lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(C(=O)c1cccnc1)c1cccc(OCC(O)CN2CCc3ccccc3C2)c1
InChIKey: BODKRUVQWYSAGB-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 417.51, ALogP 3.16, HBA 5, HBD 1, PSA 65.9, QED 0.64
Compound (SMILES): CCC(C(=O)Nc1ccc(-c2ncon2)cc1)n1cc(OC)c(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: JIVAXRBOSDSXCF-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 489.92, ALogP 4.69, HBA 8, HBD 1, PSA 123.04, QED 0.4
Compound (SMILES): NC1=N[C@](CF)(c2cc(Oc3nccc(C(F)(F)F)c3F)ccc2F)C[C@@H](C(F)(F)F)O1
InChIKey: ZKPWDRUCTOKPHI-BLLLJJGKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 9520 nM (pChEMBL 5.019999980926514)
Descriptors: MW 473.3, ALogP 5, HBA 5, HBD 1, PSA 69.73, QED 0.64
Compound (SMILES): Cc1cc(C(=O)N2Cc3cc4c(cc3C[C@H]2C(=O)N[C@@H](Cc2ccc(Oc3ccnc(C)c3C)cc2)C(=O)O)OC[C@H](c2ccc(OCc3ccc(Cl)c(Cl)c3)cc2)O4)n(C)n1
InChIKey: RIHXMPSXZWJFPB-WVSZGQIDSA-N
Target: Glucagon-like peptide 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43220
Assay: cAMP Functional Assay: The efficacy of GLP-1 receptor agonists was studied in a cAMP functional assay using HEK-293 Cells expressing the cloned human GLP-1 receptor. GLP-1-expressing cells (10,000-20,000 cells per 0.1 mL) were plated in 96-well plates in Dulbecco's modified eagles media (DMEM) containing 10% fetal bovine serum, 2 mg/mL G418 and penicillin-streptomycin. Following overnight incubation of cells, media was removed, and compounds (at concentrations ranging from 0.0001 to 100 μM) were added to monolayer cells in Iscove's modified dulbecco's medium (IMDM), 100 μM RO 20-1724 PDE inhibitor, 0.1% BSA, 2% DMSO in a final volume of 100 μL, and incubated for 30 min at 37° C. 95%02, 5% CO2 in a humidified incubator. Alternatively, cells were harvested and combined with compounds in suspension using the buffer and protocol described above. cAMP was quantified using a homogenous time-resolved fluorescence detection system (cAMP dynamic, CIS bio International).
Measurement: EC50 = 132 nM (pChEMBL 6.880000114440918)
Descriptors: MW 876.79, ALogP 8.31, HBA 10, HBD 2, PSA 154.34, QED 0.12
Compound (SMILES): c1cc(N2CCNCC2)c(Nc2ncc3ccc(-c4nccs4)nn23)cn1
InChIKey: LUJRXDRGQWULDY-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
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 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 378.47, ALogP 2.4, HBA 9, HBD 2, PSA 83.27, QED 0.56
Compound (SMILES): Cc1cccc(C)c1Oc1ccc2c(C#N)nc(C(=O)NCCCCC(=O)O)c(O)c2c1
InChIKey: QTGCKPZNEUANAZ-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 10520 nM (pChEMBL 4.980000019073486)
Descriptors: MW 433.46, ALogP 4.21, HBA 6, HBD 3, PSA 132.54, QED 0.45
Compound (SMILES): CC1(COc2nc(C(=O)NC(C)(C)c3nccs3)ccc2N2CC(F)(F)C2)COC1
InChIKey: PRWRZHFVIKSKFL-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
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 = 259.3 nM (pChEMBL 6.590000152587891)
Descriptors: MW 438.5, ALogP 3.07, HBA 7, HBD 1, PSA 76.58, QED 0.72
Compound (SMILES): COc1ccc(-c2ncn(CC(=O)O)c2-c2ccc(OC)cc2)cc1
InChIKey: GIKSVHXWHXYPFG-UHFFFAOYSA-N
