lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)Nc1cc(-n2ncc3cc(C#N)cnc32)ncc1-c1c[nH]cn1
InChIKey: UZDJXEQNYJMLGI-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 105 nM (pChEMBL 6.980000019073486)
Descriptors: MW 344.38, ALogP 2.9, HBA 7, HBD 2, PSA 108.1, QED 0.59
Compound (SMILES): CCN1C(=O)N(c2c(F)c(OC)cc(OC)c2F)Cc2cnc(-c3ccccc3)cc21
InChIKey: DEMWYUOYYYJCQV-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 425.44, ALogP 5.01, HBA 4, HBD 0, PSA 54.9, QED 0.58
Compound (SMILES): Cc1cc(-c2cn[nH]c2)c(C)cc1Nc1ccccc1C(=O)O
InChIKey: PCOYXWISXBOMSF-UHFFFAOYSA-N
Target: Alpha-ketoglutarate-dependent dioxygenase FTO (SINGLE PROTEIN), Homo sapiens
UniProt: Q9C0B1
Assay: Inhibition Assay: The present invention provides a novel class of demethylase FTO inhibitors which inhibit FTO protein activity at very low concentration (typically IC50 values is below 30 μM).
Measurement: IC50 < 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 307.35, ALogP 4.14, HBA 3, HBD 3, PSA 78.01, QED 0.68
Compound (SMILES): CCN1CCOc2c(cccc2NCC(=O)N(CCNC(C)(C)C)Cc2ccccc2C(F)(F)F)C1
InChIKey: SAQSBXUPRTYFBX-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: The assay is using the PathHunter CHO-K1 CXCR7 β-arrestin cell line from DiscoverX. The system is based on the Enzyme Fragment Complementation Technology. Two complementing fragments of the β-galactosidase enzyme are expressed within stably transfected cells. The larger portion of β-gal, termed EA for Enzyme Acceptor, is fused to the C-terminus of b-arrestin 2. The smaller fragment, termed ProLink¿ tag, is fused to CXCR7 at the C-terminus. Upon activation, b-arrestin is recruited which forces the interaction of ProLink and EA, allowing complementation of the two fragments of b-gal and the formation of a functional enzyme which is capable of hydrolysing the substrate and generating a chemiluminescent signal. CHO-K1 CXCR7 β-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicillin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1X. 5 μl/well of HBSS1X/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg). The calculated EC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. Average EC50 values from several measurements are given as geometric mean values.
Measurement: EC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 506.61, ALogP 4.75, HBA 5, HBD 2, PSA 56.84, QED 0.51
Compound (SMILES): CCCCc1nc(=O)c(S(=O)(=O)c2ccc(-c3cncc(F)c3)cc2)c(O)n1[C@@H](CC)c1cccc2c1OCO2
InChIKey: LDPKSJQHATVZNW-QHCPKHFHSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyrl)apelin-13) peptide, which was set to 100%.
