lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(C)C(=O)[C@@H](NC(=O)c1ccc(C2CC2)c(OCC2CC2)n1)c1ccccc1
InChIKey: IDETYHWNJKLEFE-FQEVSTJZSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 393.49, ALogP 3.31, HBA 4, HBD 1, PSA 71.53, QED 0.75
Compound (SMILES): CC(C)(Nc1nnc(-c2ccc3[nH]cc(-c4cncc(C5CC5)n4)c3c2)o1)c1ccccc1
InChIKey: ZBCKBGNGRYQRDG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: 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; on
Measurement: IC50 = 302.1 nM (pChEMBL 6.519999980926514)
Descriptors: MW 436.52, ALogP 5.9, HBA 6, HBD 2, PSA 92.52, QED 0.34
Compound (SMILES): CNS1(O)C[C@@](C)(c2cc(NC(=O)c3ccc(Cl)cn3)ccc2F)NC(=N)C1(C)C
InChIKey: IRHBPBKUEXETAX-FQEVSTJZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 453.97, ALogP 4.11, HBA 5, HBD 5, PSA 110.13, QED 0.48
Compound (SMILES): C[C@@H](NC(=O)c1nn(-c2ccc(Cl)cc2Cl)c2c1CCCC/C2=C\c1ccc(F)cc1)c1ccccc1
InChIKey: HSWSMMPLNSVOKF-ACXJOVJBSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Inverse agonist activity at human cannabinoid receptor 2 expressed in HEK293 cells assessed as increase in forskolin-induced cAMP production preincubated with cells followed by forskolin stimulation measured after 30 mins in presence of D2-labeled cAMP and Eu3+-TBP-NHS cryptate by HTRF assay
Measurement: EC50 = 5590 nM (pChEMBL 5.25)
Descriptors: MW 534.46, ALogP 8.08, HBA 3, HBD 1, PSA 46.92, QED 0.26
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CC[C@H]2c2nc(-c3ccccc3Cl)no2)c1
InChIKey: GLQGXTZSKHQCMH-SFHVURJKSA-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 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 420.86, ALogP 3.87, HBA 7, HBD 0, PSA 89.94, QED 0.5
Compound (SMILES): COc1ccccc1S(=O)(=O)Nc1cccc2cccnc12
InChIKey: ZXLVEUPWPBFXJP-UHFFFAOYSA-N
Target: Lactoylglutathione lyase (SINGLE PROTEIN), Homo sapiens
UniProt: Q04760
Assay: Inhibition recombinant human N-terminal 6His-tagged GLO1 (2 to 184 residues) expressed in Escherichia coli using MG as substrate preincubated for 15 to 20 mins followed by substrate addition and measured every 1 min for 8 mins
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 314.37, ALogP 3.04, HBA 4, HBD 1, PSA 68.29, QED 0.8
Compound (SMILES): C#COc1ccc2nc3ccc(=O)cc-3oc2c1
InChIKey: HFKUUXRENJLWLQ-UHFFFAOYSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: In Vitro Competitive Radioligand Binding Assay: To determine the Ki values, fixed brain tissues prepared from 15-month-old Tg2576 mice (N=5) will be incubated for 30 min with 1 uM methoxy-X34 in the presence of a serial dilution of test compounds (10-9-10-6 M, 6 concentrations/compound). Tissue sections (3 sections per reaction) will be then mounted and coverslipped. Photographs of methoxy-X34 binding images will be taken using a Nikon fluorescent microscope. The fluorescent intensity on cerebral amyloid deposits and neuritic plaques will be separately quantified using Image J software.
