lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(C)OC(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3ccnc(C(N)=O)c3)n2)CC1
InChIKey: KCMWPZJLDQAXKA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 468.52, ALogP 1.86, HBA 9, HBD 3, PSA 182.25, QED 0.57
Compound (SMILES): CC(C)(C)C[C@H]1N[C@@H](C(=O)NC2CCC(O)CC2)[C@H](c2cccc(Cl)c2F)[C@@]12C(=O)Nc1cc(F)ccc12
InChIKey: WOBLKIIJUYHOLE-YJRDPZTCSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Fluorescence-polarization Binding Assay: The binding affinity of the MDM2 inhibitors was determined using an optimized, sensitive and quantitative fluorescence polarization-based (FP-based) binding assay using a recombinant human His-tagged MDM2 protein (residues 1-118) and a fluorescently tagged p53-based peptide. The design of the fluorescence probe was based upon a previously reported high-affinity p53-based peptidomimetic compound (5-FAM-βAla-βAla-Phe-Met-Aib-pTyr-(6-Cl-LTrp)-Glu-Ac3c-Leu-Asn-NH2 (SEQ ID NO: 1)) (García-Echeverría et al., J. Med. Chem. 43: 3205-3208 (2000)). This tagged peptide is called PMDM6-F. MDM2 protein was serially double diluted in a Dynex 96-well, black, round-bottom plate, and the PMDM6-F peptide was added at 1 nM concentration. The assay was performed in the buffer: 100 mM potassium phosphate, pH 7.5; 100 μg/mL bovine gamma globulin; 0.02% sodium azide, 0.01% Triton X-100) and the polarization values were measured after 3 h of incubation using an ULTRA READER (Tecan U.S. Inc).
Measurement: IC50 = 520 nM (pChEMBL 6.28000020980835)
Descriptors: MW 546.06, ALogP 4.79, HBA 4, HBD 4, PSA 90.46, QED 0.44
Compound (SMILES): COc1ccc(Br)c(CC2CCN(CCc3ccccc3OC)CC2)c1
InChIKey: JBUHYCVPTFVUQE-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Displacement of [3H]-8-OH-DPAT from human 5-HT1A receptor expressed in CHOK1 cells after 30 mins by liquid scintillation counting analysis
Measurement: Ki = 3.03 nM (pChEMBL 8.520000457763672)
Descriptors: MW 418.38, ALogP 4.96, HBA 3, HBD 0, PSA 21.7, QED 0.63
Compound (SMILES): O=C(Nc1cn(CC(=O)N2CCC[C@H]2C(O)CO)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: YCIHLDAASPFOJI-OYKVQYDMSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.62 nM (pChEMBL 9.210000038146973)
Descriptors: MW 575.96, ALogP 2.44, HBA 10, HBD 3, PSA 147.11, QED 0.28
Compound (SMILES): Cc1ccc(C(=O)N2[C@H](Cc3cccc(-n4nccn4)c3)COC[C@H]2C)c(-n2nccn2)c1
InChIKey: FCVGUKJNRFNLDS-WIYYLYMNSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
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 = 314 nM (pChEMBL 6.5)
Descriptors: MW 443.51, ALogP 2.63, HBA 8, HBD 0, PSA 90.96, QED 0.47
Compound (SMILES): CN1CCc2c(cccc2OCC(=O)NCC(O)CN2CCc3ccccc3C2)C1
InChIKey: ZZKVUCNLXVGLLG-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 = 101 nM (pChEMBL 7)
Descriptors: MW 409.53, ALogP 1.59, HBA 5, HBD 2, PSA 65.04, QED 0.73
Compound (SMILES): Nc1cc2c3c(c1)c(C(F)(F)F)c(CCCOc1ccccc1)n3CCC2
InChIKey: XRMJXPDOJPWTHL-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Mus musculus
UniProt: Q9WVG5
Assay: Endothelial Lipase Assay: To assay for cell surface lipase activity, cells expressing human endothelial lipase (EL) or LPL were plated in CellBIND® 384-well plates (Corning, Lowell, Mass.) in 25 μL serum free medium at a density of 2000 cells/well. After 18-24 hours incubation at 37° C., the medium was removed and replaced with 15 μL assay buffer [Hank's Buffered Saline Solution with 25 mM HEPES pH 7.2] and 15 μL PLA1 substrate for a final concentration of 10 μM using a Multidrop reagent dispenser. Fluorescence signal was monitored for 30 min at 37° C. on a Safire II plate reader in kinetic mode (60 cycles, kinetic interval: 30 seconds) with an excitation wavelength of 490 nm and an emission wavelength of 515 nm.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 374.41, ALogP 5.2, HBA 3, HBD 1, PSA 40.18, QED 0.49
Compound (SMILES): COc1cc(-c2ccc(C)c(Cl)c2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: VZZOFNXRGUQVFG-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 3153 nM (pChEMBL 5.5)
Descriptors: MW 539.97, ALogP 5.56, HBA 7, HBD 1, PSA 103.43, QED 0.3
Compound (SMILES): CN1C(=N)N[C@]2(c3cc(NC(=O)c4ccc(Cl)cn4)ccc3F)COC[C@@H]2S1(=O)=O
InChIKey: UMTXXAYFIKSALN-YJBOKZPZSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3x the desired final concentration in 1Ã¿BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1Ã¿BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 uM for 4 uM for autoBACE-2) prepared in 1xBACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay.
