lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(NC(=O)c2ccc(CN3CCN(C)CC3)cc2)cc1Nc1nccc(-c2cncc(C3=NN=C(C(=O)Nc4cn[nH]c4)C3)c2)n1
InChIKey: XQKRNUCXAFMEFO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL2 (SINGLE PROTEIN), Homo sapiens
UniProt: P42684
Assay: Kinase Assay: Kinase base buffer (50 mM HEPES, pH 7.5 0.0015% Brij-35; 10 mM MgCl2 2 mM DTT) and Stop buffer, (100 mM HEPES, pH 7.5 0.015% Brij-35; 0.2% Coating Reagent (50 mM EDTA) are prepared. Test compound is diluted in 100% DMSO to 50-times the desired final inhibitor concentration (the Stock Solution) and serially diluted in half-log increments resulting in final concentrations 250 μM to 75 μM, 25 μM, 7.5 μM, 2.5 μM, 0.75 μM, 0.25 μM, 75 nM, 25 nM, 7.5 nM in DMSO. 10 μl of each compound is placed in a 96-well plate as the intermediate plate. 90 μl of Kinase Buffer is added to to each well to prepare the intermediate plate.Figure US10906896-20210202-P00001Mix the compounds in intermediate plate for 10 min on shaker. For the assay of enzyme inhibitions, 5 μl of each well from the intermediate plate is transferred to a 384-well plate in duplicates, 10. Then 10 μl of 2.5× enzyme solution is added to each well of the 384-well assay plate and incubated for 10 min. Then enzyme substrate is added as 10 μl of 2.5×FAM-labeled peptide+ATP solution to each well of the 384-well assay plate. The reaction is allowed to proceed at 28° C. and quenched with the addition of 25 μl of stop buffer.
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 668.77, ALogP 4.5, HBA 11, HBD 4, PSA 168.78, QED 0.17
Compound (SMILES): N#CCC(=O)N1CCN(c2ccc(Nc3nc(N4CCCCCC4)nc(C=N)c3C(N)=O)cc2)CC1
InChIKey: CTFYZKXJZIFWJX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of Enzymatic JAK2 Kinase Activity: Two μL of 10% DMSO in blank, substrate control and positive control wells and 2 μL of test compound in test wells was added. Thirteen μL of assay buffer in blank and substrate control wells and 13 μL of Enzyme buffer mix in positive and test wells was added. The reaction mixture was incubated for 30 minutes at RT on a plate shaker. Ultra Light-pGT substrate (5 μL) [poly Glu-Tyr (4:1) labeled with U Light TM dye, a tyrosine kinase substrate] and ATP mix was added to all wells. The reaction plate was incubated for 60 minutes at RT on a plate shaker. The reaction was stopped by adding 40 mM EDTA (10 μL) in buffer. Ten μL of antibody was added to all the wells. The plate was read in a Wallac 1420 Multilabel Counter Victor 3 instrument (Ex: 340 nm Em: 615 & 665 nm).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 489.58, ALogP 2.26, HBA 9, HBD 3, PSA 155.33, QED 0.5
Compound (SMILES): COC(=O)c1cc2c(cc1C)/C(=C(/Nc1ccc(C(=O)NOCCN3CCC(OC)CC3)cc1)c1ccccc1)C(=O)N2
InChIKey: DPFXYGUGSJIYFI-FLWNBWAVSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: FGFR3 Enzyme Inhibition: The inhibitory activities of compounds of the invention against FGFR3 (FGFR3 Kinase Enzyme System: Promega), were evaluated by mixing the FGFR3 protein (12.5 ng/mL, 2 μL), substrate (Poly (Ala6, Glu2, Lys5, Tyr1), 100 ng/mL, 2 μL) with the test compound (2 μL at either 3 μM, 0.67 μM, 0.15 μM, 0.033 μM, 0.0073 μM, 0.0016 μM, 0.0036 μM or 0.00008 μM) for 90 min at 25° C. The kinase reaction was initiated by adding ATP (50 μM, 2 μL) and the mixture was incubated for 90 min at 25° C. ADP-Glo reagent was added for 40 min (8 μL) then development reagent (16 μL) was added for 40 min prior to detection in a microplate reader (EnVision® Multilabel Reader, Perkin Elmer).