Measurement: EC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 565.62, ALogP 5.05, HBA 9, HBD 1, PSA 120.61, QED 0.3
Compound (SMILES): COC1(C(=O)N2CCOc3c(C#N)cncc3C2)CCN(c2ncc(F)c(C3CC3)n2)CC1
InChIKey: UHVABRVXDRPUTH-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Fluorescent Polarization Binding (FP Binding) Assay: Test compounds were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final). In each well of a 384-well Plate were dispensed 0.1 μL of compound solution (or DMSO for controls) followed by 5 μL of GST-hRIPK1 (8-327) at twice the final concentrations in assay buffer (50 mM HEPES pH 7.5, 10 mM NaCl, 50 mM MgCl2, 0.02% CHAPS, 0.5 mM DTT and 0.01% Pluronic F127). For negative control the enzyme addition was replaced by assay buffer only.After addition of 5 μL of fluorescentlabeled ligand at twice the final concentrations in assay buffer, the plate was incubated at RT for 30 mM. At the end, the binding was measured as FP value with the Envision (PerkinElmer) plate reader using filter for an excitation λ=531 nm FP and an emission λ=595 nm FP (S & P-pol). GST-hRIPK1 (8-327) enzyme was produced via Baculovirus expression.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 452.49, ALogP 2.17, HBA 8, HBD 0, PSA 104.47, QED 0.7
Compound (SMILES): CC(C)c1cc(-c2ccc(C[C@@H](C#N)NC(=O)[C@H]3N[C@@H]4CC[C@H]3C4)c(F)c2)ccc1[S+](C)[O-]
InChIKey: ZPKAAZKAWPZZBG-QGBJVGRCSA-N
Target: Dipeptidyl peptidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53634
Assay: Inhibition Assay of Human DPPI (Cathepsin C): Materials: Microtiterplates (Optiplate-384 F) were purchased from PerkinElmer (Prod. No. 6007270). The substrate Gly-Arg-AMC was from Biotrend (Prod.-No. 808756 Custom peptide). Bovine serum albumin (BSA; Prod. No. A3059) and Dithiothreitol (DTT; Prod. No D0632) were from Sigma. TagZyme buffer was from Riedel-de-Haen (Prod.-No. 04269), NaCl was from Merck (Prod.-No. 1.06404.1000) and morpholinoethane sulfonic acid (MES), was from Serva (Prod.-No. 29834). The DPP1 inhibitor Gly-Phe-DMK was purchased from MP Biomedicals (Prod.-No. 03DK00625). The recombinant human DPPI was purchased from Prozymex. All other materials were of highest grade commercially available. The following buffers were used: MES buffer: 25 mM MES, 50 mM NaCl, 5 mM DTT, adjusted to pH 6.0, containing 0.1% BSA; TAGZyme Buffer: 20 mM NaH2PO4, 150 mM NaCl adjusted to pH 6.0 with HCl. Assay conditions: The recombinant human DPPI was diluted in TAGZyme buffer to 1 U/ml (38.1 μg/ml, respectively), and then activated by mixing in a 1:2 ratio with a Cysteamine aqueous solution (2 mM) and incubating for 5 min at room temperature. Five uL test compound (final concentration 0.1 nM to 100 μM) in aqua bidest (containing 4% DMSO, final DMSO concentration 1%) were mixed with 10 μL of DPPI in MES buffer (final concentration 0.0125 ng/μL) and incubated for 10 min. Then, 5 μL of substrate in MES buffer (final concentration 50 μM) were added. The microtiter plates were then incubated at room temperature for 30 mM Then, the reaction was stopped by adding 10 μL of Gly-Phe-DMK in MES-buffer (final concentration 1 μM). The fluorescence in the wells was determined using a Molecular Devices SpectraMax M5 Fluorescence Reader (Ex 360 nm, Em 460 nm) or an Envision Fluorescence Reader (Ex 355 nm, Em 460 nm). Each assay microtiter plate contained wells with vehicle controls (1% DMSO in bidest+0.075% BSA) as reference for non-inhibited enzyme activity (100% Ctl; high values) and wells with inhibitor (Gly-Phe-DMK, in bidest+1% DMSO+0.075% BSA, final concentration 1 μM) as controls for background fluorescence (0% Ctl; low values). The analysis of the data was performed by calculating the percentage of fluorescence in the presence of test compound in comparison to the fluorescence of the vehicle control after subtracting the background fluorescence using the following formula: (RFU(sample)−RFU(background))*100/(RFU(control)−RFU(background)).