Measurement: Ki = 55.2 nM (pChEMBL 7.260000228881836)
Descriptors: MW 453.88, ALogP 1.52, HBA 6, HBD 3, PSA 124.48, QED 0.65
Compound (SMILES): C[C@H]1[C@@H](CC(=O)Nc2ccc(Cl)c(-c3ccccn3)c2)O[C@@H]2O[C@@]3(C)CC[C@H]4[C@H](C)CC[C@@H]1[C@@]24OO3
InChIKey: FYBFXVILVGDINR-WSGLMESZSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of Smo-mediated Hh signalling pathway in mouse Shh Light2 cells by Gli-luciferase reporter gene assay
Measurement: IC50 = 241 nM (pChEMBL 6.619999885559082)
Descriptors: MW 513.03, ALogP 5.98, HBA 6, HBD 1, PSA 78.91, QED 0.51
Compound (SMILES): CCS(=O)(=O)c1cc(Nc2ncc(F)c(N(C)c3cc(CO)ccc3C)n2)ccc1N1CCN(C)CC1
InChIKey: VPSBNPIVVHGVAY-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase PAK 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13153
Assay: Inhibition of PAK1 phosphorylation in human MCF10A cells
Measurement: IC50 = 490 nM (pChEMBL 6.309999942779541)
Descriptors: MW 528.65, ALogP 3.47, HBA 9, HBD 2, PSA 101.9, QED 0.46
Compound (SMILES): CN1CC[C@H](c2c(O)cc(O)c3c(=O)cc(-c4ccccc4F)oc23)[C@H](O)C1
InChIKey: MADUTGUALSLNOW-YVEFUNNKSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: Inhibition of human His6-tagged CDK9/cyclin T1 expressed in baculovirus infected sf9 cells using GST-CTD as substrate after 10 mins in presence of [gamma-32P]ATP by SDS-PAGE analysis
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 385.39, ALogP 2.79, HBA 6, HBD 3, PSA 94.14, QED 0.63
Compound (SMILES): CN(C)CCCOc1ccc(-c2ccc3ncc4c(c3c2)n(C2COC2)c(=O)n4C)cn1
InChIKey: WGCDXRFFSPQJBW-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Cellular Assay: Assay b) HT29 cells (ECACC #85061109) were seeded into 384 well assay plates (Costar #3712) at a density of 6000 cells/well in 40 μl EMEM medium containing 1% L glutamine and 10% FBS and allowed to adhere overnight. The following morning compound of Formula (I) in 100% DMSO was added to assay plates by acoustic dispensing. After 1 h incubation at 37° C. and 5% CO2, 40 nl of 3 mM 4NQO in 100% DMSO was added to all wells by acoustic dispensing, except minimum control wells which were left untreated with 4NQO to generate a null response control. Plates were returned to the incubator for a further 1 h. Then cells were fixed by adding 20 μl of 3.7% formaldehyde in PBS solution and incubating for 20 mins at r.t. Then 20 μl of 0.1% Triton X100 in PBS was added and incubated for 10 minutes at r.t., to permeabalise cells. Then the plates were washed once with 50 μl/well PBS, using a Biotek EL405 plate washer. Phospho-Chk1 Ser 345 antibody (Cell Signalling Technology #2348) was diluted 150 fold in PBS containing 0.05% polysorbate/Tween and 15 μl was added to