Target: Prostaglandin G/H synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35354
Assay: Inhibition of human recombinant COX-2 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 = 12570 nM (pChEMBL 4.900000095367432)
Descriptors: MW 338.36, ALogP 3.32, HBA 5, HBD 1, PSA 73.58, QED 0.75
Compound (SMILES): COC(=O)N[C@H](C(=O)N1CCC[C@H]1c1nc2ccc([C@H]3CC[C@H](c4ccc5nc([C@@H]6CCCN6C(=O)[C@@H](NC(=O)OC)C(C)C)[nH]c5c4)N3c3ccc(C(C)(C)C)cc3)cc2[nH]1)C(C)C
InChIKey: NREDIDBJPFQPKQ-UEECLIOXSA-N
Target: Nonstructural protein 5A (SINGLE PROTEIN), Hepatitis C virus
UniProt: Q5L478
Assay: Inhibition of Hepatitis C virus genotype 1a H77 replicon NS5A Y93C mutant transfected in human HuH7 cells assessed as decrease in replication of viral replicon after 4 days by luciferase reporter gene assay
Measurement: EC50 = 0.016 nM (pChEMBL 10.800000190734863)
Descriptors: MW 888.13, ALogP 8.91, HBA 9, HBD 4, PSA 177.88, QED 0.1
Compound (SMILES): O=C(O)[C@H]1CC[C@H](C(=O)N2CCC(c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3cccc(C4(F)CCC4)c3)C2)CC1
InChIKey: IAEIWATZJQYNEW-KLBFEONNSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 74 nM (pChEMBL 7.130000114440918)
Descriptors: MW 681.64, ALogP 7.12, HBA 4, HBD 1, PSA 91.75, QED 0.31
Compound (SMILES): COCCNc1cc(NC(=O)N2CCCc3cc(CN(CCN(C)C)C(C)=O)c(C=O)nc32)ncc1C#N
InChIKey: HKVAALOLNUASDA-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Biochemical Kinase Assay: Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 ul per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 16 mM MgCl2, 1122 uM ATP, 4 uM peptide (5-Fluo-Ahx-KKKKEEIYFFFG-NH2, Biosyntan GmbH) and 4.5 ul per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 16 mM MgCl2, 6 nM FGFR4 (GST-FGFR4(388-802), produced in-house by expression in insect cells and affinity chromatography). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 ul per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 522.61, ALogP 2.11, HBA 9, HBD 2, PSA 143.79, QED 0.33
Compound (SMILES): CC[C@@H](N[C@@H](C)CC(N)=O)c1ccc(Cl)c(C(=O)c2ccc3c(c2)OCCN3)c1F
InChIKey: RQZBQWDQPONYTP-BLLLJJGKSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: FRET-Based Assay: The HCV NS3 protease functions have been extensively studied and are considered as potential targets for antiviral therapy: see for example the many references listed in the introductory section of this application. Therefore, the activity of the compounds of the invention as anti-HCV agents was assessed using a full length HCV NS3 protease.The protease activity of the full length NS3/4a was measured using a FRET-based assay utilizing a peptide substrate derived from the NS4A/B cleavage site (Anaspec) and labelled at one end with a quencher (QXL520) and at the other with a fluorophore (5-FAMsp). NS3/4a (produced in-house by literature methods) was incubated with test compounds and peptide substrate in 50 mM Tris pH8, 20 mM DTT, 1% CHAPS, 10% glycerol and 5% DMSO. The reaction was followed by monitoring the change in fluorescence on a Molecular Devices Gemini plate reader for 30 minutes at room temperature.
