lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(O)CSc1nc2c(c(Nc3ccc(S(=O)(=O)C(F)(F)F)cc3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: WSSNJQAGBBKKII-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 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 607.6, ALogP 5.01, HBA 9, HBD 2, PSA 108.31, QED 0.22
Compound (SMILES): COc1cccc2sc(NC3=NCC4(CN5CCC4CC5)O3)nc12
InChIKey: CNFRLZVGNANAIK-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 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 344.44, ALogP 2.57, HBA 7, HBD 1, PSA 58.98, QED 0.91
Compound (SMILES): S=C([S-])N1CCCCC1.[Na+]
InChIKey: RPDVFFALGUJAPM-UHFFFAOYSA-M
Target: Carbonic anhydrase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P00918
Assay: Inhibition of human erythrocyte carbonic anhydrase 2 preincubated for 15 mins prior to testing by bromothymol blue-based stopped-flow CO2 hydration assay
Measurement: Ki = 30.1 nM (pChEMBL 7.519999980926514)
Descriptors: MW 161.29, ALogP 1.69, HBA 1, HBD 1, PSA 3.24, QED 0.43
Compound (SMILES): Cc1cc2ccc1[C@@H](C)COC(=O)Nc1cc(F)c(S(=O)(=O)C3CC3)c(c1)CN(C)C(=O)[C@@H]2Nc1cc2ccnc(N)c2cc1F
InChIKey: KIAGUBIJUWEWLA-RBSBEOHCSA-N
Target: Coagulation factor VII (SINGLE PROTEIN), Homo sapiens
UniProt: P08709
Assay: In Vitro Assay: Tissue kallikrein-1 (37° C) activity was determined in reactions containing 0.05 nM enzyme and 90 μM substrate (H-D-Val-Leu-Arg-AFC) in buffer (0.1M sodium phosphate pH 7.4, 0.2 M NaCl, 0.5% PEG 8000, and 1% DMSO). Assays were performed using 96-well microtiter plates (COSTAR 3600, CORNING, NY, USA) and a thermostatic temperature controlled plate reader (SPECTRAMAX® Gemini, Molecular Devices, Sunnyvale, Calif., USA). Fluorescence was monitored using 400 nm excitation and 505 nm emission wavelengths.
Measurement: Ki = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 649.72, ALogP 5.82, HBA 8, HBD 3, PSA 143.72, QED 0.25
Compound (SMILES): CC(F)(F)c1cc(NC(=O)Nc2ccc(Oc3ccnc(NC(=O)C4CC4)c3)cc2)ccc1Cl
InChIKey: YNRDEUFQOSIAMC-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 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 486.91, ALogP 6.63, HBA 4, HBD 3, PSA 92.35, QED 0.35
Compound (SMILES): Cc1[nH]c(C#N)c(CC2CCCCC2)c1-c1ccc(N2CCc3cccc(C(=O)Nc4nc5ccccc5s4)c3C2)nc1C(=O)NS(C)(=O)=O
InChIKey: IMBYTTKVQBEIBL-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: FL5.12 Cellular Assay: The efficacy of the compounds of Formula (I) can also be determined in cell-based killing assays using a variety of cell lines and mouse tumor models. For example, their activity on cell viability can be assessed on a panel of cultured tumorigenic and non-tumorigenic cell lines, as well as primary mouse or human cell populations. In one exemplary set of conditions, mouse FL5.12 cells transfected with Bcl-XL were cultured under standard conditions in RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3). For assaying the compound activity, the cells were exchanged into an IL-3-depleted deprivation media, which was identical to the growth media except for the absence of FBS and WEHI-3B conditional media, for 2 days. Then the cells were exchanged to 3% FBS assay media (RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3).