each well and incubated over night at r.t. The next morning plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and then 20 μl of secondary Ab solution, containing 500 fold diluted Alexa Fluor 488 Goat anti-rabbit IgG (Molecular Probes #A-11008) and 0.002 mg/ml Hoeschst dye (Molecular Probes #H-3570), in PBST, was added. After 2 h incubation at r.t., the plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and plates were then sealed with black plate seals until read. Plates were read using an ArrayScan VTI instrument, using an XF53 filter with 10× objective. A two laser set up was used to analyse nuclear staining with Hoeschst (405 nm) and secondary antibody staining of pChk1 (488 nm).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 433.51, ALogP 2.85, HBA 8, HBD 0, PSA 74.41, QED 0.42
Compound (SMILES): CC(C)Oc1ccc(S(=O)(=O)N2CCN(c3cc(Br)cc(OCC(=O)O)c3)CC2)cc1
InChIKey: HEGPIPBYFYKJPN-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q13258
Assay: Displacement of [3H]-PGD2 from PGD2 receptor in human platelet membranes after 2 hrs by micro beta scintillation counting analysis
Measurement: Ki = 570 nM (pChEMBL 6.239999771118164)
Descriptors: MW 513.41, ALogP 3.21, HBA 6, HBD 1, PSA 96.38, QED 0.58
Compound (SMILES): O=C(Nc1cn(CC(=O)N2CC3CCCNC3C2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: ZVECZAKPYAYDBN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.78 nM (pChEMBL 9.109999656677246)
Descriptors: MW 570.99, ALogP 3.31, HBA 9, HBD 2, PSA 118.68, QED 0.35
Compound (SMILES): COc1ccc(C(=O)NC2=C(c3ccc(C)cc3)C(F)(F)C(c3ccc(OCCCC(F)(F)F)cc3)(C(F)(F)F)NC2=O)cc1
InChIKey: AUTXDAZKDQCXTP-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT SPA Assay: MGAT2 enzyme was assayed using membranes isolated from Sf9 cells expressing the recombinant human MGAT2 cDNA with 2-monooleoylglycerol and [3H]-oleoyl-CoA as substrates as described by Seethala et al. [Anal. Biochem., 383(2):144-150 (Dec. 15, 2008)]. Briefly, the assays were conducted in 384-well plates in a total volume of 30 uL at 25° C. In each assay, 200 ng of recombinant human MGAT2 membrane was incubated with 10 uM of 2-monooleoylglycerol and 15 uM of [3H]-oleoyl-CoA in 100 mM potassium phosphate (pH 7.4) for 20 min with various concentrations of compounds delivered in DMSO. The assay was terminated by the addition of 20 ul of Stopping Solution (7.5 mg/ml Yttrium Oxide Polylysine beads, 3.3 mg/ml Fraction V BSA and 200 uM Mercuric chloride in 50 mM HEPES, pH 7.4). The signal was measured 1 h after quenching the reaction using LEADSEEKERSM for 5 minutes. To calculate the degree of inhibition, the zero level of enzyme activity (blank) was defined.
