lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#Cc1cccc(C(=O)OCCCCCc2ccccc2)n1
InChIKey: WHGQVCFQGOORRJ-UHFFFAOYSA-N
Target: N-acylethanolamine-hydrolyzing acid amidase (SINGLE PROTEIN), Homo sapiens
UniProt: Q02083
Assay: Inhibition Assay: In order to have an assay method more conducive to high-throughput screening than those published for measuring the NAE hydrolyzing activity of NAAA, we developed the fluorogenic PEA analog N-(4-methyl coumarin)palmitamide (PAMCA), which is hydrolyzed to fluorescent 7-amino-4-methyl coumarin (AMC) and palmitic acid. For three point concentration inhibition assays with hNAAA the following procedure is used. Purified activated NAAA (final concentration of 0.25 μg/mL) is incubated in assay buffer (100 mM citrate-phosphate buffer, pH 4.5, 3 mM DTT, 0.1% Triton X-100, 0.05% BSA, and 150 mM NaCl) made up to a total volume of 180 μL, followed by addition of the compound dissolved in 10 μL DMSO (along with DMSO neat for the control sample) with the final concentrations for each compound of 100, 10, and 1 μM, in triplicate on a 96 well plate. These samples are allowed to incubate for 15 min at room temperature and then 10 μL of a PAMCA stock solution in DMSO (final PAMCA concentration [5 μM]) was added. After 5 minutes of agitation on a shaking plate, the reaction is allowed to proceed at 37° C. for 120 minutes, with fluorescence readings taken every 10 minutes at a wavelength of 460 nm (using an excitation wavelength of 360 nm) on a Synergy HT Plate Reader using Gen5 software from Bio-Tek. The enzyme activity is calculated by converting the relative fluorescence units to AMC formed, using a standard curve of AMC. For compounds that inhibit hNAAA in range IC50 <1 μM full inhibition curves using eight different concentrations of inhibitor (8 point assay) are generated. The assay procedure used is the same as the three point assay. For ostensibly covalent compounds (as observed by a significant decrease in the slope of a plot of fluorescence vs. time in three point screen) samples are allowed to incubate for 2 hours at 37° C., instead of 15 minutes, before addition of 10 μL of a PAMCA stock solution in DMSO for a final PAMCA concentration of 5 μM. After 5 minutes of agitation on a shaking plate, the reaction is allowed to proceed at 37° C. for 120 minutes Inhibition constants are calculated using pro Fit software (Quantum Soft, Uetikon am See, Switzerland) and a Levenberg-Marquardt algorithm.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 294.35, ALogP 3.52, HBA 4, HBD 0, PSA 62.98, QED 0.58
Compound (SMILES): CC(C)C(=O)NCc1ccc(Cl)c(-c2nc(-c3ccc(OCC(F)(F)F)nc3)cc(=O)[nH]2)c1F
InChIKey: IIDYGPAQHWWNFL-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 498.86, ALogP 4.5, HBA 5, HBD 2, PSA 96.97, QED 0.47
Compound (SMILES): N#Cc1ccc2c(c1)cc(-c1cncs1)n2S(=O)(=O)c1cc(F)cc(F)c1O
InChIKey: TWRROGXEMSHQLF-UHFFFAOYSA-N
Target: ATP-citrate synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P53396
Assay: ADP-Glo Assay: Compounds of the present invention were evaluated in an ADP-GLO assay as follows:a) Dilute cpd 1:3 in succession in DMSO by hand for each cpds for 12 ptsb) Add 0.1 μL diluted cpd solution to assay plate, each dose with 2 replicatesc) Centrifuge 1000 RPM for 1 mind) Add 5 μL ACL working solution to 384-well assay plate, centrifuge 1000 RPM for 1 mine) Incubate at 25° C. for 15 minf) Add 5 μL substrate working solution to initiate reactiong) Final ACL reaction concentrations: 3 nM ACL, 15 μM ATP, 3 μM CoA, 300 μM Citrate, 0.01% Brij35, 4 mM DTT, 1% DMSO;h) Reference final conc: 30 uM starting Conc, 3× dilution, 11+0 points. Test cpd conc: 30/100 uM starting Conc, 3× dilution, 11+0 points.i) Incubate at 25° C. for 60 minj) Add 10 μL ADP Glo reagent, centrifuge 1000 RPM for 1 mink) Incubate at 25° C. for 40 minl) Add 20 μL kinase detection reagent, centrifuge 1000 RPM for 1 minm) Incubate at 25° C. for 40 minn) Read on Envision for US LUM as RLU.