Measurement: Ki = 4613 nM (pChEMBL 5.340000152587891)
Descriptors: MW 237.21, ALogP 2.26, HBA 4, HBD 0, PSA 52.33, QED 0.48
Compound (SMILES): CN1CCN(Cc2ccc(-c3cn4c5nc(ncc35)NCCCCCCC(O)CCCC4)cc2)CC1
InChIKey: VPDVHIDCAMWQER-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Inhibition of Tyro3 (unknown origin) by microfluidic capillary electrophoresis assay
Measurement: IC50 = 94 nM (pChEMBL 7.03000020980835)
Descriptors: MW 490.7, ALogP 4.75, HBA 7, HBD 2, PSA 69.45, QED 0.55
Compound (SMILES): CC(NCc1ccccc1)C(=O)Nc1ccc(Nc2nccc(-c3cnc(N4CC[C@H](F)C4)c(C#N)c3)n2)cn1
InChIKey: YSCOLOPYFYZWAA-BVHINDKJSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
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 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 537.6, ALogP 4.21, HBA 9, HBD 3, PSA 131.75, QED 0.29
Compound (SMILES): CN(C)c1ccc(S(=O)(=O)N[C@H]2CCN([C@H](Cc3cc4ccnc(N)c4cc3OC(F)(F)F)C(=O)O)C2=O)cn1
InChIKey: ZZCAKOUFQSCCCF-ZWKOTPCHSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
Assay: Inhibition Assay: The compounds according to the invention are tested in a range of concentrations (10pM to 10 µM final) in assay buffer (50 mM TRIS, 100 mM NaCl, 0.1 % BSA, pH 7,5) with 0.1% maximal final DMSO concentration and deposited in a proportion of 25 µl per well, on 25 µL enzyme (human coagulation factor Xa: Enzyme Research Laboratories HFXa, final concentration 0.003Ul/ml). The reactive were mixed, centrifuged and incubated 10 minutes at 37°C in a 96 well microtiter plate. The enzyme reaction was started with 50 µL substrate (S-2765, Biogenic ref 821413 in a final concentration of 62.5µM final). The time course of the reaction was monitored at 405 nm in a microtiter plate reader (Tecan M200) for 20 minutes at 37°C. The percentage of inhibition of enzymatic activity (expressed as maximum rate of cleavage of the substrate) is calculated with respect to the enzymatic activity in the absence of inhibitor.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 582.56, ALogP 1.75, HBA 9, HBD 3, PSA 168.05, QED 0.34
Compound (SMILES): O=C1NC(=S)S/C1=C\c1cc(Br)c(Cl)cc1Br
InChIKey: QMEXPPFZLBRMRS-WAPJZHGLSA-N
Target: CDGSH iron-sulfur domain-containing protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZ45
Assay: Displacement of [3H]rosiglitazone from recombinant human C-terminal His-tagged MitoNEET cytosolic domain (32 to 108 residues) expressed in Escherichia coli BL21 by Cheng-Prusoff analysis
Measurement: Ki = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 413.54, ALogP 4.35, HBA 3, HBD 1, PSA 29.1, QED 0.42
Compound (SMILES): Cn1c(=O)c2c(CC(=O)Nc3nc(-c4cccc(C(F)(F)F)c4)cs3)cccc2n(C)c1=O
InChIKey: XRWIKIYEWODHPQ-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 474.46, ALogP 3.56, HBA 7, HBD 1, PSA 85.99, QED 0.49
Compound (SMILES): Cc1ncc(NC(=O)c2cncc(N(C)C)c2)cc1-c1ccnc(N2CCOCC2)c1
InChIKey: NTPDQQPEZCMACA-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 1.449 nM (pChEMBL 8.84000015258789)
Descriptors: MW 418.5, ALogP 3, HBA 7, HBD 1, PSA 83.48, QED 0.68
Compound (SMILES): C[C@H](Nc1nc(N)nc(N)c1C#N)c1nc2cccc(Cl)c2c(=O)n1-c1cncc(C2CC2)c1
InChIKey: WVBXXFRDRUYJPZ-NSHDSACASA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of human full length PI3K p110delta/p85alpha using phosphatidylinositol 3,4,5-trisphosphate as substrate measured after 30 mins by TR-FRET assay
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 473.93, ALogP 3.31, HBA 10, HBD 3, PSA 161.42, QED 0.39