Measurement: IC50 = 1176 nM (pChEMBL 5.929999828338623)
Descriptors: MW 642.54, ALogP 7.09, HBA 4, HBD 2, PSA 76.66, QED 0.19
Compound (SMILES): Cc1c(F)cncc1-c1nc2ccc(F)cn2c1C1CC1
InChIKey: BYZQRWHNJVYODK-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Inhibition of human CYP11B1-8C7 expressed in Chinese hamster V79 cells using 11-deoxycortisol as substrate preincubated for 1 hr followed by substrate addition measured after 3 hrs by HTRF assay
Measurement: IC50 = 930 nM (pChEMBL 6.03000020980835)
Descriptors: MW 285.3, ALogP 3.86, HBA 3, HBD 0, PSA 30.19, QED 0.72
Compound (SMILES): Nc1ncc(-c2ccc(CN3CCCC3)s2)cc1C(=O)Nc1ccncc1
InChIKey: SPYTVKZEORYOEZ-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: IKKepsilon Flashplate Assay: The kinase assay is performed either as 384-well Flashplate assay (for e.g. Topcount measurement). 1 nM IKKepsilon, 800 nM biotinylated IkappaBalpha(19-42) peptide (Biotin-C6-C6-GLKKERLLDDRHDSGLDSMKDEE) SEQ ID NO: 1 and 10 uM ATP (spiked with 0.3 uCi 33P-ATP/well) are incubated in a total volume of 50 ul (10 mM MOPS, 10 mM Mg-acetat, 0.1 mM EGTA, 1 mM Dithiothreitol,0.02% Brij35, 0.1% BSA, 0.1% BioStab, pH 7.5) with or without test compound for 2 hours at 30° C. The reaction is stopped with 25 ul 200 mM EDTA. After 30 Min at room temperature the liquid is removed and each well washed thrice with 100 ul 0.9% sodium chloride solution. Non-specific reaction is determined in presence of 3 uM MSC2119074 (BX-795). Radioactivity is measured with Topcount (PerkinElmer).
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 379.49, ALogP 3.64, HBA 6, HBD 2, PSA 84.14, QED 0.71
Compound (SMILES): C=CC(=O)Nc1ccccc1[C@@H]1CCNc2c(C(N)=O)c(-c3ccc(OCC4CC4)cc3)nn21
InChIKey: SKKIKHFBSXYTQB-NRFANRHFSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assay: Compounds disclosed herein were tested for inhibition of Btk kinase activity in an assay based on time-resolved fluorescence resonance energy transfer methodology. Recombinant Btk was pre-incubated with the compounds disclosed herein at room temperature for 1 hour in an assay buffer containing 50 mM Tris pH7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DTT, 20 nM SEB, 0.1% BSA, 0.005% tween-20. The reactions were initiated by the addition of ATP (at the concentration of ATP Km) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubating at room temperature for 1 h, an equal volume of stop solution containing 50 mM HEPES pH7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, Eu cryptate-conjugated p-Tyr66 antibody and streptavidin-labeled XL665 was added to stop the reaction. Plates were further incubated at room temperature for 1 hour, and then the TR-FRET signals (ex337 nm, em 620 nm/665 nm) were read on BMG PHERAstar FS instrument. The residual enzyme activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 615 nm to that at 665 nm.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 457.53, ALogP 3.97, HBA 6, HBD 3, PSA 111.27, QED 0.44
Compound (SMILES): CC[C@H](C)[C@H](NC(=O)[C@]1(NC(=O)[C@@H]2CCCN2C(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN)CCO[C@@H]1c1ccc(Br)cc1)C(=O)N[C@@H](CC(C)C)C(=O)O
InChIKey: VBXPVGZTIVEQAY-XSQOBMHJSA-N
Target: Neurotensin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30989
Assay: Displacement of [3H]neurotensin from human NTS1 receptor expressed in CHOK1 cell membranes after 60 mins by scintillation counting
Measurement: Ki = 13000 nM (pChEMBL 4.889999866485596)
Descriptors: MW 865.91, ALogP 1.97, HBA 10, HBD 8, PSA 261.3, QED 0.07
Compound (SMILES): O=C1c2ccccc2C(O)(c2ccc3[nH]c(=O)[nH]c3c2)N1c1cccc(Cl)c1F
InChIKey: MPBLSRRIBYVXJE-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 409.8, ALogP 3.5, HBA 3, HBD 3, PSA 89.19, QED 0.47
