lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C=CCNS(=O)(=O)c1ccccc1Nc1nc(Nc2ccc(N3CCOCC3)cc2OC)nc2c1C1(CC1)CN2
InChIKey: DJMRSFSHQCMTDU-UHFFFAOYSA-N
Target: Protein-tyrosine kinase 2-beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q14289
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 563.68, ALogP 3.73, HBA 10, HBD 4, PSA 129.74, QED 0.27
Compound (SMILES): CC1(O)CN(C(=O)N2CC=C(c3cc4c(-c5cccc(N6CCOc7cc(C8CC8)cc(F)c7C6=O)c5CO)ccnc4[nH]3)CC2)C1
InChIKey: OJNINYKJMHYTKF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of recombinant human full length BTK preincubated for 10 mins followed by Tk-biotin peptide substrate addition after 90 mins by TR-FRET assay
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 637.71, ALogP 5.05, HBA 6, HBD 3, PSA 122.23, QED 0.28
Compound (SMILES): COc1cc(C(=O)NC(Cc2ccccc2)C(C)=O)ccc1-c1cn[nH]c1
InChIKey: PILIWAPJQLCXKO-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
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 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 363.42, ALogP 3.02, HBA 4, HBD 2, PSA 84.08, QED 0.68
Compound (SMILES): COc1cccc(OC)c1C1CC(F)(F)C(=O)N1Cc1cccc(-c2ccccc2)c1
InChIKey: IQQJHNDTIRISLY-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements: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 g/ml G418, 100 U/ml penicillin, 100 g/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. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 81 nM (pChEMBL 7.090000152587891)
Descriptors: MW 423.46, ALogP 5.48, HBA 3, HBD 0, PSA 38.77, QED 0.53
Compound (SMILES): CNC(=O)CCC#Cc1cn2c(-c3ccc(C(=O)NC4CC4)cc3)cnc2c(NCC(C)C)n1
InChIKey: MMDPYNFJFGHILL-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Mps-1 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 ul 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 ul of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 uM=> final conc. in the 5 ul assay volume is 10 uM) and peptide substrate (1.67 uM=> final conc. in the 5 ul assay volume is 1 uM) 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 ul assay volume). The reaction was stopped by the addition of 3 ul 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, PerkinElmer LAS, Rodgau-Jugesheim, 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-Jugesheim, Germany).
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 458.57, ALogP 3.23, HBA 6, HBD 3, PSA 100.42, QED 0.45
Compound (SMILES): COC(=O)NC(C(=O)N1CCC[C@H]1c1ncc(-c2ccc(-c3cc(F)c(-c4ccc5nc([C@@H]6CCCN6C(=O)[C@@H](NC(=O)OC)C(C)C)[nH]c(=O)c5c4)c4c3CC3(CCCC3)C4)nc2)[nH]1)C(C)C
InChIKey: WJVDLCZIVOJHQZ-UIVNZDFFSA-N
Target: Nonstructural protein 5A (SINGLE PROTEIN), Hepatitis C virus
UniProt: Q5L478
Assay: Luciferase Assay: The Huh-7 cells transfected with HCV replicons system were seeded into 96-well plates (8,000 cells in 125 μL/well) respectively; each test compound was diluted to desired concentration using 5-fold serial dilutions protocol, 10 doses in duplicate, and added to wells with POD™ 810 Plate Assembler. The plates were incubated in a CO2 incubator for 72 hours; after that, 40 μL of Luciferase assay substrate (Promega Bright-Glo) was added to each well, and detected by a chemiluminescence detection system (Topcount Microplate Scintillation and Luminescence Counter) 5 minutes later; the EC50 (half-maximal effective concentration, concentration for 50% of maximal effect) values of test compounds were analyzed by GraphPad Prism software, respectively. In this paper, experiments were repeated twice and set the holes without compounds as negative control.