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 433.91, ALogP 3.82, HBA 5, HBD 3, PSA 93.45, QED 0.55
Compound (SMILES): COc1cccc(CNC(=O)c2ccc(-c3cn[nH]c3)cc2N)c1
InChIKey: YQZUYMIRRIDTAO-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 131 nM (pChEMBL 6.880000114440918)
Descriptors: MW 322.37, ALogP 2.6, HBA 4, HBD 3, PSA 93.03, QED 0.63
Compound (SMILES): CNc1nc(NCc2ccc(NC(=O)C3CCN(Cc4ccccc4)CC3)cc2)c2cc(C)ccc2n1
InChIKey: ZAKBAQHYORKWNL-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 = 726 nM (pChEMBL 6.139999866485596)
Descriptors: MW 494.64, ALogP 5.44, HBA 6, HBD 3, PSA 82.18, QED 0.3
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: GHZCINLOSBWLDB-LYXMDHKGSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 2385.85
Compound (SMILES): CC1CCN(C(=O)C(CCn2cccc2C#N)NS(=O)(=O)c2cc(Cl)c(N)cc2Cl)CC1
InChIKey: VLUQTRJGDOZHRP-UHFFFAOYSA-N
Target: C-C chemokine receptor type 10 (SINGLE PROTEIN), Homo sapiens
UniProt: P46092
Assay: Antagonist activity at human CCR10 expressed in CHOK1 cells coexpressing aequorin/Galphaq assessed as inhibition of human CCL27-dependent calcium flux in presence of coelenterazine H by chemiluminescence assay
Measurement: IC50 = 63.1 nM (pChEMBL 7.199999809265137)
Descriptors: MW 498.44, ALogP 3.24, HBA 6, HBD 2, PSA 121.22, QED 0.57
Compound (SMILES): O=C(O)c1nccc(-c2ccc(OC3CC3)c(Cl)c2)n1
InChIKey: SKLFIIGEXZGVMW-UHFFFAOYSA-N
Target: Kynurenine 3-monooxygenase (SINGLE PROTEIN), Homo sapiens
UniProt: O15229
Assay: Inhibition of human KMO
Measurement: IC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 290.71, ALogP 3.04, HBA 4, HBD 1, PSA 72.31, QED 0.94
Compound (SMILES): CCCOc1ccc(N2CCC(Oc3ccc([C@H](C)NC(C)=O)cc3)C2)c(C)c1
InChIKey: NKNHXPOIPXMSCM-XNUZUHMRSA-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 200 μM 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.
Measurement: IC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 396.53, ALogP 4.64, HBA 4, HBD 1, PSA 50.8, QED 0.71
Compound (SMILES): O=C(Nc1cccc(CN2CCC(C(=O)NC3CCCC3)CC2)c1)c1ccc(Cl)cc1
InChIKey: LVBQKJVDHOCPMK-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 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 439.99, ALogP 4.86, HBA 3, HBD 2, PSA 61.44, QED 0.67
Compound (SMILES): CC(C)(Nc1cc(C(N)=O)nc(-c2ccc(Oc3ccc(F)cc3)cc2)n1)C(N)=O
InChIKey: BSAUUXPPXUVRQO-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: In vitro assays were performed in a recombinant cell line expressing cDNA encoding the alpha subunit (Nav1.7, SCN9a, PN1, NE) of human Nav1.7 (Accession No. NM002977). The cell line was provided by investigators at Yale University (Cummins et al, J. Neurosci. 18(23): 9607-9619 (1998)). For dominant selection of the Nav1.7-expressing clones, the expression plasmid co-expressed the neomycin resistance gene. The cell line was constructed in the human embryonic kidney cell line, HEK293, under the influence of the CMV major late promoter, and stable clones were selected using limiting dilution cloning and antibiotic selection using the neomycin analogue, G418. Recombinant beta and gamma subunits were not introduced into this cell line. Additional cell lines expressing recombinant Nav1.7 cloned from other species can also be used, alone or in combination with various beta subunits, gamma subunits or chaperones.