Compound (SMILES): Cc1nc([C@H](CC2CC2)NC(=O)c2ccc(C3CC3)c(S(=O)(=O)CC(C)C)n2)no1
InChIKey: FGECUFZKASUBKF-SFHVURJKSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 432.55, ALogP 3.35, HBA 7, HBD 1, PSA 115.05, QED 0.65
Compound (SMILES): O=C(c1cccc(Cl)c1Cl)N1CCc2c(n[nH]c2-c2cncnc2)C1
InChIKey: GXNIYQCVNZERQO-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 374.23, ALogP 3.37, HBA 4, HBD 1, PSA 74.77, QED 0.75
Compound (SMILES): O=C(c1cc(F)ccc1-c1ncccn1)N1CCOC1Cn1ccc(-c2ccc(F)cn2)n1
InChIKey: LZWJHKGAHUOHTO-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Antagonistic Activity Assay: The antagonistic activities of the test compounds on human orexin-1 receptor (hOX1R) and orexin-2 receptor (hOX2R) were measured by modifying from the method described in literature (Toshikatsu Okumura et al., Biochemical and Biophysical Research Communications 280, 976-981, 2001). Chinese hamster ovary (CHO) cells forcibly expressing the hOX1R and hOX2R were seeded into a 96 well Black clear bottom plate (Nunc) at 20,000 cells per well, which were cultured in Ham's F-12 medium containing 0.1 mM MEM non-essential amino acids, 0.5 mg/ml G418, 10% fetal bovine serum (all by Invitrogen) for 16 hours under the conditions of 37° C., 5% CO2. After removing the medium, 100 uL of 0.5 uM Fluo-4AM ester (Dojin) in an assay buffer (25 mM HEPES (Dojin), Hank's balanced salt solution (Invitrogen), 0.1% bovine serum albumin, 2.5 mM probenecid, 200 ug/ml Amaranth (all by Sigma-Aldrich), pH 7.4) was added and the cells were incubated for 60 minutes at 37° C., 5% CO2. After removing the medium, 100 uL of 0.5 uM Fluo-4AM ester (Dojin) in an assay buffer (25 mM HEPES (Dojin), Hank's balanced salt solution (Invitrogen), 0.1% bovine serum albumin, 2.5 mM probenecid, 200 ug/ml Amaranth (all by Sigma-Aldrich), pH 7.4) was added and the cells were incubated for 60 minutes at 37° C., 5% CO2. After removing the assay buffer containing fluo-3AM ester, the test compound was dissolved in dimethyl sulfoxide to be 10 mM and diluted with the assay buffer, 150 uL of which was added and incubated for 30 minutes.
Measurement: IC50 = 71.7 nM (pChEMBL 7.139999866485596)
Descriptors: MW 448.43, ALogP 3.18, HBA 7, HBD 0, PSA 86.03, QED 0.47
Compound (SMILES): Cc1cc(Nc2cc(C3CC3)n[nH]2)nc(NCc2c(-c3ccccc3)noc2C)n1
InChIKey: GNZKEJZKCVRRIT-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) tri-phosphorylated enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 401.47, ALogP 4.7, HBA 7, HBD 3, PSA 104.55, QED 0.41
Compound (SMILES): CC(C)C[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)CNC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1ccccc1)NC(=O)CCC(=O)NCCCOCCOCCOCCCNC(=O)CCCC[C@@H]1SC[C@@H]2NC(=O)N[C@@H]21)C(C)C)C(C)C)C(N)=O
InChIKey: DYHJOUGZIZFHEP-LZJRUSSCSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Inhibition of C-terminus 10x-His-taged human TFPI-1 (29 to 282 residues) assessed as inhibition of factor 10a using S-2222 substrate incubated for 115 mins by micro-plate reader based assay
Measurement: EC50 = 1890 nM (pChEMBL 5.71999979019165)
Descriptors: MW 2991.61
Compound (SMILES): O=C(OCc1ccccc1)N1CCCC(c2ncn3cnc4[nH]ccc4c23)C1
InChIKey: NNOBCIHRMOGXAE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 57 nM (pChEMBL 7.239999771118164)
Descriptors: MW 375.43, ALogP 3.73, HBA 5, HBD 1, PSA 75.52, QED 0.59
Compound (SMILES): CN1CCC(COC(=O)C2(C)CCN(C(=O)Cn3cc(NC(=O)c4cnn5cccnc45)c(-c4cc(Cl)ccc4OC(F)F)n3)CC2)CC1
InChIKey: NGVBKFCFPGVNPI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 2.16 nM (pChEMBL 8.670000076293945)
Descriptors: MW 699.16, ALogP 4.61, HBA 11, HBD 1, PSA 136.19, QED 0.24
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCN(Cc2cc3ccccn3n2)CC1