Measurement: EC50 = 0.022 nM (pChEMBL 10.65999984741211)
Descriptors: MW 930.09, ALogP 7.93, HBA 10, HBD 4, PSA 204.6, QED 0.1
Compound (SMILES): Nc1ncc(-c2ccc(CN3CCOCC3)s2)cc1C(=O)Nc1ccncc1
InChIKey: BXMGOFSMDOETKZ-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 = 450 nM (pChEMBL 6.349999904632568)
Descriptors: MW 395.49, ALogP 2.87, HBA 7, HBD 2, PSA 93.37, QED 0.69
Compound (SMILES): Cc1c(C(=O)N2Cc3nnn(-c4cc[nH]n4)c3CC2C)cccc1C(F)(F)F
InChIKey: DFBQVWUVWWXOKB-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 = 10.2 nM (pChEMBL 7.989999771118164)
Descriptors: MW 390.37, ALogP 2.9, HBA 5, HBD 1, PSA 79.7, QED 0.73
Compound (SMILES): CCC(C)Oc1cc(C(=O)Nc2ccc(O)cc2C)ncn1
InChIKey: PVQOFJVNOFWCTM-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: 35S-GTPgammaS Binding: Measurements of 35S-GTPgammaS Binding: Membranes (1 to 10 ug protein) prepared as described above, were incubated in 96-well Scintiplates (PerkinElmer) with test compounds diluted in DMSO, in 180 ul of 20 mM HEPES, pH 7.4, 10 mM MgCl2, 2 ug/well Saponin, 0.2% fatty acid free BSA (Assay buffer), 140 mM NaCl and 1.7 uM GDP. The assay was initiated with the addition of 20 ul of 1.5 nM [35S]-GTPgammaS (1100 Ci/mmol; GE Healthcare) in assay buffer. After 60 min incubation at 30° C. on a shaker, plates were centrifuged for 10 min at 2000 RPM. Supernatant was discarded and membrane bound radioactivity was measured on a PerkinElmer 1450 MicroBeta counter. Triplicate samples were averaged and expressed as % response relative to S1P activation in absence of compound (n=2).
Measurement: EC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 301.35, ALogP 2.92, HBA 5, HBD 2, PSA 84.34, QED 0.83
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COC/C=C/c4ccccc4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: NRWBLDJHRKXQPL-ADQUDHGXSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Binding Assay: Binding assay of certain compounds of this invention to the opioid receptor was determine using radioligand binding assay (screening and dose-displacement).
Measurement: Ki = 4.35 nM (pChEMBL 8.359999656677246)
Descriptors: MW 527.71, ALogP 5.65, HBA 5, HBD 0, PSA 40.16, QED 0.39
Compound (SMILES): C[C@@H]1CN(Cc2ccccc2)c2cc(S(C)(=O)=O)ccc2[C@H](C)N1C(=O)Nc1cccnc1
InChIKey: BORYATIHUWWRHT-MOPGFXCFSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Channel blocking activity at recombinant human CaV 3.2 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 > 8000 nM (pChEMBL 5.0970001220703125)
Descriptors: MW 464.59, ALogP 4.49, HBA 5, HBD 1, PSA 82.61, QED 0.62
Compound (SMILES): COC(=O)[C@H](CO)NC(=O)[C@H](CCCCNC1C2CC3CC(C2)CC1C3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)NC(=O)CNC(=O)c1ccccc1
InChIKey: UCRSNRWWVDEHLO-JXGGDCLASA-N
Target: Chromobox protein homolog 7 (SINGLE PROTEIN), Homo sapiens
UniProt: O95931
Assay: Antagonist activity at recombinant human C-terminal His-tagged CBX7 chromodomain (8 to 62 residues) expressed in Escherichia coli Rosetta BL21(DE3)pLysS using biotin-labeled H3K9me3 measured after 30 mins by AlphaScreen assay
Measurement: IC50 > 60000 nM (pChEMBL 4.2220001220703125)