Measurement: IC50 = 10250 nM (pChEMBL 4.989999771118164)
Descriptors: MW 417.42, ALogP 3.86, HBA 7, HBD 1, PSA 95.98, QED 0.55
Compound (SMILES): NS(=O)(=O)c1cccc(-c2ccc(C3CCN(CC4CCNC4)CC3)cc2)c1-c1nnn[nH]1
InChIKey: MUOJKGZDIBVZHX-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 11.7 nM (pChEMBL 7.929999828338623)
Descriptors: MW 467.6, ALogP 1.97, HBA 7, HBD 3, PSA 129.89, QED 0.51
Compound (SMILES): NC(=O)/C=C/CC[C@H](NC(=O)OCCO)C(=O)Nc1cccn(Cc2nc3ccccc3[nH]2)c1=O
InChIKey: IATFNKLJUOHTIR-COKXLDBZSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 μl/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 482.5, ALogP 0.62, HBA 8, HBD 5, PSA 181.43, QED 0.25
Compound (SMILES): O=C(O)c1ccc(N(Cc2ccc(C3CCCCC3)cc2)C(=O)[C@H]2CCCN2S(=O)(=O)c2c(F)cc(F)cc2F)cc1O
InChIKey: JHSGFWLJGBMLTB-HHHXNRCGSA-N
Target: Signal transducer and activator of transcription 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P40763
Assay: In Vitro Assay: Nuclear extract preparations and DNA-binding activity/electrophoretic mobility shift assay (EMSA) were carried out as previously described (Zhang X, et al., (2010) Biochem Pharmacol 79:1398-409; Zhang X, et al., (2012) Proc Natl Acad Sci USA 109:9623-8). The 32P-labeled oligonucleotide probes used were hSIE (high affinity sis-inducible element from the c-fos gene, m67 variant, 5′-AGCTTCATTTCCCGTAAATCCCTA) (SEQ ID NO: 1) that binds Stat1 and Stat3 and MGFe (mammary gland factor element from the bovine β-casein gene promoter, 5′-AGATTTCTAGGAATTCAA) (SEQ ID NO: 2) for Stat1 and Stat5 binding. Except where indicated, nuclear extracts were pre-incubated with compound for 30 min at room temperature prior to incubation with the radiolabeled probe for 30 min at 30° C. before subjecting to EMSA analysis. Where appropriate, bands corresponding to Stat3:DNA complexes were scanned and quantified for each concentration of compound using ImageJ and plotted as percent of control (DMSO) against concentration of compound, from which the IC50 values were derived.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 616.66, ALogP 5.94, HBA 5, HBD 2, PSA 115.22, QED 0.32
Compound (SMILES): Nc1nn2cc(Cl)cnc2c1C(=O)Nc1cnccc1N1CCN(C2COC2)CC1
InChIKey: IDIFLJSMIIRFPL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK) (SEQ ID NO:1).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 454 of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 428.88, ALogP 1.13, HBA 9, HBD 2, PSA 113.91, QED 0.64
Compound (SMILES): CC(C)c1ccccc1-c1ncc2c(n1)N(Cc1ccc(C(=O)N3CC(F)C3)cc1)C(=O)C2
InChIKey: ZKHSQLBDQAWANO-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: HTS Assay: The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2) (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (I-785, GG670, 671AA)/UAF1 (I-677)-Flag; Viva) in the assay was 100 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<<Km. 10 μL of 2× enzyme was added to assay plates (pre-stamped with compound) either simultaneously with 2×Ub-Rh110 or preincubated with USP1 40 minutes prior to the addition of 10 μL of 2×Ub-Rh110 to compound plates. Plates were incubated stacked for 45 minutes at room temperature before fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 444.51, ALogP 4.15, HBA 4, HBD 0, PSA 66.4, QED 0.59
Compound (SMILES): Cc1nc(Cn2ncc3ncc(-c4cccc(C(F)(F)F)c4)cc32)no1