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCC4(CCNCC4)C3)n2)n1
InChIKey: SDHGIPREFAWIKS-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 13.7 nM (pChEMBL 7.860000133514404)
Descriptors: MW 426.53, ALogP 3.16, HBA 8, HBD 1, PSA 84.65, QED 0.54
Compound (SMILES): Cc1ccc(C(=O)Nc2sc(-c3ccncc3)nc2C(N)=O)cc1
InChIKey: HCRZWDGAZRWFKY-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: IKKε Kinase Assay: The kinase assay is performed either as 384-well Flashplate assay (for e.g. Topcount measurement). 1 nM IKKε, 800 nM biotinylated IκBα(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 = 5300 nM (pChEMBL 5.28000020980835)
Descriptors: MW 338.39, ALogP 2.86, HBA 5, HBD 2, PSA 97.97, QED 0.76
Compound (SMILES): Cc1n[nH]c2ccc(C(=O)N3CCC[C@H]3c3ccc(Cl)cc3)cc12
InChIKey: QJIOPOJQZXHHEW-SFHVURJKSA-N
Target: Cyclin-dependent kinase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: P49336
Assay: Inhibition of CDK8 in human 7dF3 cells preincubated for 2 hrs followed by beta-oestradiol addition measured after 24 hrs by luciferase reporter gene assay
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 339.83, ALogP 4.5, HBA 2, HBD 1, PSA 48.99, QED 0.75
Compound (SMILES): O=C(N[C@H]1CC[C@H](CCN2CCN(c3nsc4ccccc34)CC2)CC1)C1CC1
InChIKey: LBNGJZBDUWZGLH-UAPYVXQJSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]methyl-spiperone from recombinant human D3 receptor expressed in CHO cell membranes after 60 mins by scintillation counting method
Measurement: Ki = 0.21 nM (pChEMBL 9.680000305175781)
Descriptors: MW 412.6, ALogP 3.89, HBA 5, HBD 1, PSA 48.47, QED 0.78
Compound (SMILES): CC(C)Nc1ccc(CS(=O)(=O)N2CCCCC2C(=O)OCCCc2cccnc2)cc1
InChIKey: YMJPQAJUULWTTC-UHFFFAOYSA-N
Target: Outer membrane protein MIP (SINGLE PROTEIN), Legionella pneumophila
UniProt: Q70YI1
Assay: Inhibition of Mip PPIase activity in Legionella pneumophila
Measurement: IC50 = 44668.36 nM (pChEMBL 4.349999904632568)
Descriptors: MW 459.61, ALogP 3.76, HBA 6, HBD 1, PSA 88.6, QED 0.43
Compound (SMILES): NS(=O)(=O)c1ccc(SCc2ccc(C(F)(F)F)cc2)s1
InChIKey: XRXMPNGFKACJMA-UHFFFAOYSA-N
Target: Carbonic anhydrase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00915
Assay: Inhibition of human recombinant cytosolic carbonic anhydrase 1 preincubated for 10 mins prior to testing by stopped-flow CO2 hydration assay
Measurement: Ki = 4250 nM (pChEMBL 5.369999885559082)
Descriptors: MW 353.41, ALogP 3.71, HBA 4, HBD 1, PSA 60.16, QED 0.85
Compound (SMILES): Cc1cccc(C)c1N1C[C@](C(=O)Nc2cc(C(F)(F)F)cc(C(F)(F)F)c2)(C(C)C)CC1=O
InChIKey: RQNXFRJAXYBPFE-QFIPXVFZSA-N
Target: Chemerin-like receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99788
Assay: Displacement of [125I]-chemerin from ChemR23 (unknown origin) transfected in mouse BaF3 cells after 3 hrs by scintillation counting method
Measurement: IC50 <= 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 486.46, ALogP 6.36, HBA 2, HBD 1, PSA 49.41, QED 0.51
Compound (SMILES): COC(=O)c1cnc(N2CCc3nc(SC)nc(Nc4ccc(C(F)(F)F)cc4)c3CC2)c(Cl)c1
InChIKey: SNSPFVJWZAGRBI-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O35433
Assay: Functional Assay: The assay was performed as described below, using HEK293 cells transfected with rat TRPV1 (rTRPV1/HEK293). These cells had a geneticin selection marker and were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, penicillin/streptomycin (50 units/mL), and 500 μg/mL geneticin in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 > 6670 nM (pChEMBL 5.176000118255615)