InChIKey: RAMNFCPRAKUYEY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 163 nM (pChEMBL 6.789999961853027)
Descriptors: MW 473.52, ALogP 2.81, HBA 9, HBD 2, PSA 130.16, QED 0.39
Compound (SMILES): O=C(O)CC[C@H](NC(=O)N[C@@H](CCCCn1cc(COCCNc2ccc([N+](=O)[O-])cc2[N+](=O)[O-])nn1)C(=O)O)C(=O)O
InChIKey: BNQBVBFTPMOTDQ-ROUUACIJSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: PMSA Inhibition Assay: For IC50 measurements, inhibitors were dissolved in Reaction Buffer containing 40 μM NAAG to a final volume of 100 μL. Then, 25 μL of this solution was transferred to each of three wells in a microtiter plate, and 5 μL aliquots were serially diluted into 20 μL of solution containing 40 μM NAAG over 10 wells (5-fold dilutions). Inhibitor concentration therefore ranged over 6 orders of magnitude in these experiments. rhPSMA (20 ng/mL in Reaction Buffer, 20 μL, R&D Research) was then added to each well. The plate was incubated at room temperature for 15 min, and then heated to 95 °C. for 3 minutes. The plate was allowed to cool to room temperature, and glutamic acid levels were measured using a commercially available Amplex® − Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen). Fluorescence intensities were measured using a Synergy 2 multiwell plate reader (Biotek), fitted with excitation and emission filters of 545 nm and 590 nm, respectively. Ki values were obtained from IC50 values using the Cheng-Prusoff equation, a substrate concentration of 20 μM, and a Km value of 0.925 μM.
Measurement: IC50 = 1.05 nM (pChEMBL 8.979999542236328)
Descriptors: MW 610.54, ALogP 0.96, HBA 13, HBD 6, PSA 291.28, QED 0.07
Compound (SMILES): COCCOc1cccc(N2C(=O)c3ccccc3C2(O)c2ccc3[nH]c(NC(=O)OC)nc3c2)c1
InChIKey: WFEJKSYCJXOFAL-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 488.5, ALogP 3.62, HBA 7, HBD 3, PSA 126.01, QED 0.34
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3ccc(-c4cnn(C)c4)cc3)nn2[C@H]2CCOC2)C1
InChIKey: ZNIQPKLUCOYTFF-IBGZPJMESA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (6×His disclosed as SEO ID NO: 3) (Perkin Elmer AD0110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 406.49, ALogP 2.89, HBA 7, HBD 1, PSA 77.21, QED 0.72
Compound (SMILES): NC(=O)OCC(CNC(=O)Cn1nc(-c2ccc(Cl)cc2)n(C[C@H](O)C(F)(F)F)c1=O)c1ccccc1Cl
InChIKey: HHPVBOHKYBGQSP-IBYPIGCZSA-N
Target: Vasopressin V2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30518
Assay: Cellular in Vitro Assay: On the day before the assay, the cells are plated out in culture medium (DMEM, 10% FCS, 2 mM glutamine, 10 mM HEPES) in 384-well microtitre plates and kept in a cell incubator (96% humidity, 5% v/v CO2, 37° C.). On the day of the assay, the culture medium is replaced by a Tyrode solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM glucose, 20 mM HEPES), which additionally contains the cofactor coelenterazine (50 μM), and the microtitre plate is then incubated for a further 3-4 hours. The test substances in various concentrations are placed for 10 to 20 minutes in the wells of the microtitre plate before the agonist [Arg8]-vasopressin is added, and the resulting light signal is measured immediately in the luminometer.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 576.36, ALogP 2.94, HBA 8, HBD 3, PSA 141.47, QED 0.34
Compound (SMILES): O=C(O)c1ccccc1S(=O)(=O)Nc1ccc(-c2nc3cc(NS(=O)(=O)c4ccccc4C(=O)O)ccc3o2)cc1
InChIKey: INRTVDROQCXLEM-UHFFFAOYSA-N
Target: HSP60/HSP10 (PROTEIN COMPLEX), Homo sapiens
UniProt: P61604
Assay: Inhibition of human mitochondrial HSP60/HSP10 expressed in Escherichia coli DH5alpha/BL21 (DE3) assessed as inhibition of denatured MDH refolding preincubated for 10 mins followed by ATP addition measured after 40 to 60 mins by fluorescence based assay