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 409.42, ALogP 2.85, HBA 6, HBD 3, PSA 133.22, QED 0.55
Compound (SMILES): NCCC[C@@H]1N[C@H](CNC(=O)C2CCN(c3ccccc3)C2=O)CCN(CC(c2ccccc2)c2ccccc2)C1=O
InChIKey: XSOVPZBZTYQPSS-XTBGFXFQSA-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 < 10000 nM (pChEMBL 5)
Descriptors: MW 567.73, ALogP 3.29, HBA 5, HBD 3, PSA 107.77, QED 0.31
Compound (SMILES): CC(C)Oc1ccc(Nc2ncc(NC(=O)C3CCOCC3)c(=O)n2Cc2ccc(Cl)cc2)cc1Cl
InChIKey: HCXCTBUQSPWVBS-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 in M MgCl2, 5.0 mM CaCl2, 5.6 D-glucose, 2.5 mM probenecid, 1.0% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence inte
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 531.44, ALogP 5.49, HBA 7, HBD 2, PSA 94.48, QED 0.4
Compound (SMILES): Cn1nc(N2CCCC2)nc1CCc1nc2c(Cl)cc(C(F)(F)F)cn2n1
InChIKey: MKVGWNBIJDGDDF-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Scintillation Proximity Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 μl contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. # A-7906), 50 nM cGMP (Sigma, cat. # G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. # TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat # SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 μl of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate solution (stop reagent). After 1 h under shaking, the plate was centrifuged one minute at 170 g to allow beads to settle. Afterwards, radioactive counts were measured on a Perkin Elmer Top-Count Scintillation plate reader.
Measurement: IC50 = 104.77 nM (pChEMBL 6.980000019073486)
Descriptors: MW 399.81, ALogP 2.92, HBA 7, HBD 0, PSA 64.14, QED 0.67
Compound (SMILES): Cc1cc(Nc2cc(C3CC3)n[nH]2)nc(Nc2ccc(CCC(=O)N(C)C)cc2)n1
InChIKey: LZTZNACINFBFAT-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) un-phosphorylated apo-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 405.51, ALogP 3.89, HBA 6, HBD 3, PSA 98.83, QED 0.53
Compound (SMILES): COCC1(C)CN(C(=O)c2ccc3cc(Oc4ccc(C(F)(F)F)cn4)ccc3n2)C1
InChIKey: PMADMDHWOUCDAR-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 2550 nM (pChEMBL 5.590000152587891)
Descriptors: MW 431.41, ALogP 4.55, HBA 5, HBD 0, PSA 64.55, QED 0.59
Compound (SMILES): N[C@H](C(=O)O)C(NS(=O)(=O)c1ccc(Cl)cc1)C(=O)O
InChIKey: PHSLITQWZPTNLO-JAMMHHFISA-N
Target: Excitatory amino acid transporter 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P43005
Assay: Inhibition of [3H]-D-Asp uptake at human EAAT3 expressed in HEK293 cells measured after 3 mins by TopCount method
Measurement: IC50 = 977.24 nM (pChEMBL 6.010000228881836)
Descriptors: MW 322.73, ALogP -0.52, HBA 5, HBD 4, PSA 146.79, QED 0.55
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(OC(F)(F)F)c2)cc1-c1cc(N2CCOCC2)nc(N2CCOCC2)n1
InChIKey: ZPMMEQLJGQJSKJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 543.55, ALogP 4.28, HBA 8, HBD 1, PSA 89.05, QED 0.49
Compound (SMILES): O=C(NC1CCC(O)CC1)[C@@H]1NC2(CCCCCC2)[C@@]2(C(=O)Nc3cc(Cl)ccc32)[C@H]1c1cccc(Cl)c1F
InChIKey: UVWMOYGSAUBCGS-NGYPPSJLSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Binding Assay: The binding affinity of the MDM2 inhibitors disclosed herein was determined using a 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 called PMDM6-F (García-Echeverria et al., J. Med. Chem. 43: 3205-3208 (2000)).