Measurement: IC50 = 11.5 nM (pChEMBL 7.940000057220459)
Descriptors: MW 467.61, ALogP 4.04, HBA 4, HBD 2, PSA 87.98, QED 0.6
Compound (SMILES): C#C[C@@]1(O)[C@@H](COC(Cc2ccccc2)(C(=O)O)C(=O)O)O[C@@H](n2cnc3ccc(C(F)(F)F)nc32)[C@@H]1O
InChIKey: OFPBDKVSUCHWJM-ZRAXVTTISA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Inhibition of the CD73 Enzyme In Vitro: For measurements of soluble CD73 enzyme activity, recombinant CD73 was obtained from R&D Systems, Cat. No. 5795-EN-010. Serial dilutions of test compounds were incubated with recombinant CD73 and AMP in reaction buffer (25 mM Tris HCl pH7.5, 5 mM MgCl2, 50 mM NaCl, 0.25 mM DTT, 0.005% Triton X-100). The final reaction volume was 25 μL and the final concentrations of recombinant CD73 and AMP were 0.05 nM and 50 μM, respectively. Reactions were allowed to proceed for 1 hour at 37° C. before the addition of 100 μL Malachite Green (Cell Signaling Technology, Cat. No. 12776). After 5 minutes at room temperature, absorbance at 630 nm was determined on a microplate spectrophotometer.
Measurement: IC50 = 235 nM (pChEMBL 6.630000114440918)
Descriptors: MW 535.43, ALogP 1.24, HBA 9, HBD 4, PSA 164.23, QED 0.24
Compound (SMILES): CCCCc1nc(=O)c(S(=O)(=O)c2ccc(-c3ccncc3C)cc2)c(O)n1[C@@H](COC)c1ccccc1
InChIKey: FAPOQYJEIYJCLZ-VWLOTQADSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyrl)apelin-13) peptide, which was set to 100%.
Measurement: EC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 533.65, ALogP 4.73, HBA 8, HBD 1, PSA 111.38, QED 0.31
Compound (SMILES): O[C@@H]1[C@H](O)CS[C@H]1c1nnc2c(N[C@H]3C[C@@H]3c3ccccc3)nccn12
InChIKey: UNQRJNKDMHISGL-XLWJZTARSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Assay for Binding Affinity for Adenosine Receptor A3: HEK-293 cell membrane homogenates (32 μg protein), in which A3 adenosine receptors were expressed, are incubated for 120 min at 22° C. with 0.15 nM [1251]AB-MECA in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA and 2 UI/ml ADA. Nonspecific binding is determined in the presence of 1 μM IB-MECA. Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is IB-MECA, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 369.45, ALogP 1.6, HBA 8, HBD 3, PSA 95.57, QED 0.64
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1ccc2c(c1)C1OC2c2ccc(Nc3ccccc3F)cc21
InChIKey: QUCVQDXHGNPHLI-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Plasma Kallikrein Inhibition Assay: The chromogenic substrate D-Pro-Phe-Arg-pNa, 2HCl (BIOPHEN CS-31(02) from Hyphen BioMed, Neuville-Sur-oise, France) was dissolved in 5 mL deionized water and stored at 4° C. Concentration was determined in the spectrophotometer at 342 nm using an extinction coefficient of 8270. All other chemicals were of analytical grade.Human plasma kallikrein was purchased from Enzyme Research Labs (South Bend, Ind., USA, batch HPKa 2830). A stock solution of 7 μM in 50% glycerol was stored at −20° C.Enzyme reactions were conducted in assay buffer comprised of 20 mM HEPES at pH 7.4, 150 mM NaCl, 0.1% PEG-8000 and 0.01% Triton X-100.Both enzyme and substrate were diluted in assay buffer.The compound solutions as well as the enzyme and the substrate solutions were transferred to 96-well plates (Clear, UV-Star, Flat-bottom, Half-Area plates; cat. No. 675801 Greiner Bio-one, purchased from VWR International, Arlington Heights, Ill., USA) using a Rainin LTS 96-channel pipettor (Rainin, Columbus, Ohio, USA). Plate measurements were conducted using a SPECTROStar Nano reader (BMG Labtech, San Francisco, Calif., USA). The SPECTROStar Nano is a spectrophotometer and absorbance was measured at 405 nm. We used discrete wavelength, precise, kinetic reads of 15 cycles with a 60 sec cycle time.Determination of IC50 valuesFor the determination of IC50 values, the assays were performed at room temperature in 96-well plates with a total assay volume of 85 μL per well. The test compound was dissolved in 100% DMSO. The compounds were serially diluted in DMSO in a 7 point dose response. For the assays, 66.5 μL of protease solution (protease in assay buffer) was added per well followed by the addition of 8.5 μL of compound in 100% DMSO. The final assay concentration of the human plasma kallikrein was 250 μM. After 30 min. incubation at room temperature on an orbital shaker, the reactions were started by the addition of 10 μL substrate solution (in assay buffer, final assay concentration was 600 uM). After the addition of the substrate solution the final DMSO concentration was 10%. The plate was placed again on the shaker for 5 sec, spun at 2000 rpm for 5 sec and read on the spectrophotometer. The effect of the compound on the enzymatic activity was obtained from the linear part of the progress curves and determined after 15 minutes.