InChIKey: YMHSYYWXJIPKCI-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: Effects of Test Articles on Cloned Human NR1/GuN2B Ion Channels Expressed in Mammalian Cells: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty-four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed, and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 202 nM (pChEMBL 6.699999809265137)
Descriptors: MW 359.31, ALogP 3.86, HBA 6, HBD 0, PSA 69.63, QED 0.56
Compound (SMILES): C/C=C/Cc1cn(-c2ccc(C(C)(C)O)cc2OC)c2cc[nH]c2c1=O
InChIKey: ZUOHLDHVUOZNKI-AATRIKPKSA-N
Target: Bromodomain-containing protein 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H8M2
Assay: IC50 measurements for inhibitors using BRD9 AlphaLisa Binding Assay: His/Flag epitope tagged BRD9134-239 was cloned, expressed, and purified to homogeneity. BRD9 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-11/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD9 (50 nM final) was combined with peptide (3 nM final) in 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.8% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature AlphaLisa Streptavidin Acceptor Beads (Perkin-AL125C) and AlphaLisa Nickel donor beads (Perkin AS 10 ID) were added to a final concentration of 15 ug/mL each. After ninety minutes of equilibration in the dark, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 352.43, ALogP 3.67, HBA 4, HBD 2, PSA 67.25, QED 0.69
Compound (SMILES): C[C@@H]1Cc2ccccc2CN1C(=O)c1cc2c(cc1-c1cc(C(=O)N(C)c3ccc(O)cc3)c3n1CCCC3)CN(C(=O)Oc1ccccc1)C2
InChIKey: DMWXIVGITPVVOV-HHHXNRCGSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, labelled (histag-Bcl-2 such that Bcl-2 corresponds to the UniProtKB® primary accession number: P10415), 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 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 680.81, ALogP 7.58, HBA 6, HBD 1, PSA 95.32, QED 0.21
Compound (SMILES): C=CC(=O)N[C@@H]1CCCC[C@@H]1Nc1nc(Nc2cc(C(N)=O)ccc2C)ncc1Cl
InChIKey: XCFZEEAABFYNEX-CVEARBPZSA-N
Target: Mitogen-activated protein kinase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P27361
Assay: Omnia Assay: The protocol below describes continuous-read kinase assays to measure potency of compounds against activated ERK1 enzyme. The mechanics of the assay platform are best described by the vendor (Invitrogen, Carlsbad, Calif.) on their website at the following URL: invitrogen.com/site/us/en/home.html.Briefly, a 1.25× stock of ERK1 enzyme (14-439-K) from Millipore (Billerica, Mass.), 5×ATP (AS001A) and ST17-Sox conjugated peptide substrate (KNZ1171C) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT. 10 μL of ATP/ST17-sox peptide substrate mix was combined with 0.5 μL volume of 100% DMSO and serially diluted compounds were prepared in 100% DMSO in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.). Kinase reactions were started with the addition of 40 μL of ERK1 solution and monitored every 71 seconds for 30-240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30+ minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes or seconds) and then plotted against inhibitor concentration to estimate AppIC50 from log [Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 428.92, ALogP 3.31, HBA 6, HBD 4, PSA 122.03, QED 0.5
Compound (SMILES): O=C(NC1(c2ccccn2)CC1)c1ccc(-c2cnc3ncc(C4(c5ccc6ncccc6c5)CC4)n3c2)cc1F