Descriptors: MW 874.09, ALogP 1.9, HBA 10, HBD 8, PSA 233.16, QED 0.06
Compound (SMILES): O=S(=O)(Nc1ccc(F)c(Nc2ncccc2-c2ncnc3[nH]cnc23)c1F)c1ccsc1
InChIKey: YSBVTNXKLLPITL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Cell Activity Inhibition Assay: To check the B-Raf cell activity inhibitory capability of the compounds of the present invention, the following experiments were conducted in A375P cell line (ATCC). The A375P cell line (ATCC) was derived from a patient with human melanoma, and had V600E mutant of B-Raf gene. A375 cell was maintained in DMEM supplemented with 10% fetal bovine serum, glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 ug/mL). The cell was maintained at 37° C., and in 5% CO2 and 100% humidity. To conduct the growth inhibition test, the cell was plated by using a white 384-well microplate, with 1000 cells/20 Î¼l plated per 1 well. After 24 hours, 5 ul of 5x stock solution of drug was added. All the drugs were initially prepared with 200x stock solution in DMSO, and the final concentration of DMSO was 0.5%. The cell was incubated at 37° C. for 72 hours. For MTT assay, 'CellTiter 96 (R) Non-Radioactive Cell Proliferation Assay (G4100)â¿¿ kit by Promega Corp. was used.
Measurement: EC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 485.5, ALogP 4.3, HBA 8, HBD 3, PSA 125.55, QED 0.33
Compound (SMILES): COc1cc(C(=O)N2CC[C@H]2c2noc(-c3ccccc3OC(F)(F)F)n2)c(-n2nccn2)cc1Cl
InChIKey: CXSOMSKKVALFEI-HNNXBMFYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Intracellular Calcium 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 μg/mL G418, 100 U/mL penicillin, 100 μg/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. On the day of the assay, 50 μL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 μM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μL/well, incubated for 120 min and finally 10 μL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 520.86, ALogP 4.47, HBA 9, HBD 0, PSA 108.4, QED 0.37
Compound (SMILES): CCN(c1ccccc1)c1c(Cl)nc2ccc(C(O)(c3ccc(Cl)cc3)c3cncn3C)cc2c1Cl
InChIKey: QKBAPBUOFNHEOM-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: RORγt Reporter Assay: The reporter assay was performed by transiently transfecting HEK293T cells with 5 μg of pBIND-RORγt LBD or pBIND-RORγt LBD-AF2 and 5 μg pGL4.31 (Promega Cat no. C935A) using Fugene 6 (Invitrogen Cat no. E2691) at a 1:6 ratio of DNA: Fugene 6 in a T-75 flask in which cells were at least 80% confluent. Twenty four hours after bulk transfection, cells were plated into 96-well plates at 50,000 cells/well in phenol-red free DMEM containing 5% Lipid Reduced FCS and Pen/Strep. Six hours after plating, cells were treated with compounds for 24 hours. Media was removed and cells were lysed with 50 μL 1x Glo Lysis Buffer (Promega). Dual Glo Luciferase Reagent (50 μL/well) was then added and firefly luciferase luminescence was read on an Envision after a ten minute incubation. Finally, Stop and Glo reagent (50 μL/well) was added and renilla luciferase luminescence was read on an Envision after a ten minute incubation.