Descriptors: MW 523.97, ALogP 5.4, HBA 8, HBD 1, PSA 80.24, QED 0.27
Compound (SMILES): COc1cccnc1N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CC1
InChIKey: WAYVPUXWZPVQRA-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 = 559 nM (pChEMBL 6.25)
Descriptors: MW 449.49, ALogP 3.18, HBA 8, HBD 2, PSA 122.09, QED 0.57
Compound (SMILES): COC(=O)C(C)(C)Nc1nc2c(-c3cc4c([nH]3)CCNC4=O)cccc2nc1C
InChIKey: ZDHNAPJCCZPPCX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): 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 384-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 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 54.3 nM (pChEMBL 7.260000228881836)
Descriptors: MW 393.45, ALogP 2.58, HBA 6, HBD 3, PSA 109, QED 0.59
Compound (SMILES): CC1(C)CCCN1c1cnc2cccc(-c3cc4c([nH]3)CCNC4=O)c2n1
InChIKey: JYNVODGAIHNSGN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): 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 384-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 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 361.45, ALogP 3.29, HBA 4, HBD 2, PSA 73.91, QED 0.73
Compound (SMILES): COc1cc(OC(C)C)cc(N(CC2CC2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: XIUDTKCWPZRTTG-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 443.55, ALogP 5.37, HBA 7, HBD 0, PSA 65.3, QED 0.36
Compound (SMILES): CNC(=O)N1CC2(C1)C(=N)N[C@](C)(c1cc(NC(=O)c3ccc(OC)cn3)ccc1F)CS2(=O)=O
InChIKey: PXSZYCDQHYLPTG-NRFANRHFSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3x the desired final concentration in 1Ã¿BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1xBACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 uM for 4 uM for autoBACE-2) prepared in 1xBACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 us delay on a RUBYstar HTRF plate reader (BMG Labtechnologies). Raw RFU data is normalized to maximum (1.0 nM BACE/DMSO) and minimum (no enzyme/DMSO) RFU values. IC50s are determined by nonlinear regression analysis (sigmoidal dose response, variable slope) of percent inhibition data with minimum and maximum values set to 0 and 100 percent respectively. Similar IC50s are obtained when using raw RFU data. The Ki values are calculated from the IC50 using the Cheng-Prusoff equation.
Measurement: Ki = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 504.54, ALogP 1.09, HBA 7, HBD 4, PSA 153.58, QED 0.49
Compound (SMILES): Cn1ccc2cnc(-c3cccc(OCC(O)CN4CCc5ccccc5C4)c3)nc21
InChIKey: NNEHUZIKNKWOGK-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 = 101 nM (pChEMBL 7)
Descriptors: MW 414.51, ALogP 3.43, HBA 6, HBD 1, PSA 63.41, QED 0.52
Compound (SMILES): Cc1cccc(Cl)c1Nc1nc2cc(C(=O)N[C@H]3CC[C@H](C(F)(F)F)CC3)c3c(c2[nH]1)OCCO3
InChIKey: UHGUYSAHGCWJSP-HDJSIYSDSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition of human recombinant mPGES-1 expressed in CHO cells assessed as reduction in conversion of PGH2 to PGE2 incubated for 10 mins followed by substrate addition measured after 1 min in presence of GSH
Measurement: IC50 = 18.66 nM (pChEMBL 7.730000019073486)
Descriptors: MW 508.93, ALogP 5.89, HBA 5, HBD 3, PSA 88.27, QED 0.41
Compound (SMILES): CCC[C@H](Nc1nc(N)nc(N)c1C#N)c1nc2cccc(Cl)c2c(=O)n1-c1ccc(N)nc1
InChIKey: BSJOHMALVPGZIA-HNNXBMFYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Time-Resolved Fluorescence Resonance Energy transfer (TR-FRET) Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoforms: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 10lps delay and 500 μs read window).