Measurement: EC50 = 97 nM (pChEMBL 7.010000228881836)
Descriptors: MW 707.88, ALogP 6.49, HBA 9, HBD 3, PSA 160.94, QED 0.16
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3cc(OC)c4nnn(Cc5ccc(F)cc5)c(=O)c4c3)cc12
InChIKey: YQSZHQDSWAVRIF-UHFFFAOYSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Non-nucleoside inhibition of NS5B polymerase in HCV genotype 1b by Taqman probe based RT-PCR analysis
Measurement: EC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 659.67, ALogP 4.96, HBA 9, HBD 1, PSA 136.63, QED 0.24
Compound (SMILES): CC(C)c1nc2c(c(NCCc3ccc(Cl)c(Cl)c3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: WTMRTCFXRGNTCE-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 = 940 nM (pChEMBL 6.03000020980835)
Descriptors: MW 524.42, ALogP 6.58, HBA 5, HBD 1, PSA 53.94, QED 0.39
Compound (SMILES): CC(C)Nc1nnc(-c2ccc3[nH]cc(-c4nccc(C(F)(F)F)n4)c3c2)o1
InChIKey: QPOKFUGFTPMYSP-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 = 25.25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 388.35, ALogP 4.51, HBA 6, HBD 2, PSA 92.52, QED 0.53
Compound (SMILES): CC(C)(C)OC(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3cccc(-c4ccccc4O)c3)n2)CC1
InChIKey: BFASBHGEATWKMQ-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 = 182 nM (pChEMBL 6.739999771118164)
Descriptors: MW 516.6, ALogP 4.74, HBA 8, HBD 3, PSA 146.5, QED 0.43
Compound (SMILES): O=C(O)CC1CCC(c2ccc(-c3ccc4nc(C(=O)Nc5ccccc5C(F)(F)F)cn4c3)cc2)CC1
InChIKey: DEHCFYLXTWYSDY-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q9Z2A7
Assay: Inhibition Assay: Â Assay buffer [20 mM HEPES (pH 7.4), 100 mM MgCl2 and 0.04% bovine serum albumin (BSA)] containing 500 uM of enzyme substrate (didecanoyl glycerol) and 7.5 uM radiolabeled acyl-CoA substrate ([14C]decanoyl-CoA) was added to each well of a phospholipid-coated radiometric assay plate. A small aliquot of cell lysate having human DGAT1 activity (5 ug/well) obtained in above, of human liver microsomes having human DGAT1 activity (5 ug/well), or of mouse liver microsomes having mouse DGAT1 activity (5 ug/well) was added to the assay plate to start the reaction, and the reaction was allowed to proceed for 60 minutes at 25° C. The reaction was terminated upon the addition of an equal volume (100 uL) of isopropanol. The plates were sealed, incubated overnight and counted the next morning on a liquid scintillation counter and luminometer.
Measurement: IC50 < 450 nM (pChEMBL 6.3470001220703125)
Descriptors: MW 521.54, ALogP 7.02, HBA 4, HBD 2, PSA 83.7, QED 0.29
Compound (SMILES): O=S(=O)(Nc1cscn1)c1ccc2c(c1)OCCN2c1ccc(C(F)(F)F)cc1Cl
InChIKey: AJOXHXYQBXGWPM-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 12600 nM (pChEMBL 4.900000095367432)
Descriptors: MW 475.9, ALogP 5.15, HBA 6, HBD 1, PSA 71.53, QED 0.56
Compound (SMILES): COC(=O)Nc1nc2cc(C3c4ccccc4C(=O)N3c3cccc(Br)c3)ccc2[nH]1
InChIKey: RXMIRTCFHZUDPO-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 477.32, ALogP 5.25, HBA 4, HBD 2, PSA 87.32, QED 0.42
Compound (SMILES): CNCCN(c1cc(OC)cc(OCCO)c1)c1ccc2ncc(-c3cnn(C)c3)nc2c1.Cl
InChIKey: XRWLZQITSKSQMM-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 448.53, ALogP 2.77, HBA 9, HBD 2, PSA 97.56, QED 0.38
Compound (SMILES): CCc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(C(F)(F)F)c3)cn2CC2CCN2)CC1
InChIKey: WHMIUKFQZDKPNG-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 = 280 nM (pChEMBL 6.550000190734863)
Descriptors: MW 503.55, ALogP 4.39, HBA 7, HBD 2, PSA 84.89, QED 0.49
Compound (SMILES): O=C(CCCCCCNC(=O)c1nc(-c2nc3c(s2)CCCC3)sc1C1CC1)NO