Measurement: Ki = 1 nM (pChEMBL 9)
Descriptors: MW 574.52, ALogP 5.59, HBA 4, HBD 4, PSA 90.46, QED 0.38
Compound (SMILES): Cc1ccc(C2CN(c3ccc(C(F)(F)F)cn3)CCN2C(=O)C2CCS(=O)(=O)CC2)s1
InChIKey: RHTKJTFNZXGBRS-UHFFFAOYSA-N
Target: Sodium- and chloride-dependent glycine transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48067
Assay: Cytostar-T Assay: In detail, SK-N-MC cells are seeded into 96-well Cytostar-T assay plates at a density of 200,000 cells/well and grown for 16-18 hours to confluence in growth medium as recommended by ATCC. Before starting the assay, cells are washed once with HBSS (Hank's buffered salt solution; Sigma, H8264) cont. 5 mM alanine (referred in here as HBSS/Ala) and afterwards the following reagents are added: 1.80 ul/well HBSS/A1a 2. 20 ul/well of HBSS/A1a containing 6x the concentration of compound in 6% DMSO 3. approx. 5-10 min incubation 4. 20 ul/well 3 uM glycine (3H-glycine (Perkin Elmer, NET004001MC, specific activity: 52 Ci/mmol; diluted 1:1 with unlabelled glycine) in HBSS/A1a. In the final assay, glycine concentration is 500 nM (250 nM derived from the 3H-glycine Perkin Elmer, 250 nM unlabelled glycine), DMSO concentration is 1%. The assay plate is immediately after addition of the 3H-glycine placed into a Micro-Beta Counter (Perkin Elmer) and the signal is recorded over 60 min.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 487.57, ALogP 3.69, HBA 6, HBD 0, PSA 70.58, QED 0.66
Compound (SMILES): CCNC(=O)c1ccc(-n2cnc3c(NCCC(F)(F)F)cc(Oc4ccc(O)cc4)cc32)cc1C
InChIKey: XQIRZFSXJQCQPG-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Kinase Assay: For the assay 50 nL of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Mps-1 in assay buffer [0.1 mM sodium-ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μl of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μl assay volume is 10 μM) and peptide substrate (1.67 μM=>final conc. in the 5 μl assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 μl assay volume). The reaction was stopped by the addition of 3 μl of a solution of HTRF detection reagents (100 mM Hepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nM anti-phospho(Ser/Thr)-Europium-antibody [#AD0180, Perkin Elmer LAS, Rodgau-Jügesheinn, Germany]. The resulting mixture was incubated 1 h at 22° C. to allow the binding of the phosphorylated peptide to the anti-phospho(Ser/Thr)-Europium-antibody. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Europium-labelled anti-phospho(Ser/Thr) antibody to the Streptavidin-XLent. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a Viewlux TR-FRET reader (PerkinElmer LAS, Rodgau-Jügesheinn, Germany).
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 498.51, ALogP 5.95, HBA 6, HBD 3, PSA 88.41, QED 0.28
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4cnc(O[C@@H]5CCN(C(CF)CF)C5)c(Cl)c4)c(Oc4cccc5[nH]ncc45)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: VQVZZSKWOXRCEN-UUWRZZSWSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 0.052 nM (pChEMBL 10.279999732971191)
Descriptors: MW 908.9, ALogP 8.72, HBA 10, HBD 2, PSA 132.99, QED 0.11
Compound (SMILES): NC(=O)c1cccc([C@H]2C[C@H]3CC[C@@H](C2)N3CCN(Cc2c(F)cccc2F)C(=O)[C@H](O)CO)c1.O=C(O)C(F)(F)F
InChIKey: XWEAOTYPNQQVDL-JXNOJSNNSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Cavia porcellus
UniProt: P41144
Assay: Displacement of [3H]-diprenorphine from guinea pig kappa opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 487.55, ALogP 2.16, HBA 5, HBD 3, PSA 107.1, QED 0.5