Measurement: IC50 = 490 nM (pChEMBL 6.309999942779541)
Descriptors: MW 537.88, ALogP 7.37, HBA 5, HBD 1, PSA 54.18, QED 0.23
Compound (SMILES): c1nc2cc(NC3=NCC4(CN5CCC4CC5)O3)ccc2s1
InChIKey: GIXVQDNIPFHYIN-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: Antagonist activity at human 5-HT3A expressed in HEK293 cells assessed as reduction in acetylcholine-induced activity preincubated for 30 mins followed by addition of acetylcholine measured for 2 mins by Fluo-4AM dye based FLIPR assay
Measurement: IC50 = 41000 nM (pChEMBL 4.389999866485596)
Descriptors: MW 314.41, ALogP 2.56, HBA 6, HBD 1, PSA 49.75, QED 0.88
Compound (SMILES): O=C(O)c1cccc(NCCOc2ccc3cc2COC/C=C/COCc2cccc(c2)-c2ccnc(n2)N3)c1
InChIKey: RLNHDPUVCRDNQW-OWOJBTEDSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Inhibition of full length human recombinant N-terminal GST-tagged HDAC6 (1 to 1215 residues) expressed in sf9 cells preincubated with enzyme followed by fluorogenic Arg-His-Lys-Lys(Ac)-AMC substrate addition measured after 2 hrs by fluorescence assay
Measurement: IC50 = 53000 nM (pChEMBL 4.28000020980835)
Descriptors: MW 538.6, ALogP 5.68, HBA 8, HBD 3, PSA 114.83, QED 0.21
Compound (SMILES): COc1cc2c(cc1OC)CN(CC(O)COc1cccc(CNC3CCCC3)c1)CC2
InChIKey: DWUWWPGCKJJQRU-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of human full length FLAG-tagged PRMT5/human His6-tagged MEP50 expressed in baculovirus-infected Sf9 cells assessed as reduction in tritium incorporation into peptide substrate using human Histone H4 (1 to 15 residues) and [3H]SAM as substrate preincubated with enzyme and H4 for 30 mins followed by [3H]SAM addition and measured after 120 mins by radioactive flashplate assay
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 440.58, ALogP 3.53, HBA 6, HBD 2, PSA 63.19, QED 0.59
Compound (SMILES): CN(C)C(=O)CN1CCC(CN2CCN(C(=O)Cn3cc(NC(=O)c4cnn5cccnc45)c(-c4cc(Cl)ccc4OC(F)F)n3)CC2)CC1
InChIKey: RRMJKQNETHUSEU-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 = 2.51 nM (pChEMBL 8.600000381469727)
Descriptors: MW 713.19, ALogP 3.04, HBA 11, HBD 1, PSA 133.44, QED 0.25
Compound (SMILES): CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)N(C)Cc1ccc(C(=O)Oc2ccc(C(=N)N)cc2F)s1
InChIKey: HLHMUJJGITWCBH-JXFKEZNVSA-N
Target: Serine protease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P07477
Assay: Inhibition Assay: Using a 96 well plate (#3915, Costar), a test compound (25 μL), 400 mM Tris-HCl buffer (pH 8.0, 25 μL) and 0.5 mg/mL fluorescence enzyme substrate (Gly-Asp-Asp-Asp-Asp-Lys-β-Naphtylamide, 25 μL) were mixed, and recombinant human enteropeptidase (R&D Systems, Inc., 25 μL) was added. Using a fluorescence plate reader fmax (Molecular Devices, Inc.), the reaction rate was measured from the time-course changes at excitation wavelength 320 nm and fluorescence wavelength 405 nm. The Ki value was calculated from the concentration of the test compound, reciprocal of reaction rate, and Km value of the enzyme substrate, and by using Dixon plot.