InChIKey: JJSALMKKOIUDFE-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: In Vitro c-Met Kinase Enzyme Assay: Compounds were screened in vitro for their ability to inhibit c-Met kinase activity. The IC50 values of compounds for the inhibition of c-Met kinase were determined as described in the literature with some modifications (Wang, X. et al, Mol. Cancer Ther. 2003, 2(11):1085-1092; Calic, M. et al., Croatica Chemical ACTA. 2005, 78(3):367-374). Briefly, histidine-tagged c-Met catalytic domain fusion protein (Invitrogen, #PV3143) was used for the assay. IC50 measurements were based on the degree of phosphorylation of poly Glu-Tyr (Sigma-Aldrich, #P0275) that was coated (0.01 mg/per well) on 96-well microplates (R&D systems, #DY990). The reaction was carried out in a 50 μL solution containing 50 mM HEPES (pH 7.5), 10 mM MnCl2, 10 mM MgCl2, 0.5 mM DTT, 100 μM Na3VO4, 5 μM ATP (Cell Signaling Technology, #9804) and serial dilutions of individual compounds. The reaction lasted for 25 minutes at 30° C. After the reaction was completed, the contents of the plates was discarded. Plates were then washed with TBS-T (250 μL/well, 5×) and then blocked with TBS-T containing 1% BSA for 2 hours. The contents of the plates was discarded, and 100 μL (per well) of peroxidase-labeled anti-phospho-tyrosine antibody (Sigma, #A5964) diluted (1:60,000) in 1% BSA containing TBS-T were then added and incubated for 1 hour. Plates were washed with TBS-T (250 mL/well, 5×) and followed by the color reaction using 100 μL (1:1 mixture) of H2O2 and tetramethylbenzidine (R&D Systems, #DY999). The reaction was stopped in minutes with 100 μL of 2 N H2SO4. The optical density was measured immediately using a microplate reader at 450 nm with wavelength correction at 540 nm. IC50 values were calculated with the GraphPad Prism software. The linear range (i.e., the time period over which the rate remained equivalent to the initial rate) was determined for the kinase and IC50 determinations were performed within this range.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 540.6, ALogP 5.98, HBA 6, HBD 1, PSA 85.07, QED 0.28
Compound (SMILES): O=C(Nc1nnc(CCc2ccc(Cl)cc2)s1)[C@@H]1CC(=O)N(c2ccc(CN3CCOCC3)cc2)C1
InChIKey: DSOWBRKRKUDOAF-HXUWFJFHSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Quanta Red Assay: Measuring ATX activity using an enzyme coupled Quanta Red assay (Thermo Scientific Pierce Protein Research Products, Product #15159) was determined as follows. 8 μL human recombinant ATX (final concentration 0.8 μg/mL) in 1× Assay buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM CaCl2 was added to an opaque black flat-bottom 384-well plate (Corning, #3575) containing 2 μL test compound in 40% DMSO (4% final DMSO concentration). 10 μL of Quanta Red, Horseradish peroxidase (HRP), Choline Oxidase (CO), Rac-1-Palmitoyl-glycero-3-phosphocholine solution (final concentration 1:250 for Quanta Red, 0.5units/ml HRP, 0.5units/ml CO, 15 μM Rac-1-Palmitoyl-glycero-3-phosphocholine) in 1× assay buffer (as described previously) was added to each well to start the reaction and the plate was incubated at room temperature for 2 hours. The reaction was stopped after 2 hours with a 20 μL addition of Quanta Red Stop solution (1:20 dilution in distilled water). The above-described mixture with DMSO alone was used as a positive control whereas that with DMSO alone without ATX was taken as a negative control. For each test compound, ten concentrations were measured covering a range of 6.1 nM to 120 μM to determine IC50 values. The top concentration was decreased to 1.2 μM when a test compound's IC50 value was evaluated in low nanomolar range. Fluorescence was determined in a BMG Labtech Pherastar plus plate reader (λ emission=540 nm, λ excitation=590 nm). Data were analysed using Excel fit software.