Measurement: IC50 = 5248 nM (pChEMBL 5.28000020980835)
Descriptors: MW 480.54, ALogP 5.62, HBA 5, HBD 3, PSA 89.27, QED 0.35
Compound (SMILES): CCn1c(=O)[nH]/c(=N\c2ccc(Oc3ncc(C(=O)O)s3)cc2)n(Cc2ccc(F)cc2)c1=O
InChIKey: XXYQLQJKSIWCRR-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 483.48, ALogP 2.72, HBA 9, HBD 2, PSA 131.57, QED 0.42
Compound (SMILES): CN1CCN(c2ccc(-c3cc4c(-c5ccc(OC6CCOCC6)c(C#N)c5)ncnc4[nH]3)cc2)CC1
InChIKey: SRGAHDUUSDDCQN-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 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 494.6, ALogP 4.47, HBA 7, HBD 1, PSA 90.3, QED 0.44
Compound (SMILES): CC(C)COc1nc(-c2ccc(NS(=O)(=O)c3ccccc3F)cc2)nc2[nH]nc(N)c12
InChIKey: NIAXDMOXVZLFJX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Sgk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00141
Assay: Enzymatic Activity Assay: The compounds were tested for serum and glucocorticoid-regulated kinase 1 (SGK-1) inhibitory activity in a substrate phosphorylation assay designed to measure the ability of the isolated enzyme to catalyze the transfer of phosphate from ATP to serine/threonine residues in a fluorescein-labeled substrate peptide, using recombinant human SGK-1 enzyme produced in a baculovirus system (Biomol, Hamburg, Germany, Cat. No. 4-331). The synthesized fluorescent labeled peptide substrate contained (5(6)-Carboxyfluorescein)-RPRAATF-NH2. The phosphorylated substrate peptide and non-phosphorylated substrate peptide were separated with caliper life science's lab-chip technology based on a micro fluidics method. All fluid flow was established on the chip by applying a vacuum of a few psi to the waste well transporting fluid from various sources through interconnecting channels. Because the phosphoryl group is doubly negatively charged, under the pressure-driven hydrodynamic flow and the voltage-driven flow within the electric field, the fluorescent labeled peptide substrate and its phosphorylation product appear at different times in the detection window to the detection point. Substrate turnover can thus be determined as the ratio of the product peak area and the sum of substrate peak area and product peak area.The enzyme reaction was carried out in a buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM MnCl2, 2 mM DTT, and 0.03% bovine serum albumine. The enzyme was pre-incubated with the test compound for 30 min at 24° C. The kinase reaction was initiated by addition of the substrate mixture containing the peptide substrate (final concentration 1 μM) and ATP (final concentration 10 μM). After 60 min incubation at 37° C., the enzyme reaction was terminated by adding a buffer containing 100 mM Hepes (pH 7.4) and 35 mM EDTA.