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 593.6, ALogP 4.49, HBA 8, HBD 4, PSA 192.97, QED 0.19
Compound (SMILES): CC1=CC(=O)N(Nc2nc(-c3cccs3)nc3sc(-c4ccc(OCCNCc5ccccc5)cc4)c(C)c23)C1=O.Cl
InChIKey: BXZSDODQTBHLDB-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Inhibition of human FLT3 D835Y mutant preincubated for 15 mins followed by poly(Glu:Tyr) substrate addition for 30 mins in presence of [gamma-32P]ATP by TR-FRET assay
Measurement: IC50 = 9.478 nM (pChEMBL 8.020000457763672)
Descriptors: MW 581.72, ALogP 6.21, HBA 9, HBD 2, PSA 96.45, QED 0.15
Compound (SMILES): C[C@@H](Nc1ncnc2[nH]c(-c3ccc(NC(=O)CCCCC(=O)NO)cc3)cc12)c1ccccc1
InChIKey: BNNHALXHKHKDOT-QGZVFWFLSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of N-terminal GST-tagged human HER2 (Lys676 to Val1255 residues) expressed in baculovirus expression system using biotinylated FLT3 at Tyr589 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA method
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 472.55, ALogP 4.8, HBA 6, HBD 5, PSA 132.03, QED 0.13
Compound (SMILES): N[C@H]1CCCN(c2ccncc2Nc2ncc3ccc(-c4ccccc4Cl)nn23)C1
InChIKey: OYFIIXZMOKZCBZ-HNNXBMFYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: PIM Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 419.92, ALogP 4.12, HBA 7, HBD 2, PSA 84.37, QED 0.52
Compound (SMILES): COc1cccc(C(=O)NCC(c2ccc(C(F)(F)F)nc2)N2CCOCC2)c1C
InChIKey: FZSHDJQQXJOSSR-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO42H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates. 93-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1X); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes. Two sets of drug plates were prepared: A) Mixtures of compound plus agonist were prepared as follows, in order to determine close response: BzATP: 11 point ½ log, diluted in buffer, starting from 1 mM. Testing compounds: 11 point ½ log, diluted in 2% DMSO buffer starting from 10 μM. B) Agonist only mixture was prepared with BzATP at a single concentration in buffer (concentration determined by dose response). Compound mixtures (A) were added to assay plates containing cells and placed at room temperature for 30 minutes, then BzATP (B) was added. Fluorescence was read using the Tetra FLIPR® (Molecular Devices, Inc., Sunnyvale, Calif., USA) and IC50 values were calculated by standard methods to determine antagonist activity.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 423.44, ALogP 3.22, HBA 5, HBD 1, PSA 63.69, QED 0.77
Compound (SMILES): CS(=O)(=O)C1(c2nc(-c3ccc(F)c4[nH]ccc34)nc3c2OC[C@@H]2COCCN32)CC1
InChIKey: RVIXVVFPDQOMBH-LBPRGKRZSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: ADP Hunter Plus Assay: The kinase activity was measured by using the commercial ADP Hunter Plus assay available from DiscoveRx (#90-0083), which is a homogeneous assay to measure the accumulation of ADP, a universal product of kinase activity. The enzyme, DNA-PK was purchased from Promega (#V5811). The assay was done following the manufacturer recommendations with slight modifications: Mainly the kinase buffer was replace by 15 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM NaCl, 1 mM EGTA, 0.1 mg/mL BGG, 0.02% Tween 20. The DNA-PK was assayed in a titration experiment to determine the optimal protein concentration for the inhibition assay. To calculate the 1050 of the Compounds, serial 1:3 dilutions of the compounds were added to the enzyme at a fixed concentration (2 U/uL). The enzyme was preincubated with the inhibitor and 200 uM DNA substrate and then ATP was added to a final 75 uM concentration. Reaction was carried out for 1 hour at 37° C. Reagent A and B were sequentially added to the wells and plates were incubated for 30 min at RT. Fluorescence counts were read in a EnVision instrument (Perkin Elmer) with the recommended settings (550 and 590 nm as excitation and emission wavelengths, respectively). Values were normalized against the control activity included for each enzyme (i.e., 100% DNA_PK kinase activity, without compound). These values were plotted against the inhibitor concentration and were fit to a sigmoid dose-response curve by using the model sigmoidal Four-Parameter Logistc implement for Activity base - software.