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 526.06, ALogP 3.8, HBA 7, HBD 1, PSA 87.66, QED 0.48
Compound (SMILES): Cc1nc(F)ccc1[C@H](Nc1cc(Cl)c2ncc(C#N)c(NCC(C)(C)C)c2c1)C1=CN([C@@H](C)CF)NN1
InChIKey: VWHXVZNSMRQEJE-HAWMADMCSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 541.05, ALogP 5.74, HBA 8, HBD 4, PSA 100.93, QED 0.27
Compound (SMILES): C[C@@H](Nc1cc(=O)n(C)c2ccc(Nc3ccnc(Cl)c3C#N)cc12)C(=O)NC1CCCC1
InChIKey: HFXLBWNSMJYTRE-CQSZACIVSA-N
Target: B-cell lymphoma 6 protein (SINGLE PROTEIN), Homo sapiens
UniProt: P41182
Assay: HTRF Assay: Each 15 μL HTRF reaction in a 384-well black Proxiplate (Perkin Elmer) contained either 1 nM (data in table 1b) or 10 nM (data in table 1a) Trx-6xHis-BCL6 (in house-produced, human BCL6 BTB domain covering amino-acid sequence 5-129), 300 nM BCOR-AF633 peptide (RSEIISTAPSSWWPGP-Cys-AlexaFluor 633-amide, Cambridge Research Biochemical) and 0.5 (data in table 1b) or 1 nM (data in table 1a) anti-6xHis-Terbium cryptate (CisBio Bioassays, France), in assay buffer (25 mM Hepes pH8, 100 mM NaCl, 0.05% Tween20, 0.5 mM TCEP, 0.05% bovine serum albumin). Test compounds in DMSO or DMSO alone were added to the wells using an ECHO550 acoustic dispenser (Labcyte Inc) to give the appropriate test concentration in 0.7% v/v DMSO final. After 2 hours incubation at room temperature the plate was read on an Envision plate reader (Perkin Elmer) with 337 nm laser excitation, a first emission filter APC 665 nm and a second emission filter Europium 615 nm. The % inhibition at each concentration was calculated by normalising FRET ratio to the appropriate high (DMSO with all reagents) and low (DMSO without BCL6) controls. The compound IC50S were determined using GraphPad Prism 6.0 or Dotmatics (Bishops Storford, UK) software by fitting the normalised data to a sigmoidal four-parameter logistic fit equation.
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 464.96, ALogP 4.06, HBA 7, HBD 3, PSA 111.84, QED 0.47
Compound (SMILES): CSc1cc(C)[nH]c(=O)c1CNC(=O)c1c(C)n([C@H](C)C2CCC(N3CC(OCC(F)F)C3)CC2)c2ncccc12
InChIKey: SNWTYORWWYVIOU-KMFTYDHNSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: EZH2 biochemical assay (IC50): Compound potencies were assessed through incorporation of 3H-SAM into a biotinylated H3 peptide. Specifically, 30 pM PRC2 containing wt EZH2 (pentameric complex prepared in-house) was pre-incubated with 450 nM SAM, 450 nM 3H-SAM, 2 μM H3K27me3 activating peptide (H2N-RKQLATKAAR(Kme3)SAPATGGVKKP-amide) and compounds (as 10 point duplicate dose response titrations in DMSO, final assay 0.8% DMSO (v/v)) for 3-5 h in 50 mM Tris (pH 8.5), 1 mM DTT, 0.07 mM Brij-35, 0.1% BSA, and 0.8% DMSO in a total volume of 12.5 μl. Reaction was initiated with biotinylated H3 substrate peptide (H2N-RKQLATKAAR(Kme1)SAPATGGVKKP-NTPEGBiot) as a 2 μM stock in 12.5 μl of buffer and allowed to react at room temperature for 18-22 h. Quenching was accomplished by addition of 20 μl of STOP solution (50 mM Tris (pH 8.5), 200 mM EDTA, 2 mM SAH). 35 μl of the quenched solution was transferred to streptavidin coated FlashPlates (PerkinElmer), incubated 1-2 h, washed, and read in a TopCount Reader (PerkinElmer). IC50s were calculated in Genedata Screener using non-linear least square four parameter fits, where the four parameters were IC50, Hill slope, pre-transitional baseline (0% INH), and post-transitional baseline (100% INH).