Measurement: IC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 456.5, ALogP 3.58, HBA 7, HBD 3, PSA 135.88, QED 0.39
Compound (SMILES): C=CC(=O)N1CCN(c2cnc3[nH]cc(C(=O)NC(C)C)c3n2)C[C@@H]1CC
InChIKey: LLXOSVZFPKHOAS-ZDUSSCGKSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.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, 4 μM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. 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 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 370.46, ALogP 1.71, HBA 5, HBD 2, PSA 94.22, QED 0.78
Compound (SMILES): CNCc1cc(C)c(-c2nc3c(-c4cnc(N5CCOCC5)nc4)n[nH]c3cc2C#N)c(F)c1
InChIKey: FCKUHZYRLWZXSH-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μl of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech). Ki values were determined.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 458.5, ALogP 2.96, HBA 8, HBD 2, PSA 115.64, QED 0.47
Compound (SMILES): COC(=O)Nc1cc2c(cn1)OCc1c-2ccc(OC[C@@](C)(N)CC(C)C)c1Cl
InChIKey: CQXZSQUEJBKQNM-NRFANRHFSA-N
Target: AP2-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q2M2I8
Assay: AAK1 Kinase Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2 and ATP) and test compounds in assay buffer (10 mM Tris-HCL pH 7.4, 10 mM MgCl2, 0.01% Tween-20 and 1.0 mM DTT). The reactions were initiated by the combination of bacterially expressed, GST-Xa-hAAK1 with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA buffer to each sample. The reactions were analyzed on the Caliper LabChip 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to EDTA quenched control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assays are ATP, 22 μM; (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2, 1.5 μM; GST-Xa-hAAK1, 3.5 nM; and DMSO, 1.6%. Dose response curves were generated to determine the concentration required inhibiting 50% of 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 = 0.81 nM (pChEMBL 9.09000015258789)
Descriptors: MW 419.91, ALogP 4.62, HBA 6, HBD 2, PSA 95.7, QED 0.71
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H](CS(=O)(=O)N2CCC(C(F)F)C2)C1
InChIKey: LZYADDZYJYUBGG-ZIHWECLZSA-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 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 399.47, ALogP 2.09, HBA 5, HBD 1, PSA 82.19, QED 0.8
Compound (SMILES): COc1ncc(-c2ccnc(NC(C)=O)c2)c(C)c1NS(=O)(=O)c1ccc(F)cc1F
InChIKey: PXDXNGXOBANEFM-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: VPS34 Enzyme Assays: 100 nL compounds in DMSO are added to wells of a 384 well microtitre plate (Greiner 780076). At room temperature: 5 ul VPS34 reaction buffer (Invitrogen Assay Buffer Q (diluted 1 in 5 with nanopure water) plus 2 mM DTT and 2 mM MnCl2) containing ATP (20 uM, Promega) and 200 uM PI-PS substrate (Invitrogen PV5122) is added followed immediately by 5 ul VPS34 reaction buffer (as above) containing VPS34 (5 nM, Millennium Protein Sciences Group) and the mixture is incubated with shaking at room temperature for 1 hour. Then 5 ul VPS34 stop-detect mix (as per Invitrogen Adapta Assay kit (PV5009) instructions (contains kinase quench buffer, TR-FRET buffer, Adapta Eu anti-ADP antibody and Alexa Fluor 647 ADP tracer)) is added to quench the reaction. The plates are then incubated for 30 minutes at room temperature with shaking and then read on a BMG PheraStar Plus reader.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 448.45, ALogP 3.5, HBA 6, HBD 2, PSA 110.28, QED 0.6
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@@H]3C[C@@H]3C[C@H]2C(=O)N[C@@H]2C[C@H]2c2ccccc2Cl)c2ncccc12
InChIKey: NEQUYRBLOORVCJ-FHBRLQHDSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition Assay: Method 1: Recombinant human factor D (expressed in E. coli and purified using standard methods) at 10 nM concentration is incubated with test compound at various concentrations for 1 hour at room temperature in 0.1 M Hepes buffer, pH 7.5, containing 1 mM MgCl2, 1 M NaCl and 0.05% CHAPS. A synthetic substrate Z-Lys-thiobenzyl and 2,4-dinitrobenzenesulfonyl-fluoresceine are added to final concentrations of 200 μM and 25 μM, respectively. The increase in fluorescence is recorded at excitation of 485 nm and emission at 535 nm in a microplate spectrofluorimeter. IC50 values are calculated from percentage of inhibition of complement factor D-activity as a function of test compound concentration.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 477.95, ALogP 2.95, HBA 6, HBD 1, PSA 97.19, QED 0.55