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 476.93, ALogP 2.8, HBA 11, HBD 4, PSA 187.44, QED 0.32
Compound (SMILES): COc1n[nH]c2cc(NC(=O)N[C@@H](c3ccccc3)[C@@H](O)C(C)C)ncc12
InChIKey: ZLVGVXSUBMGOAD-IRXDYDNUSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 58.5 nM (pChEMBL 7.230000019073486)
Descriptors: MW 369.43, ALogP 2.85, HBA 5, HBD 4, PSA 112.16, QED 0.53
Compound (SMILES): Cn1cc(-c2nnc(SCCCN3CC[C@]4(C[C@@H]4c4ccc(C(F)(F)F)cc4)C3)n2C)cn1
InChIKey: KIDLIVLNXRZSJP-KNQAVFIVSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Displacement of [3H]-spiperone from human dopamine D2 receptor expressed in CHO-K1 cell membranes coexpressing Galpha16 after 120 mins by liquid scintillation counting
Measurement: Ki = 537.03 nM (pChEMBL 6.269999980926514)
Descriptors: MW 476.57, ALogP 4.6, HBA 7, HBD 0, PSA 51.77, QED 0.37
Compound (SMILES): COCCOCCOCCOCCOCCOCCO[C@H]1C=CC2C3Cc4ccc(OC)c5c4[C@@]2(CCN3C)C1O5
InChIKey: IKEAYGVEBHZCFT-LXMWPHLLSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.
Measurement: EC50 = 97381 nM (pChEMBL 4.010000228881836)
Descriptors: MW 577.72, ALogP 2.25, HBA 10, HBD 0, PSA 86.31, QED 0.17
Compound (SMILES): CCC(CC)(NC(=O)c1ccc(C(F)(F)F)c(OCC2CC2)n1)C(=O)OC
InChIKey: QDFPOVRGHXOWDO-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.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 388.39, ALogP 3.35, HBA 5, HBD 1, PSA 77.52, QED 0.69
Compound (SMILES): C=CS(=O)(=O)Nc1cccc(Cc2nn(C(C)(C)C)c3ncnc(N)c23)c1
InChIKey: GDBDFXMMJSKVKY-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: In vitro Kinase Assay: In vitro kinase assays for c-Src variants were performed in 50 mM Tris (pH 8.0), 10 mM MgCl2 and 1 mg/mL BSA. When obtaining kinetic parameters (kcat, Km) kinase and peptide substrate (IYGEFKKK) (SEQ ID NO:51) concentrations were 2 nM and 500 μM, respectively, while ATP concentrations ranged from 2000-0.655 μM. Addition of nonradioactive ATP supplemented with 32P ATP (3,000 Ci/mmol, NEN) was used to initiate kinase reactions. Time points were selected such that product formation never exceeded 10%. Reactions were quenched by spotting 3 μL quantities onto phosphocellulose sheets (P81, Whatman). Afterwards, the sheets were washed 3×5 minutes in 0.5% phosphoric acid and dried. Radioactivity was measured by phosphorimaging and recorded on a Typhoon fluorescence imager (Molecular Dynamics).