InChIKey: HEXCVAOOHDWROK-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: Enzyme Assay: Enzyme activity of HDAC1,3 is determined using the substrate Ac-Lys-Tyr-Lys(Ac)-AMC while the enzyme activity of HDAC6 is assayed using the substrate Boc-lys(Ac)-AMC. The reaction is carried out in flat-bottom, 96-well, or 384-well microplates. After the substrate was deacetylated by HDACs, the product AMC which was obtained from hydrolysis by trypsin can generate fluorescence signal. Measurement is taken in using an multilabe plate reader a 355 nm excitation filter and a 460 nm emission filter. The initial rate of fluorescence should accurately reflect the rate of product formation and enzyme activity. Sample processing: The sample was dissolved in DMSO and kept at a low temperature. DMSO in the final system is limited under a low concentration which won't affect the enzyme activity.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 448.61, ALogP 4.21, HBA 7, HBD 3, PSA 104.21, QED 0.29
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3C(C(=O)NC(C)C)O4)cc12
InChIKey: YQJWYQSWZYNOTE-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 = 4.3 nM (pChEMBL 8.369999885559082)
Descriptors: MW 699.74, ALogP 6.23, HBA 8, HBD 2, PSA 135.77, QED 0.2
Compound (SMILES): CC1c2nnn(-c3ncccn3)c2CCN1C(=O)c1ccc(Cl)cc1Cl
InChIKey: QCYUCLKQXJJFRJ-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
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 = 4.7 nM (pChEMBL 8.329999923706055)
Descriptors: MW 389.25, ALogP 3.12, HBA 6, HBD 0, PSA 76.8, QED 0.67
Compound (SMILES): Cc1ccc(F)c2nc([C@H](C)Nc3ncnc(N)c3C#Cc3ccc(F)cn3)n(-c3cccc(C#N)c3)c(=O)c12
InChIKey: YSECYDOLTBGCQD-KRWDZBQOSA-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: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 534.53, ALogP 4.18, HBA 9, HBD 2, PSA 135.4, QED 0.33
Compound (SMILES): COc1ccc(-n2ncc3cnc(-c4cncc(C)n4)cc32)nc1N1CCCNC1=O
InChIKey: YNACXPCSBHKFII-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 = 181 nM (pChEMBL 6.739999771118164)
Descriptors: MW 416.45, ALogP 2.51, HBA 8, HBD 1, PSA 110.95, QED 0.54
Compound (SMILES): CN(C)C(=O)c1ccc2c(c1)c(Sc1c(Cl)cccc1Cl)nn2-c1ccc(C(=O)O)cc1
InChIKey: KEZFSQIBHDEVNP-UHFFFAOYSA-N
Target: Retinoic acid receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P13631
Assay: Inverse agonist activity at GAL4 DBD-fused RARgamma LBD (unknown origin) expressed in human HG5LN cells after 18 hrs by luciferase reporter gene assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 486.38, ALogP 5.88, HBA 5, HBD 1, PSA 75.43, QED 0.38
Compound (SMILES): O=C(c1cc(-c2cc(Cl)ccc2C(=O)N2Cc3ccccc3C[C@H]2CN2CCOCC2)n2c1CCCC2)N(c1ccc(O)cc1)c1ccc2c(c1)CCN2
InChIKey: WLPFJTKZRREUJX-DHUJRADRSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
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 742.32, ALogP 7.7, HBA 7, HBD 2, PSA 90.28, QED 0.18
Compound (SMILES): CN1CCN(c2ccc(-c3ccc4[nH]c5nccc(/C=C/c6ccc(C(F)(F)F)cc6)c5c4c3)cc2)CC1
InChIKey: XDWXZZIZKQCZCZ-GORDUTHDSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: ELISA-Based In Vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad).
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 512.58, ALogP 7.32, HBA 3, HBD 1, PSA 35.16, QED 0.27
Compound (SMILES): N[C@]1(C(=O)O)[C@@H]2[C@@H](C(=O)O)[C@@H]2[C@H](F)[C@H]1OCc1ccc(Cl)c(Cl)c1
InChIKey: UDWSIQSDTWFRQU-ANYOLZDRSA-N
Target: Metabotropic glutamate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14832
Assay: Antagonist activity at recombinant human mGlu3 receptor expressed in hamster AV12 cells co-expressing human EAAT1 assessed as reduction of DCG-4-inhibited forskolin-stimulated cAMP formation after 20 mins by HTRF assay
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 378.18, ALogP 1.96, HBA 4, HBD 3, PSA 109.85, QED 0.72
Compound (SMILES): COc1cc2c(cc1OC)C(=O)/C(=C/c1cccc(O)c1)CO2
InChIKey: NCRSSNXOAYGHAY-WUXMJOGZSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Inhibition of human recombinant MAO-B using kynuramine as substrate measured after 30 mins by fluorescence assay
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 312.32, ALogP 3.07, HBA 5, HBD 1, PSA 64.99, QED 0.88
Compound (SMILES): COc1cccc(C#Cc2nc3c(N4CCOCC4)ccnn3c2-c2ccc(C(=O)O)cc2)n1