Measurement: Ki = 0.38 nM (pChEMBL 9.420000076293945)
Descriptors: MW 536.58, ALogP 2.28, HBA 8, HBD 5, PSA 183.11, QED 0.11
Compound (SMILES): Cn1cc(-c2ccc3nc(NC(=O)C4CCOCC4)cn3n2)c(-c2ccc(F)cc2)n1
InChIKey: OVVDOOKBJRZOAR-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 5.4 nM (pChEMBL 8.270000457763672)
Descriptors: MW 420.45, ALogP 3.3, HBA 7, HBD 1, PSA 86.34, QED 0.55
Compound (SMILES): CCn1nc(C)cc1C(=O)Nc1cc(Nc2ccc3c(/C=C/c4ccccn4)n[nH]c3c2)c(F)cc1C
InChIKey: MGMPLTIIDFSKQV-PKNBQFBNSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Inhibition of human phosphorylated FGFR1 L457V/C488A/C584S mutant expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using poly(E4Y1) as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 495.56, ALogP 6.1, HBA 6, HBD 3, PSA 100.52, QED 0.25
Compound (SMILES): CN(C)C1(c2ccccc2)CCC(c2cccs2)(N(C)C)CC1
InChIKey: KAHACFHOOARSEP-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Binding Assay: The affinity to the human μ-opiate receptor was determined in a homogeneous preparation in microtiter plates. For this, dilution series of the respective compound to be tested were incubated for 90 minutes at room temperature with a receptor membrane preparation (15-40 mg of protein per 250 μl of incubation batch) of CHO-K 1 cells, which express the human μ-opiate receptor (RB-HOM receptor membrane preparation of NEN, Zaventem, Belgium), in the presence of 1 nmol/l of the radioactive ligand [3H'-naloxone (NET719, NEN, Zaventem, Belgium) and of 1 mg WGA-SPA beads (wheat germ agglutinin SPA beads from Amersham/Pharmacia, Freiburg, Germany) in a total volume of 250 μl. 50 mmol/l of tris-HCl supplemented by 0.05% by wt. of sodium azide and 0.06% by wt. of bovine serum albumin was used as incubation buffer. 25 μmol/l of naloxone were additionally added to determine the non-specific bond. After the ninety-minute incubation time had ended, the microtiter plates were centrifuged for 20 minutes at 1000 g and the radioactivity measured in a β-counter (Microbeta-Trilux, PerkinElmer Wallac, Freiburg, Germany).
Measurement: Ki = 445 nM (pChEMBL 6.349999904632568)
Descriptors: MW 328.53, ALogP 4.54, HBA 3, HBD 0, PSA 6.48, QED 0.81
Compound (SMILES): CC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(C)C)C(=O)O)[C@@H](C)O
InChIKey: GWCBAFZZIRQCKW-PKNHJFCNSA-N
Target: Kelch-like ECH-associated protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14145
Assay: Binding affinity to Keap1 (unknown origin) Kelch domain by ITC assay
Measurement: Kd = 46.5 nM (pChEMBL 7.329999923706055)
Descriptors: MW 1078.14
Compound (SMILES): O=C(Cc1ccc(SCc2ccccc2C(=O)O)cc1)OCc1ccc(C(F)(F)F)cc1
InChIKey: CLZRKELBHAVUBB-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P37231
Assay: Reporter Gene Assay: Transactivation Assay Expression vectors were prepared by inserting the ligand binding domain cDNA (complementary DNA) of human PPARalpha (amino acid 168-468) and human PPARgamma (amino acid 205-505), 3′ to and in frame with, the yeast GAL4 transcription factor DNA binding domain and the nuclear localization signal from the T-antigen of Polyoma Virus into the mammalian expression vector pSGS (Stratagene). The resulting expression vectors pSGGAL-PPARalpha and pSGGAL-PPARgamma were used in co-transfection experiments together with a modified pGL3 promoter plasmid (Promega) containing five copies of the UAS GAL4 recognition site. 2.5 μg pSGGAL-PPARalpha or pSGGAL-PPARgamma were mixed with 25 μg pGL3p 1×UAS and 22.5 μg pBluescript (Stratagene) in 0.95 mL ice cold PBS containing between 9-12 million U-2 OS (human osteosarcoma) cells. The cell/DNA mixture was incubated on ice for 5 minutes and then divided between two 0.4 cm cuvettes and electroporated at 960 μF, 2