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 444.49, ALogP 2.4, HBA 7, HBD 1, PSA 97.41, QED 0.66
Compound (SMILES): O=C(O)Cn1cnc(-c2ccccc2)c1-c1ccccc1
InChIKey: UFTBOKJDSUBCER-UHFFFAOYSA-N
Target: Prostaglandin G/H synthase 1 (SINGLE PROTEIN), Ovis aries
UniProt: P05979
Assay: Inhibition of ovine COX-1 assessed as reduction in oxidation of TMPD using arachidonic acid as substrate preincubated for 5 mins followed by substrate and TMPD addition measured after 5 mins by colorimetric assay
Measurement: IC50 = 670 nM (pChEMBL 6.170000076293945)
Descriptors: MW 278.31, ALogP 3.3, HBA 3, HBD 1, PSA 55.12, QED 0.8
Compound (SMILES): CCc1sc2c(c1CC(=O)Nc1nc(-c3cc(F)c(OC(F)F)c(F)c3)cs1)c(=O)n(C)c(=O)n2C
InChIKey: ZLRQOPRFOLBNTA-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 542.54, ALogP 4.05, HBA 9, HBD 1, PSA 95.22, QED 0.36
Compound (SMILES): COc1ccc(-c2ccccc2N2CCN(CCCCCC(=O)NCc3ccc(C#N)cc3)CC2)cc1
InChIKey: HGCNXEAQMMJLQX-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Displacement of [3H]-8-OH-DPAT from human 5-HT1A receptor expressed in HEK293 cell membranes after 1 hr by microbeta counting analysis
Measurement: Ki = 126 nM (pChEMBL 6.900000095367432)
Descriptors: MW 496.66, ALogP 5.23, HBA 5, HBD 1, PSA 68.6, QED 0.37
Compound (SMILES): Cc1ccc(NC(=O)c2cncc(C(C)(C)C)c2)cc1-c1cc(N2CCOCC2)nc(N2CCOCC2)n1
InChIKey: LDTUEQNAYCVAAO-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 0.562 nM (pChEMBL 9.25)
Descriptors: MW 516.65, ALogP 4.07, HBA 8, HBD 1, PSA 92.71, QED 0.54
Compound (SMILES): COc1cc2c(Nc3cc(Cl)c(Cl)cc3F)ncnc2cc1O[C@@H]1C[C@H]2OC[C@H](OC)[C@H]2O1
InChIKey: JIBJCRRXQHEKJX-ANULTFPQSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: Inhibition of recombinant human N-terminal His6-tagged EphA2 (N598 to R890 residues) expressed in baculovirus using poly-EY as substrate after 180 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 496.32, ALogP 4.74, HBA 8, HBD 1, PSA 83.96, QED 0.49
Compound (SMILES): NC(=O)c1c(-c2ccc(Oc3ccccc3)cc2)nn2c1NCC(N)C2
InChIKey: IOIPGNPFRJOTEC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assay: Compounds disclosed herein were tested for inhibition of Btk kinase activity in an assay based on time-resolved fluorescence resonance energy transfer methodology. Recombinant Btk was pre-incubated with the compounds disclosed herein at room temperature for 1 hour in an assay buffer containing 50 mM Tris pH7.4, 10 mM MgCl2, 2 mM MnCl2, 0.1 mM EDTA, 1 mM DTT, 20 nM SEB, 0.1% BSA, 0.005% tween-20. The reactions were initiated by the addition of ATP (at the concentration of ATP Km) and peptide substrate (Biotin-AVLESEEELYSSARQ-NH2). After incubating at room temperature for 1 h, an equal volume of stop solution containing 50 mM HEPES pH7.0, 800 mM KF, 20 mM EDTA, 0.1% BSA, Eu cryptate-conjugated p-Tyr66 antibody and streptavidin-labeled XL665 was added to stop the reaction. Plates were further incubated at room temperature for 1 hour, and then the TR-FRET signals (ex337 nm, em 620 nm/665 nm) were read on BMG PHERAstar FS instrument. The residual enzyme activity in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 615 nm to that at 665 nm.
Measurement: IC50 = 451 nM (pChEMBL 6.349999904632568)
Descriptors: MW 349.39, ALogP 2.19, HBA 6, HBD 3, PSA 108.19, QED 0.67