InChIKey: LEGAYXMQQHFMEH-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6Ã¿His sequence following the start Met to allow metal affinity purification of the recombinant protein. Cells were harvested and the phosphodiesterase protein was purified by metal chelate chromatography on Ni-sepharose 6FF.The affinity of the compounds of Formula (I) for phophodiesterases (PDE) was measured by a scintillation proximity assay (SPA). PDE Yttrium Silicate SPA beads allow PDE activity to be measured by direct binding of the primary phosphate groups of non-cyclic AMP or GMP to the beads via a complex iron chelation mechanism. The amount of bound tritiated product ([3H]-AMP) is measured by liquid scintillation counting.The compounds were dissolved and diluted in 100% DMSO in polystyrene plates to a concentration of 100-fold the final concentration in the assay. Human PDE10A enzyme solution (10 uL) was added.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 455.47, ALogP 2.73, HBA 8, HBD 1, PSA 102.08, QED 0.47
Compound (SMILES): O=C(Nc1cccc(Oc2ccc3c(/C=C/c4ccccc4)n[nH]c3c2)c1)c1ccccc1
InChIKey: JNEHFOFHMXRLFR-SAPNQHFASA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of human 50 residues truncated cytoplasmic domain of phosphorylated VEGFR2 E990V 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 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 431.5, ALogP 6.78, HBA 3, HBD 2, PSA 67.01, QED 0.31
Compound (SMILES): Cn1nc(-c2ccc(F)cc2)c(-c2ccc3nc(NC(=O)c4ccnc(F)c4)cn3n2)c1N1CCN(CCO)CC1
InChIKey: RYKONOKMNSTJAM-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 = 0.1 nM (pChEMBL 10)
Descriptors: MW 559.58, ALogP 2.84, HBA 10, HBD 2, PSA 116.71, QED 0.29
Compound (SMILES): Nc1ccccc1NC(=O)CCCCCNC(=O)c1ccc2nonc2c1
InChIKey: LAYURGNLJVCMOR-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 1153 nM (pChEMBL 5.940000057220459)
Descriptors: MW 367.41, ALogP 2.73, HBA 6, HBD 3, PSA 123.14, QED 0.42
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1NC1(C(F)(F)F)CC1
InChIKey: ZHCCGWZBLZBPDY-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 = 0.338 nM (pChEMBL 9.470000267028809)
Descriptors: MW 401.39, ALogP 3.73, HBA 4, HBD 3, PSA 82.7, QED 0.62
Compound (SMILES): NC1=N[C@](CF)(c2cc(NC(=O)c3ncc(C(F)(F)F)cc3Cl)ccc2F)C[C@@H](C(F)(F)F)O1
InChIKey: NPEULXQYTNVDQM-SUMWQHHRSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 40K cells/well in 96 well plates (Costar). The cells were cultivated for 24 hours at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. The test compounds were then added to cells in 10-point dose response concentrations with the starting concentration being either 100 uM or 10 uM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.1%. After 24 h of incubation with the test compounds the supernatant conditioned media was collected and the Aβ 40 levels were determined using a sandwich ELISA.
Measurement: IC50 = 88 nM (pChEMBL 7.059999942779541)
Descriptors: MW 516.78, ALogP 4.98, HBA 5, HBD 2, PSA 89.6, QED 0.56
Compound (SMILES): CN1CCC[C@H]1C(=O)N1CCC(CCOc2ccc(-c3nc(C#N)nc4c3ncn4CC3CCOC3)cc2C(F)(F)F)CC1
InChIKey: QEUOOSDCGFNGIZ-FHZUCYEKSA-N
Target: Cathepsin S (SINGLE PROTEIN), Mus musculus
UniProt: O70370
Assay: Inhibition Assay: First, spleen cells collected from C57BL/6 mice were seeded in a 96-well plate so as to be a 1× 105 cells/well, then, 5-fold dilution of 10 mM DMSO solution of the test compound was serially performed with RPMI 1640 medium (including 10% fetal bovine serum (FCS), 5×10^−5 M 2-mercaptoethanol, 50 IU/mL penicillin and 50 μg/mL streptomycin) nine times such that the final concentration becomes 0.026 nM to 10 μM, and the resultant products were added thereto (the final DMSO concentration was 0.1%). At the same time, LPS (Sigma L4005) was added to the wells such that the final concentration becomes 2 μg/mL, and culture was performed at 37° C. for 48 hours in 5% CO2. After culturing, the cells were stained at 4° C. for 20 minutes with PE-labeled streptavidin (BD BIOSCIENCE 554061) and biotin-labeled YAe antibody (EBIOSCIENCE 13-5741-85) together with FITC-labeled anti-mouse B220 antibody (BD BIOSCIENCE 553088), and an expression level (fluorescence intensity of YAe-bioti