Measurement: IC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 587.74, ALogP 5.08, HBA 7, HBD 2, PSA 92.25, QED 0.32
Compound (SMILES): Cc1cc2c(C(=O)n3nc(N)c4c3CN(C(=O)OC(C)(C)C)CC4)cccc2[nH]1
InChIKey: GVFPRHVKUVRFCT-UHFFFAOYSA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Factor XIIa (FXIIa) Inhibitory Activity: In a 96-well clear bottom plate, 80 μl of assay buffer was added to each well. Assay buffer consists of 0.5× Hank's Balanced Salt Solution (Invitrogen), buffered with 25 mM HEPES pH 7.4 (Invitrogen) and 0.5× Tris-buffered saline with Tween-20 0.05% (Santa Cruz Biotechnology). Test compounds were first dissolved in DMSO (Sigma) and then 4 μl were added to test wells containing assay buffer. Serial dilutions using an automated multi-channel pipette were used to generate a concentration range of approximately 1-100 μM. Human FXIIa (Enzyme Research Labs) was diluted in assay buffer to a final concentration of 12.5 nM. 80 μl of this enzyme solution was added to the assay wells. The enzyme/compound mixtures were incubated for 10 minutes at room temperature. Chromogenic substrate (Pefachrome XIIa; Enzyme Research Labs) was added to assay wells at a final concentration of 400 μM. The assay plate was spun for 1 minute at 1500 g and then read at 37° C. in a SpectraMax M2 plate reader (λ=405 nm). Activity was quantified as the rate of change in absorbance, which corresponds to the rate of substrate cleavage. IC50 values were determined as the concentration of inhibitor that produced 50% of the rate of change of control wells without any inhibitor. For compounds with activity <1 μM, the assay was repeated with a lower concentration range, typically from 10-1000 nM.
Measurement: IC50 = 50000 nM (pChEMBL 4.300000190734863)
Descriptors: MW 395.46, ALogP 3.24, HBA 6, HBD 2, PSA 106.24, QED 0.66
Compound (SMILES): CCN(C[C@@]12CC[C@@H](c3cc(-c4c(F)cccc4F)nnc31)C2(C)C)C(=O)CN1C(=O)CNC1=O
InChIKey: CVJVUMPJURDCQX-MQNRADLISA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro RORc Ligand Binding Assay: Consumable Supplier and product code GFB Unifilter plates Perkin Elmer 6005177 3-[(3- Sigma C5070 Cholamidopropyl)dimethylammonio]- 1-propanesulfonate (CHAPS) 96-well polypropylene U-bottom Nunc 267245 assay plate HEPES buffer, 1M Sigma H3375 Magnesium chloride (MgCl2) Sigma M8266 D,L-Dithiothreitol (DTT) Sigma D0632 Sodium chloride (NaCl) Sigma 71382 Bovine serum albumin (BSA) Sigma A7030 [lyophilized powder, ≥98% (agarose gel electrophoresis), Essentially fatty acid free, essentially globulin free] 25-hydroxycholesterol Sigma H1015 25-[26,27-3H]hydroxycholesterol Perkin Elmer NET674250UC American Radiolabeled Chemicals ART0766 RORc ligand binding domain Genentech (e.g., PUR 28048), expressed in E. coli Plate seals Perkin Elmer 6005185 Microscint 0 Perkin Elmer 6013611. Final concentrations were as follows: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2, 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present.