Measurement: EC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 460.47, ALogP 5.98, HBA 4, HBD 1, PSA 63.6, QED 0.33
Compound (SMILES): Cc1cc(NC(=O)Nc2cc(C(C)(F)F)ccc2F)ccc1Oc1ccnc(NC(=O)C2CC2)c1
InChIKey: UPJMWOZAGHFAMT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition Assay: [Experimental method]: Use ADP-GLO™ assay kit from Promega to evaluate the inhibitory activity of compounds at B-Raf. [Instrument]: PerKinElmer's ENVISION plate reader. [Material]: Optiplate-384 well plate (PerKinElmer), kinase buffer (50 mM Hepes pH7.5, 1 mM EGTA, 2 mM DTT, 10 mM Mg2+, 0.05% BSA), B-Raf kinase (Millipore, catalogue #14-530-K), GST-MEK1 substrate (Carna, catalogue #07-141-10), Super pure ATP (Promega), ADP-GLO™ assay kit (Promega, catalogue # V9102), DMSO (Sigma, catalogue #34869), purified water (Millipore, type: Milli-Q). [Study conditions]: Mix B-Raf kinase (final concentration: 5 nM) and compound (DMSO final concentration: 0.25%) and preincubate at 30° C. for 20 min, then add ATP (final concentration: 6 μM) and reaction substrate (final concentration: 30 nM), React for 2 hrs at 30° C. After reaction, add ADP-GLO™ reagent, react for 40 min at room temperature, then add ADP-GLO™ detecting agent, react for 30 min at room temperature. After that, use ENVSION to measure the fluorescence.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 484.48, ALogP 6.43, HBA 4, HBD 3, PSA 92.35, QED 0.36
Compound (SMILES): CC(C)n1ccc2cc(N(C(=O)c3cc(-c4cc5c(cc4C(=O)N4Cc6ccccc6C[C@H]4CN4CCOCC4)OCO5)n4ccccc34)c3ccc(O)cc3)ccc21
InChIKey: XVFNJRDHJHJWOR-QNGWXLTQSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 787.92, ALogP 8.45, HBA 9, HBD 1, PSA 101.12, QED 0.17
Compound (SMILES): CN1CC2(CCN(c3cncc(N4CC(C)(C)C4)n3)CC2)CC1=O
InChIKey: SHSBDKQUNMMHAY-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 for 3 hrs by HTRF assay
Measurement: IC50 = 4460 nM (pChEMBL 5.349999904632568)
Descriptors: MW 329.45, ALogP 1.77, HBA 5, HBD 0, PSA 52.57, QED 0.83
Compound (SMILES): O=C(NC(c1ccccn1)C(F)(F)F)c1cc(OCC2CC2)c(C2CC2)cn1
InChIKey: RKWJRVCFEIUUOB-UHFFFAOYSA-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×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° C. for 30 min. Compounds were added to a final assay volume of 100 μl 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 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM 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 as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 391.39, ALogP 4.18, HBA 4, HBD 1, PSA 64.11, QED 0.77
Compound (SMILES): C[C@]12CCC(=O)C=C1C=C[C@@H]1[C@@H]2CC[C@]2(C)C(c3cncc(F)c3)=CC[C@@H]12
InChIKey: WSFZIYLNMPMOAE-NHFPKVKZSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human testicular microsomes using 17alpha-hydroxypregnenolone as substrate assessed as decrease in DHEA levels after 90 mins by ELISA
Measurement: IC50 <= 11 nM (pChEMBL 7.959000110626221)
Descriptors: MW 363.48, ALogP 5.52, HBA 2, HBD 0, PSA 29.96, QED 0.66
Compound (SMILES): COc1ccc(Br)c(CC2CCN(CCc3ccc(Cl)cc3)CC2)c1