Compound (SMILES): CN(CCO)Cc1ccc(Nc2nccc(-c3cnc(N4CC[C@H](F)C4)c(C#N)c3)n2)cn1
InChIKey: JOPZYMVVPBJUBQ-SFHVURJKSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 448.51, ALogP 2.52, HBA 9, HBD 2, PSA 114.09, QED 0.54
Compound (SMILES): C#CCN1CCC(CN(CCOC)S(=O)(=O)c2ccc3ccccc3c2)CC1
InChIKey: GWYNEKZKKCPWHH-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P06276
Assay: Inhibition of human recombinant BuChe using butyrylthiocholine as substrate preincubated for 5 mins followed by substrate addition measured for 1 min by Ellman's method
Measurement: IC50 = 384 nM (pChEMBL 6.420000076293945)
Descriptors: MW 400.54, ALogP 2.82, HBA 4, HBD 0, PSA 49.85, QED 0.64
Compound (SMILES): CC1CC1COc1ccc(S(C)(=O)=O)cc1-c1cn(C)c(=O)c2[nH]ccc12
InChIKey: BYLLNEGCIQBVEM-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in 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) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 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). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 386.47, ALogP 2.97, HBA 5, HBD 1, PSA 81.16, QED 0.73
Compound (SMILES): CN(C)C(=O)N[C@H]1CC[C@H](CCN2CCN(c3nsc4ccccc34)CC2)CC1
InChIKey: KWNJJPLLFRQMRI-IYARVYRRSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Displacement of [3H]8-OH-DPAT from recombinant human 5-HT1A receptor expressed in HEK293 cell membranes after 60 mins by scintillation counting method
Measurement: Ki = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 415.61, ALogP 3.64, HBA 5, HBD 1, PSA 51.71, QED 0.81
Compound (SMILES): O=C(CBr)OCCn1c(=O)oc2cc3ncnc(Nc4ccc(NC(=O)N5CCCCC5)cc4)c3cc21
InChIKey: UUBGEOAHQOBCDH-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of His-tagged human HER2 (676 to 1255 residues) expressed in baculovirus after 1 hr by Z-lyte assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 569.42, ALogP 4.24, HBA 9, HBD 2, PSA 131.59, QED 0.25
Compound (SMILES): COc1c(CC(=O)O)cccc1-c1ccc(F)c2c1CN(C(=O)OCc1ccccc1)CC2
InChIKey: MLJQGBVUWPKEGD-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 449.48, ALogP 4.82, HBA 4, HBD 1, PSA 76.07, QED 0.58
Compound (SMILES): Cc1cc(SCc2nn(-c3cccc(C(F)(F)F)c3)nc2C)ccc1OCC(=O)O
InChIKey: BCVWBOLLORFZHW-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor alpha (SINGLE PROTEIN), Mus musculus
UniProt: P23204
Assay: Transactivation of yeast GAL4 DNA binding domain-tagged mouse PPARalpha expressed in HEK293T cells incubated for 20 hrs by luciferase reporter gene assay
Measurement: EC50 <= 10000 nM (pChEMBL 5)
Descriptors: MW 437.44, ALogP 4.66, HBA 6, HBD 1, PSA 77.24, QED 0.54
Compound (SMILES): CC(=O)N1CCC(C(=O)N2CC[C@](c3ccc(C(Oc4cccc5ccccc45)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: QCGFUUMLVJRVFJ-UMSFTDKQSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 196 nM (pChEMBL 6.710000038146973)
Descriptors: MW 750.73, ALogP 7.54, HBA 5, HBD 0, PSA 83.99, QED 0.14
Compound (SMILES): NCCOCCOc1ccc2nc(-c3cn(-c4ccc(O)c(F)c4)nn3)ccc2c1.O=C(O)C(F)(F)F
InChIKey: IBAPHOHCKTXOSS-UHFFFAOYSA-N
Target: Macrophage migration inhibitory factor (SINGLE PROTEIN), Homo sapiens
UniProt: P14174
Assay: Inhibition of recombinant human MIF tautomerase activity expressed in Escherichia coli assessed as borate-enol complex formation using 4-hydroxyphenyl pyruvic acid as substrate preincubated for 20 mins followed by substrate addition measured for 175 sec by fluorescence based analysis
Measurement: Ki = 144 nM (pChEMBL 6.840000152587891)
Descriptors: MW 409.42, ALogP 2.68, HBA 8, HBD 2, PSA 108.31, QED 0.43