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 611.67, ALogP 4.52, HBA 9, HBD 0, PSA 109.4, QED 0.37
Compound (SMILES): C=CCc1ccccc1NCC1=NCCN1
InChIKey: IYFFDNHPGFKOCO-UHFFFAOYSA-N
Target: Alpha-2B adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P18089
Assay: Agonist activity at human alpha2b-AR expressed in CHO cells assessed as rate of acidification after 240 mins by cytosensor microphysiometric analysis
Measurement: EC50 = 2511.89 nM (pChEMBL 5.599999904632568)
Descriptors: MW 215.3, ALogP 1.83, HBA 3, HBD 2, PSA 36.42, QED 0.74
Compound (SMILES): COc1cc(F)ccc1-c1ccnc(NC(=O)[C@@H]2CC[C@H](NC(C)=O)C2)c1
InChIKey: FUMJKSZLZJFCAQ-ZBFHGGJFSA-N
Target: CDK3/Cyclin E (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Inhibition of recombinant human CDK3/cyclin E expressed in baculovirus infected Sf9 insect cells after 80 mins in presence of [33P]ATP by microbeta scintillation counting analysis
Measurement: IC50 = 913 nM (pChEMBL 6.039999961853027)
Descriptors: MW 371.41, ALogP 3.14, HBA 4, HBD 2, PSA 80.32, QED 0.85
Compound (SMILES): CN[C@@H](C)C(=O)N[C@@H]1C(=O)N(Cc2cc(Cl)ccc2OC)c2ccccc2N(C(=O)CS(C)(=O)=O)[C@H]1C
InChIKey: PMTFHDHYPKXXAD-KXNSMUHISA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA ("Peptide A") was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA as the substrate for the TR-FRET assay. Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 mM incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 > 54800 nM (pChEMBL 4.261000156402588)
Descriptors: MW 551.07, ALogP 1.75, HBA 7, HBD 2, PSA 125.12, QED 0.51
Compound (SMILES): Cc1ccc(S(=O)(=O)NC(O)C(=O)c2cccs2)cc1
InChIKey: QOCJWEAHGGNLGM-UHFFFAOYSA-N
Target: Aldehyde dehydrogenase, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P05091
Assay: Inhibiton Assay: IC50 values were determined for CB29 and its analogs using propionaldehyde as the substrate for ALDH1A1 and ALDH2 or benzaldehyde as the substrate for ALDH3A1. The assays were performed on a Beckman DU-640 spectrophotometer at various concentrations of inhibitors ranging from 50 nM to 250 μM following a 1 minute pre-incubation. There was no pre-incubation time-dependence to the inhibition. All reactions were initiated by the addition of the aldehyde substrate. The inhibition curves were fit to the Logistic four parameter IC50 equation using the SigmaPlot (v11, StatSys). We characterized the mode of inhibition using steady-state kinetics through co-variation of inhibitor and substrate concentrations. The steady state kinetic measurements were performed in 100 mM Na2HPO4 buffer, pH 7.5. The reaction mixture contained 10 nM ALDH3A1, varied benzaldehyde (50 μM-800 μM; fixed NADP+, 1.5 mM) and varied inhibitor concentrations. In all cases−including the control reactions lacking inhibitors, the final reaction mixture contained 2% (v/v) DMSO. The reaction was initiated by addition of substrate and the initial rate of product formation was determined on a Beckman DU-640.
Measurement: IC50 = 2200 nM (pChEMBL 5.659999847412109)
Descriptors: MW 311.38, ALogP 1.54, HBA 5, HBD 2, PSA 83.47, QED 0.65
Compound (SMILES): C[C@@H]1CN(Cc2ccccc2)c2nnccc2[C@H](C)N1C(=O)NCc1ccccc1
InChIKey: HWAVFSHXEYWHQR-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 = 695 nM (pChEMBL 6.159999847412109)
Descriptors: MW 401.51, ALogP 4.16, HBA 4, HBD 1, PSA 61.36, QED 0.71
Compound (SMILES): N#Cc1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1F
InChIKey: NCOLKQKPKSFUQG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 375.41, ALogP 3, HBA 6, HBD 1, PSA 85.9, QED 0.59
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1-c1cnccc1Cl
InChIKey: ZWDQRWWMKAKDAQ-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 = 65.6 nM (pChEMBL 7.179999828338623)
Descriptors: MW 389.85, ALogP 3.93, HBA 4, HBD 2, PSA 83.56, QED 0.54