 3, PSA 135.63, QED 0.32
Compound (SMILES): CCCN(CCC)C(=O)C1=Cc2ccc(C(=O)Nc3cncc(/C=C/CN)c3)cc2N=C(N)C1
InChIKey: PVSXEDOIPKCQMR-AATRIKPKSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Pharmacological Assay: For TLR8 and TLR7 activity testing, HEK-Blue human TLR8 or TLR7 cells (Invivogen, San Diego, Calif., USA) are used, respectively. These cells are designed for studying the stimulation of human TLR8 or TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-κB and AP-1-binding sites. Therefore the reporter expression is regulated by the NF-κB promoter upon stimulation of human TLR8 or TLR7 for 20 hours. The cell culture supernatant SEAP reporter activity was determined using Quanti Blue kit (Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple/blue in the presence of alkaline phosphatase.
Measurement: EC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 460.58, ALogP 3.73, HBA 6, HBD 3, PSA 126.7, QED 0.53
Compound (SMILES): CCN(CCF)C[C@@]1(C(=O)Nc2cc(C(F)(F)F)cc(C(F)(F)F)c2)CN(c2c(C)cccc2C)C(=O)[C@H]1C
InChIKey: XOQUMUSAHJKNHI-IQGLISFBSA-N
Target: Chemerin-like receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99788
Assay: Displacement of [125I]-chemerin from ChemR23 (unknown origin) transfected in mouse BaF3 cells after 3 hrs by scintillation counting method
Measurement: IC50 <= 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 561.54, ALogP 6.24, HBA 3, HBD 1, PSA 52.65, QED 0.39
Compound (SMILES): Cn1c(NCCc2ccc(-c3ccc(F)cc3)cc2)nc2cc(Cl)ccc2c1=O
InChIKey: ZLDQEPMFWJQWSR-UHFFFAOYSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of CYP3A4 in human liver microsomes assessed as 6-beta-hydroxytestosterone metabolite formation using testosterone as substrate incubated for 30 mins by HPLC-MS method
Measurement: IC50 = 18000 nM (pChEMBL 4.75)
Descriptors: MW 407.88, ALogP 5.05, HBA 4, HBD 1, PSA 46.92, QED 0.5