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 483.52, ALogP 2.98, HBA 5, HBD 1, PSA 95.5, QED 0.64
Compound (SMILES): COc1cc(S(=O)(=O)C[C@H]2C[C@@H](N(C)c3ncnc4[nH]ccc34)C2)c(C)cn1
InChIKey: RZVBCGFEXPDAAN-OKILXGFUSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 0.019 nM (pChEMBL 10.720000267028809)
Descriptors: MW 401.49, ALogP 2.36, HBA 7, HBD 1, PSA 101.07, QED 0.68
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(-c3cnc(N4CC(C)(O)C4)nc3)nn2-c2ccccc2)[C@H](c2ccnc(F)c2)O1
InChIKey: QIIVNLIYMCBNDT-BVAGGSTKSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: Seven point five μL per well of human TrkA (PV3144, Lifetechnologies, final concentration: 1 nmol/L) suspended in the assay buffer (100 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20 and 1 mmol/L dithiothreitol (DTT)) was applied in a 384 well plate and the plate was pre-incubated for 15 min at room temperature with 0.4 μL of each compound (final concentration: 200 μmol/L-1 pmol/L) dissolved in DMSO. Then, fluorescent substrate (FL-peptide 27, 760424, PerkinElmer, final concentration: 1.5 μmol/L) and ATP (final concentration 500 μmol/L) dissolved in the assay buffer was added in each well. After the incubation of 120 min at 37° C., fifteen μL of termination buffer (100 mmol/L HEPES, 40 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20, 1 mmol/L DTT and 0.16 vol % Coating Reagent 3) was added in each well to stop the enzyme reaction. Fluorescent intensities (FI) of phosphorylated and non-phosphorylated fluorescent substrates were measured by LabChip EZReader II (Caliper LifeSciences, Inc.), and conversion ratio (CR) was calculated by the following formula-1. The CR in the well applied with DMSO alone was used as a negative control and the CR in the well without applying TrkA was used as a positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 603.66, ALogP 2.87, HBA 11, HBD 3, PSA 142.79, QED 0.24
Compound (SMILES): Cc1nc2cnc(N(C)C3CCN(CCC(C)(F)F)CC3)c(C#N)c2c(=O)n1CC#Cc1ccc(Cl)cc1-c1ccnc2c(C(=O)NS(C)(=O)=O)csc12
InChIKey: FMPOGOXAJFKKDS-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 15.8 nM (pChEMBL 7.800000190734863)
Descriptors: MW 793.32, ALogP 5.59, HBA 12, HBD 1, PSA 154.18, QED 0.19
Compound (SMILES): Cc1ncc(NC(=O)c2cnnc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)n2nccc2n1
InChIKey: DEBLQFYQFFNANE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 484.44, ALogP 3, HBA 9, HBD 1, PSA 110.43, QED 0.47
Compound (SMILES): COc1ccnc(N2CCC(F)(F)CC2)c1C(=O)Nc1ccnc(S(N)(=O)=O)c1
InChIKey: WZHJGEYRHKQJEN-UHFFFAOYSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y9
Assay: Biological Assay: The following buffers were used for the Qube® recordings: External buffer for Nav1.8 Qube® recording: 150 NaCl, 2 CaCl2, 5 KCl, 1 Mg Cl2, 10 HEPES, 12 Dextrose; External buffer for Qube® Nav1.5 recording: 120 N-Methyl-D-Glucamine, 40 NaCl, 1 KCl, 2.7 CaCl2), 5 HEPES, 0.5 MgCl2; and Internal buffer for Qube® recording: 120 CsF, 30 CsCl, 10 EGTA, 5 HEPES, 5 NaF, 2 MgCl2.
Measurement: IC50 = 1627 nM (pChEMBL 5.789999961853027)
Descriptors: MW 427.43, ALogP 1.62, HBA 7, HBD 2, PSA 127.51, QED 0.74
Compound (SMILES): O=C(O)CCCN1CCc2cccc(OCCCc3ccccc3)c2CC1
InChIKey: WABNRGRXPRMIQU-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H228
Assay: Evaluation of S1P5 Receptor Agonist Activities: CHO cells which were made to overexpress human S1P5 (EDG-8) gene were cultured in Ham's F12 Medium (manufactured by Gibco-BRL) containing 10% FBS (fetal bovine serum), penicillin/streptomycin and geneticin (0.25 mg/mL). The medium was removed from the cultured cells, the cultured cells were washed once with phosphate-buffered saline, and the cultured cells were treated with a vehicle (DMSO) solution or a compound solution each of which was diluted with Buffer (Hanks' balanced salt solution containing 20 mmol/L HEPES, 0.1 or 0.2% BSA, 1 mmol/L IBMX and 5 μmol/L forskolin) at 37° C. for 30 minutes. Thereafter, the cultured cells were washed once with phosphate-buffered saline, lysis of the cells, and the concentration of cyclic AMP in the cell lysate were measured by using cAMP Assay Kit (Cisbio Bioassays).