Compound (SMILES): CC(C)S(=N)(=O)N1CC(CC#N)(n2cc(-c3ncnc4[nH]ccc34)cn2)C1
InChIKey: WYNRCGNGBRKEDO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Activity Inhibitory Assay: Kinase Assay:Preparation of buffers, the buffer included 50 mM HEPES (pH 7.5), 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA.After the buffer was formulated, the enzyme and substrate were mixed with different concentrations of the compound prepared in advance, and placed at room temperature for 15 minutes. ATP was added to start reaction, and the reaction solution was incubated at room temperature for 90 minutes (positive and negative controls were set). 10 μL of reaction system included 2.5 μL compound, 5 μL mixture of enzyme and substrate, and 2.5 μL ATP. After the reaction was completed, the antibody was added to test and incubated at room temperature for 60 min, then was detected by Evnvision and data was collected. Data analysis and mapping was conducted with XLfit5 software.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 384.47, ALogP 2.12, HBA 7, HBD 2, PSA 127.34, QED 0.7
Compound (SMILES): CCN1C(=O)N=C(Nc2cc(OC)c(OC)nc2C)N2C1=NCC2c1ccc(F)cc1
InChIKey: QDQBAGPEZRZAGU-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Inhibition Assay: Evaluation of a human P2X7 receptor inhibitory activity Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1× HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 426.45, ALogP 3.18, HBA 7, HBD 1, PSA 91.65, QED 0.79
Compound (SMILES): Nc1ncnc(N2CCC(C(F)(F)F)C(N3CC=CC[C@@H](Nc4cc(F)cc(Cl)c4)C3=O)C2)c1F
InChIKey: MUKHOOXXWYLWNH-AQFXKWCLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro BTK Kinase Assay: The purpose of the BTK in vitro assay is to determine compound potency against BTK through the measurement of IC50. Compound inhibition is measured after monitoring the amount of phosphorylation of a fluorescein-labeled polyGAT peptide (Invitrogen PV3611) in the presence of active BTK enzyme (Upstate 14-552), ATP, and inhibitor. The BTK kinase reaction was done in a black 96 well plate (costar 3694). For a typical assay, a 24 μL aliquot of a ATP/peptide master mix (final concentration; ATP 10 μM, polyGAT 100 nM) in kinase buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 200 μM Na3PO4, 5 mM DTT, 0.01% Triton X-100, and 0.2 mg/ml casein) is added to each well. Next, 1 μL of a 4-fold, 40× compound titration in 100% DMSO solvent is added, followed by adding 15 uL of BTK enzyme mix in 1× kinase buffer (with a final concentration of 0.25 nM). The assay is incubated for 30 minutes before being stopped with 28 μL of a 50 mM EDTA solution. Aliquots (5 μL) of the kinase reaction are transferred to a low volume white 384 well plate (Corning 3674), and 5 μL of a 2× detection buffer (Invitrogen PV3574, with 4 nM Tb-PY20 antibody, Invitrogen PV3552) is added. The plate is covered and incubated for 45 minutes at room temperature. Time resolved fluorescence (TRF) on Molecular Devices M5 (332 nm excitation; 488 nm emission; 518 nm fluorescein emission) is measured. IC50 values are calculated using a four parameter fit with 100% enzyme activity determined from the DMSO control and 0% activity from the EDTA control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 516.9, ALogP 4.02, HBA 6, HBD 2, PSA 87.38, QED 0.47
Compound (SMILES): Cc1ccc(F)c(S(=O)(=O)Nc2ccc(-c3nc(C4CC4)c4c(N)n[nH]c4n3)cc2)c1
InChIKey: OAXGOWGLPGCUEA-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Sgk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00141