Measurement: IC50 = 618 nM (pChEMBL 6.210000038146973)
Descriptors: MW 386.48, ALogP 2.64, HBA 7, HBD 2, PSA 115.79, QED 0.7
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(N(Cc2cnn(C)c2)S(=O)(=O)CC)ccc1Oc1ccc(F)cc1F
InChIKey: XLPQNCAZXWYEED-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method C: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.047 mM to 0.78 nM or 5-fold serial dilution from Assay method A. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 17.3 nM (pChEMBL 7.760000228881836)
Descriptors: MW 558.61, ALogP 4.61, HBA 8, HBD 0, PSA 95.66, QED 0.28
Compound (SMILES): CN(c1cc2oc(-c3ccc(F)cc3)c(C(N)=O)c2cc1-c1ccc2c(n1)-c1cc3c(F)cccc3n1CO2)S(C)(=O)=O
InChIKey: KJHAQVYWJBEAIH-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: ELISA-Based Assay: Measurement of inhibition by compounds was performed using the HCV replicon system. Several different replicons encoding different HCV genotypes or mutations were used. In addition, potency measurements were made using different formats of the replicon assay, including different ways of measurements and different plating formats. See Jan M. Vrolijk et al., A replicons-based bioassay for the measurement of interferons in patients with chronic hepatitis C, 110 J. VIROLOGICAL METHODS 201 (2003); Steven S. Carroll et al., Inhibition of Hepatitis C Virus RNA Replication by 2'-Modified Nucleoside Analogs, 278(14) J. BIOLOGICAL CHEMISTRY 11979 (2003). However, the underlying principles are common to all of these determinations, and are outlined below.Stable neomycin phosphotransferase encoding replicons-harboring cell lines were used, so all cell lines were maintained under G418 selection prior to the assay. Potency was determined using a cell ELISA assay with an antibody to the replicons encoded NS3/4a protease. See Caterina Trozzi et al., In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor, 77(6) J. Virol. 3669 (2003).
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 600.6, ALogP 5.91, HBA 7, HBD 1, PSA 120.66, QED 0.26
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2ccccc2)C(=O)N[C@@H](Cc2ccc(Cl)c(Cl)c2)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: MZNWXAJMLAFTSG-AJQTZOPKSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: In Vitro Inhibition Assay: KLK1 inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human KLK1 (Callbiochem) was incubated at 37° C. with the fluorogenic substrate H-DVal-Leu-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 647.6, ALogP 5.24, HBA 5, HBD 4, PSA 122.55, QED 0.15
Compound (SMILES): CC(C)Oc1ccc(S(=O)(=O)N2CCN(c3cc(F)cc(OCC(=O)O)c3)CC2)cc1
InChIKey: WXRZPAHNFNTHBC-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q13258
Assay: Displacement of [3H]-PGD2 from PGD2 receptor in human platelet membranes after 2 hrs by micro beta scintillation counting analysis
Measurement: Ki = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 452.5, ALogP 2.59, HBA 6, HBD 1, PSA 96.38, QED 0.66
Compound (SMILES): COc1cccc(OC)c1C1CCCC(=O)N1Cc1cccc(OC(F)F)c1
InChIKey: NARHBIYXYMDLPF-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 = 78 nM (pChEMBL 7.110000133514404)
Descriptors: MW 391.41, ALogP 4.56, HBA 4, HBD 0, PSA 48, QED 0.69
Compound (SMILES): COc1cc(N2CCN(C(C)=O)CC2)ccc1Nc1ncc(F)c(Nc2ccc(N3CCN(S(C)(=O)=O)CC3)cc2OC)n1
InChIKey: JHCIRNYNOIUODQ-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 235 nM (pChEMBL 6.630000114440918)
Descriptors: MW 628.73, ALogP 2.87, HBA 11, HBD 2, PSA 132.47, QED 0.36
Compound (SMILES): CCNc1nc(-c2nc3cnc(N4CCN(C)CC4)cc3n2Cc2ccccc2)cn2c(C)nnc12
InChIKey: NSHYAULTAMQMBT-UHFFFAOYSA-N
Target: Histone H4 (SINGLE PROTEIN), Homo sapiens
UniProt: P62805
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 uM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 ul of the mix is added to the compound dilutions. Subsequently, 6 ul of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 ug/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer. Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 482.6, ALogP 3.08, HBA 10, HBD 1, PSA 92.3, QED 0.39