Measurement: EC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 367.49, ALogP 3.96, HBA 3, HBD 1, PSA 49.77, QED 0.68
Compound (SMILES): CN(C(=O)c1cc2c(F)c(F)ccc2[nH]1)[C@@H](CC(C)(C)C)C(=O)N1C[C@]2(C[C@H]1C#N)C(=O)Nc1ccccc12
InChIKey: PURYATKYWJIRON-HGUQOZNYSA-N
Target: Replicase polyprotein 1ab (SINGLE PROTEIN), Severe acute respiratory syndrome coronavirus 2
UniProt: P0DTD1
Assay: Biological Activity: SARS-CoV-2 3C-like (3CL) protease fluorescence assay (FRET): Recombinant SARS-CoV-2 3CL-protease was expressed and purified. TAMRA-SITSAVLQSGFRKMK-Dabcyl-OH peptide 3CLpro substrate was synthesized. Black, low volume, round-bottom, 384 well microplates were used. In a typical assay, 0.85 μL of test compound was dissolved in DMSO then incubated with SARS-CoV-2 3CL-protease (10 nM) in 10 μL assay buffer (50 mM HEPES [pH 7.5], 1 mM DTT, 0.01% BSA, 0.01% Triton-X 100) for 30 min at RT. Next, 10 μL of 3CL-protease substrate (40 μM) in assay buffer was added and the assays were monitored continuously for 1 h in an Envision multimode plate reader operating in fluorescence kinetics mode with excitation at 540 nm and emission at 580 nm at RT. No compound (DMSO only) and no enzyme controls were routinely included in each plate. All experiments were run in duplicate. Data Analysis: SARS-CoV-2 3CL-protease enzyme activity was measured as initial velocity of the linear phase (RFU/s) and normalized to controlled samples DMSO (100% activity) and no enzyme (0% activity) to determine percent residual activity at various concentrations of test compounds (0-10 μM). Data were fitted to normalized activity (variable slope) versus concentration fit in GraphPad Prism 7 to determine IC50.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 533.58, ALogP 4.34, HBA 4, HBD 2, PSA 109.3, QED 0.52
Compound (SMILES): Cc1nc(Br)n2ncn(Cc3nc([C@@H]4CO[C@@H](c5ccc(F)cc5)C4)no3)c(=O)c12
InChIKey: ANNUHVXNCFVLIP-GXTWGEPZSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (90 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 90 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC80 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 475.28, ALogP 2.78, HBA 9, HBD 0, PSA 100.34, QED 0.45
Compound (SMILES): COc1cc(-c2cc3c(-c4ccc(OC5CCNCC5)c(C#N)c4)ncnc3[nH]2)ccc1N1CCOCC1
InChIKey: AZBYFLYSUMYTKS-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Biological Assay for TBK1 and IKKs: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phopshorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKs were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2× Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 21.2 nM (pChEMBL 7.670000076293945)
Descriptors: MW 510.6, ALogP 4.14, HBA 8, HBD 2, PSA 108.32, QED 0.4
Compound (SMILES): CCn1cc(CN2CCN(c3ccc(-c4cc(-c5cnn(C)c5)cn5ncc(C#N)c45)cn3)CC2)cn1
InChIKey: BCRFYXFULKEKIH-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 10.4 nM (pChEMBL 7.980000019073486)
Descriptors: MW 492.59, ALogP 3.21, HBA 10, HBD 0, PSA 96.1, QED 0.36
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CN(C(=O)c2ccc(NC(C)=O)cc2)c2ccccc2N(Cc2c(OC)ccc3cc(Br)ccc23)C1=O
InChIKey: LZVAKEDICCBHKT-MMTVBGGISA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay for BIR2 and BIR3: 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-(c-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. 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. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 672.58, ALogP 4.86, HBA 6, HBD 3, PSA 120.08, QED 0.25