Assay: Enzymatic Activity Assay: The compounds were tested for serum and glucocorticoid-regulated kinase 1 (SGK-1) inhibitory activity in a substrate phosphorylation assay designed to measure the ability of the isolated enzyme to catalyze the transfer of phosphate from ATP to serine/threonine residues in a fluorescein-labeled substrate peptide, using recombinant human SGK-1 enzyme produced in a baculovirus system (Biomol, Hamburg, Germany, Cat. No. 4-331). The synthesized fluorescent labeled peptide substrate contained (5(6)-Carboxyfluorescein)-RPRAATF-NH2. The phosphorylated substrate peptide and non-phosphorylated substrate peptide were separated with caliper life science's lab-chip technology based on a micro fluidics method. All fluid flow was established on the chip by applying a vacuum of a few psi to the waste well transporting fluid from various sources through interconnecting channels. Because the phosphoryl group is doubly negatively charged, under the pressure-driven hydrodynamic flow and the voltage-driven flow within the electric field, the fluorescent labeled peptide substrate and its phosphorylation product appear at different times in the detection window to the detection point. Substrate turnover can thus be determined as the ratio of the product peak area and the sum of substrate peak area and product peak area.The enzyme reaction was carried out in a buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM MnCl2, 2 mM DTT, and 0.03% bovine serum albumine. The enzyme was pre-incubated with the test compound for 30 min at 24° C. The kinase reaction was initiated by addition of the substrate mixture containing the peptide substrate (final concentration 1 μM) and ATP (final concentration 10 μM). After 60 min incubation at 37° C., the enzyme reaction was terminated by adding a buffer containing 100 mM Hepes (pH 7.4) and 35 mM EDTA.
Measurement: IC50 < 1.5 nM (pChEMBL 8.824000358581543)
Descriptors: MW 438.49, ALogP 3.73, HBA 6, HBD 3, PSA 126.65, QED 0.44
Compound (SMILES): O=C(CN1C(=O)C2(CCN(C(=O)c3ccc4[nH]ncc4c3)CC2)c2c(Br)cccc21)N1CCN2CCCC2C1
InChIKey: ZHIRCLAJYQRTOC-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: In Vitro Binding Assay for DDR1: An in vitro binding competition assay was performed to evaluate the effect of the compounds of present invention on DDR1 protein.Binding Competition Assay:This assay is based on the intracellular domain of the DDR1 protein which contains the kinase active site. The recombinant protein additionally carries a GST-tag that can be recognized by an Eu-labeled anti-GST antibody. A tracer compound binding to the active site is labeled with a dye so that a FRET donor acceptor pair can be formed. Excitation energy absorbed by the Europium complex (350 nm flash light or pulsed laser) is transferred to a suitable fluorescent dye, if it is in close proximity. Compounds binding competitively with the tracer molecule will displace the bound tracer molecules and reduce the FRET signal in a dose dependent manner. Due to the long lifetime of the Eu excited state, the emission of the donor and the acceptor can be measured in time-gated mode such that most of the intrinsic fluorescence contributions have already decayed. This results in high sensitivity, excellent reproducibility and high data quality. This sensitive detection method enables protein concentrations below 20 nM.Protein, tracer and labeled antibody were obtained from commercial sources. The assay was performed in 384 low volume microtiter-plates with a final volume of 15 al. Dose response curves were generated from 16 compound dilutions in DMSO as solvent compound dilutions, a solution containing protein and labeled antibody, and a solution containing the tracer which is added in the last step. The fluorescence of donor and acceptor were then measured after one hour incubation at room temperature. Every assay run was quality-controlled with dose response curves for two reference compounds.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 591.51, ALogP 3.15, HBA 5, HBD 1, PSA 92.85, QED 0.51
Compound (SMILES): COC(=O)c1cc2c(s1)S(=O)(=O)CC(=O)N2Cc1cccc(C(F)(F)F)c1
InChIKey: VIEDHLDJXFLRBZ-UHFFFAOYSA-N
Target: Intermediate conductance calcium-activated potassium channel protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15554