InChIKey: UOKAAMJAKVREDW-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 = 1.34 nM (pChEMBL 8.869999885559082)
Descriptors: MW 422.79, ALogP 5.61, HBA 2, HBD 0, PSA 12.47, QED 0.6
Compound (SMILES): CCCc1nn(C)c2c(=O)[nH]c(-c3cc(N4CCC5(CCN(c6ncc(C(=O)NO)cn6)CC5)C4)ccc3OCC)nc12
InChIKey: FPSASUFGWQEDLR-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of full length recombinant human C-terminal His/Flag-tagged HDAC1 expressed in baculovirus infected Sf9 cells using fluorogenic HDAC substrate after 30 mins by fluorescence analysis
Measurement: IC50 = 443 nM (pChEMBL 6.349999904632568)
Descriptors: MW 587.69, ALogP 3.08, HBA 11, HBD 3, PSA 154.39, QED 0.21
Compound (SMILES): Cc1c(C(=O)Nc2cccc(C(F)(F)F)c2)cc(-c2ccccc2)n1CCCN1CCNC(=O)C1
InChIKey: DNWIDOSOXAGTSD-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 3: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) Frozen cells were thawed and subcultured with the medium (MEM-α, 10% FBS, 200 mmol/L Glutamine, 50 unit/mL penicillin, 50 μg/mL streptomycin, 1 mg/mL G418). (3) The 96-well plates which hTRPV4/CHO cells were seeded at densities of 2×10^4 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for 24 h was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 1 mol/L HEPES, 250 mmol/L probenecid, pH7.4). (5) Fluo-3, fluorescent dye for Ca influx assay was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour (final conc.; 5 μmmol/L Fluo-3). (6) The assay plate was washed with the assay buffer, and the buffer was remained at 30 μL/well. Then the assay plate was incubated for 10 min at 37° C. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the fluorescence analysis system FDSS 3000 (Hamamatsu Photonics). (8) 50 μL of 4α-PDD solution (concluding 0.1% Pluronic F-127) was applied to each well of assay plate and mixed. (9) The fluorescent intensity was measured by FDSS 3000 for 8 min from the point of time addition the compound solution, at Ex 480 nm, Em 540 nm wavelength.
Measurement: Ki = 196 nM (pChEMBL 6.710000038146973)
Descriptors: MW 484.52, ALogP 4.56, HBA 4, HBD 2, PSA 66.37, QED 0.52
Compound (SMILES): COc1cccc(OC)c1C1CCCC(=O)N1Cc1csc(Oc2ccccc2)n1
InChIKey: CKSSPOFRDLEYLY-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements: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 g/ml G418, 100 U/ml penicillin, 100 g/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. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 172 nM (pChEMBL 6.760000228881836)
Descriptors: MW 424.52, ALogP 5.21, HBA 6, HBD 0, PSA 60.89, QED 0.52
Compound (SMILES): CN1CCN(c2ccc(C(=O)Nc3n[nH]c4cc(OC/C=C/c5ccccc5)ccc34)cc2)CC1
InChIKey: QACWGMJPPZJZTM-VMPITWQZSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Assay: The assay has been performed using KIT cytoplasmic domain product and purified in house as GST fused protein. The KIT protein (4.5 microM) was pre activated with 300 microM ATP for 1 hour at 28*C in order to obtain a linear kinetic. Kinase buffer was composed of 50 mM HEPES pH 7.9 containing 5 mM MgCl2, 1 mM MnCl2, 10 mM DTT, 3 microM Na3VO4, and 0.2 mg/mL BSA. The KIT kinase assay was run with a final pre activated enzyme concentration of 4 nM, in the presence of 4.4 microM ATP (residual ATP from KIT pre activation step is negligible), 3.9 nM 33P-γ-ATP and 2.5 microM of substrate BioDB n*138 (Aminoacidic sequence: KWEEINGNNYVYIDPTQLPYDHKWEFPRNR- SEQ ID NO: 2).