Assay: KCa3.1 Assay: HEK293 cells over-expressing human KCa3.1 are used to measure the effects of compounds in inhibiting the KCa3.1 channel function. The assay is based on measuring the influx of Tl+ through KCa3.1 using a FLIPR dye assay (FLIPR Potassium Ion Channel Assay Kit #R8154 by Molecular Devices). The day before the assay HEK293/KCa3.1 cells are seeded on a 384 well plate. On the day of the assay, media is removed and replaced with Molecular Devices Potassium Ion Channel dye then incubated for 1 hour according to manufacturer's recommendations. The cells are then treated with compounds or DMSO control for 15 min, followed by addition of thallium (final concentration=1.5 mM) and ionomycin (final concentration=1 uM). The plate is read on HAMAMATSU 60000 measuring fluorescence signal for 50 seconds using (Em: 565-625 nm). Data analysis: HAMAMATSU 6000 data are exported using max-min value for frame 10 and 40 and corrected by spatial uniformity correction. Percent of control (POC) is calculated as 100×(max-minFlsample−max-minFllow)/(max-minFlhi−max-minFllow). Max-min Flsample is max-min value of each sample well containing test compound. Max-min Fllow is average max-min value of wells containing reference inhibitory compound.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 419.4, ALogP 2.87, HBA 6, HBD 0, PSA 80.75, QED 0.72
Compound (SMILES): COc1nc2ccc(C(O)(c3ccc(C)nc3C)c3cnnn3C)cc2c(Cl)c1OCCN1CCOCC1
InChIKey: VTLQXQGPKKWFQP-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor is a fluorescence based assay that estimates ligand binding affinities by measuring the effect of a ligand on protein thermal stability (Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R. (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6, 429-40, and Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258-66). This approach is applicable to a wide variety of systems, and rigorous in theoretical interpretation through quantitation of equilibrium binding constants (KD).In a ThermoFluor experiment where protein stability is monitored as the temperature is steadily increased, an equilibrium binding ligand causes the midpoint of an unfolding transition (Tm) to occur at a higher temperature. The shift in the melting point described as a ΔTm is proportional to the concentration and affinity of the ligand. The compound potency may be compared as a rank order of either ΔTm values at a single compound concentration or in terms of KD values, estimated from concentration response curves.
Measurement: Kd = 370 nM (pChEMBL 6.429999828338623)
Descriptors: MW 539.04, ALogP 3.03, HBA 10, HBD 1, PSA 107.65, QED 0.36
Compound (SMILES): COc1ncc(-c2cc(CN3CC(F)(F)C3)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)[C@H]2CC2(F)F)C[C@@H]1F
InChIKey: RQOJCGWNCGGUDS-IXDOHACOSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 4300 nM (pChEMBL 5.369999885559082)
Descriptors: MW 580.52, ALogP 1.77, HBA 9, HBD 2, PSA 130.98, QED 0.4
Compound (SMILES): Oc1ccc2c(c1)CC[C@H](c1ccccc1)[C@@H]2c1ccc(OCCN2CC(CCl)C2)cc1
InChIKey: ISXBBWWHVBTMKM-IAPPQJPRSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Biological Evaluation: The relative efficacies of Formula I compounds as inhibitors of an enzyme activity (or other biological activity) can be established by determining the concentrations at which each compound inhibits the activity to a predefined extent and then comparing the results. Typically, the preferred determination is the concentration that inhibits 50% of the activity in a biochemical assay, i.e., the 50% inhibitory concentration or IC50. Determination of IC50 values can be accomplished using conventional techniques known in the art. In general, an IC50 can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of the inhibitor under study. The experimentally obtained values of enzyme activity then are plotted against the inhibitor concentrations used. The concentration of the inhibitor that shows 50% enzyme activity (as compared to the activity in the absence of any inhibitor) is taken as the IC50 value. Analogously, other inhibitory concentrations can be defined through appropriate determinations of activity.
Measurement: EC50 = 50.9 nM (pChEMBL 7.289999961853027)
Descriptors: MW 448.01, ALogP 5.8, HBA 3, HBD 1, PSA 32.7, QED 0.46