Measurement: IC50 = 528 nM (pChEMBL 6.28000020980835)
Descriptors: MW 467.57, ALogP 4.66, HBA 5, HBD 2, PSA 73.49, QED 0.41
Compound (SMILES): CCCc1n[nH]c2c1C(c1ccc(SC)cc1)c1c(nc3c(c1N)CCCC3)O2
InChIKey: AQRLJJMSRUKJPW-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Inhibition of electric eel AChE using acetylthiocholine iodide as substrate incubated for 5 mins followed by substrate addition measured for 1 min by Ellman's method
Measurement: IC50 = 201 nM (pChEMBL 6.699999809265137)
Descriptors: MW 406.56, ALogP 5.23, HBA 5, HBD 2, PSA 76.82, QED 0.45
Compound (SMILES): CCOc1cc(F)ccc1[C@@H]1C[C@H](F)C(=O)N1Cc1ccc(OC(F)(F)F)cc1
InChIKey: XQFFUGYJZYHPFU-IRXDYDNUSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements: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 g/ml G418, 100 U/ml penicillin, 100 g/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. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 392 nM (pChEMBL 6.409999847412109)
Descriptors: MW 415.36, ALogP 4.93, HBA 3, HBD 0, PSA 38.77, QED 0.63
Compound (SMILES): Cn1cc(C(=O)N2CCC(CC#N)(n3cc(C(N)=O)c(Nc4ccnc(F)c4)n3)CC2)cn1
InChIKey: LKAJQLLNOOSKLG-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 = 63 nM (pChEMBL 7.199999809265137)
Descriptors: MW 451.47, ALogP 1.54, HBA 9, HBD 2, PSA 147.75, QED 0.54
Compound (SMILES): CSCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)CNC(=O)[C@H](CSCNC(C)=O)NC(=O)[C@H](CO)NC(=O)CN(C)C)C(C)C)C(N)=O
InChIKey: RKHYXBBBRBSHDU-IENTYHPUSA-N
Target: Neuromedin-B receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P24053
Assay: Displacement of [125I]-NMB from NMB receptor in rat C6 cells
Measurement: IC50 = 7.9 nM (pChEMBL 8.100000381469727)
Descriptors: MW 1400.65
Compound (SMILES): Cn1nc(-c2ccccc2Oc2ccccc2)c2cc[nH]c2c1=O
InChIKey: FNTQVTSIICHTJX-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 = 183 nM (pChEMBL 6.739999771118164)
Descriptors: MW 317.35, ALogP 3.72, HBA 4, HBD 1, PSA 59.91, QED 0.63
Compound (SMILES): CC(c1nnc(CNS(N)(=O)=O)o1)(c1nc2ccc(-c3ccccc3)cc2s1)S(=O)(=O)Cc1ccccc1
InChIKey: FRPQODFRMAEVIN-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Biological Assay: Endothelial lipase activity was measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 285 uL, of 1 mM DMPG in a 1:1 mixture of MeOH and CHCl3 with 15 uL, of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen and resuspended in 150 uL, of 50 mM HEPES pH 8.0 buffer containing 100 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at rt for 15 min and then was sonicated 3x4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.05 for the FRET substrate.The enzymatic assay was measured using white, opaque 96-well half area plates. Each well contained 60 uL, of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 2 ul of a DMSO solution containing compound of interest. Conditioned media obtained from HT-1080 cells.
Measurement: IC50 = 713 nM (pChEMBL 6.150000095367432)
Descriptors: MW 569.69, ALogP 3.52, HBA 9, HBD 2, PSA 158.14, QED 0.27
Compound (SMILES): C[C@]12CC[C@H](O)CC1=CC[C@@H]1[C@@H]2CC[C@]2(C)C(c3cncc(C(F)F)c3)=CC[C@@H]12
InChIKey: ISKOZWLGLDMBON-VUYFIVHNSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human H295R cells assessed as decrease in testosterone levels after 24 hrs by ELISA
Measurement: IC50 <= 15 nM (pChEMBL 7.823999881744385)
Descriptors: MW 399.53, ALogP 6.34, HBA 2, HBD 1, PSA 33.12, QED 0.59