Measurement: Ki = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 584.67, ALogP 4.49, HBA 8, HBD 3, PSA 118.23, QED 0.14
Compound (SMILES): CC1CCC(NC(=O)c2c(O)c3cc(-c4ccc(OC(F)F)cc4)cnc3n(CCN3CCOCC3)c2=O)CC1
InChIKey: ISZULGQYLMYQHU-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: CB1 and CB2 Receptor Binding Assay: CB1R binding protocol involves the use of the same solution buffer used for both incubation and washing reaction (Tris-HCl, 50 mM; EDTA, 2.5 mM; MgCl2, 2.5 mM; BSA, 0.5 mg/mL at pH 7.4), 0.4 nM for [3H]CP-55,940, test compounds (concentrations from 0.001 to 10 μM), and finally 8 μg/sample membrane in a total volume of 200 μL. CB2R binding assays were carried out with two different buffers: incubation buffer (Tris-HCl, 50 mM; MgCl2, 5 mM; CaCl2 1 mM; BSA, 0.2% at pH 7.4) and washing buffer (Tris-HCl, 50 mM; NaCl 500 mM; BSA, 0.1% at pH 7.4). The assay mixture contained incubation buffer, 0.4 nM [3H]CP-55,940, test substances (concentrations from 0.001 to 10 μM), and 4 μg/sample membrane in a total assay volume of 200 μL. Assays were performed in duplicate and incubated for 120 mM at 37° C. After the incubation, the assay mixture is filtered through 96 GF/C filter plates (Perkin Elmer #6005174) using Perkin Elmer Filtermate Harvester, and then washed four times with ice-cold washing buffer.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 556.61, ALogP 4.01, HBA 8, HBD 2, PSA 105.92, QED 0.43
Compound (SMILES): NC(=O)c1c(-c2cccc(F)c2)nn2c1CN(C(=O)Nc1ccc(F)cc1)CC2
InChIKey: NHQNHJBTEGWIEH-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 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 unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 397.39, ALogP 2.97, HBA 4, HBD 2, PSA 93.25, QED 0.71
Compound (SMILES): CC[C@H](Cn1ccc2cnc(Nc3cnn(C4CC(C)(C)OC4=O)c3)nc21)C(C)C
InChIKey: CSYBUEKYVZJVJN-NNJIEVJOSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK/TYK2 Assay: 10 mM test compound stock or 1 mM control compound stock (tofocitinib, ruxolitinib or staurosporine) in DMSO was diluted to 0.4 mM in DMSO. A 3-fold series dilution was then performed in DMSO to generate 10 different compound concentrations. The assay was carried out in 384-well white plate. 0.5 uL of 40× compound DMSO solution at different concentrations was mixed with 10 uL 2× enzyme prepared in reaction buffer (20 mM HEPES, 10 mM MgCl2, 0.01% Tween, 1 mM DTT, pH 7.5). 10 uL 2× substrate mixture prepared in reaction buffer was then added to start the reaction. A short spin was done to settle down all solutions to the bottom of the plate. Final concentrations of test compound in the reaction mixture were 10000, 3333, 1111, 370, 123, 41.2, 13.7, 4.57, 1.52and 0.51 nM. Concentrations of control compound were ten times less. Enzymatic reaction was conducted at 25° C. for 1-2 hours. 10 uL of Kinase Glo Reagents was added to stop the reaction and generate the luminescent signal which was measured using Envision. Luminescence signal was inversely related to kinase activity. Reaction mixture which did not contain enzyme served as negative control. The mixture without any compound was the positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 410.52, ALogP 4.32, HBA 8, HBD 1, PSA 86.86, QED 0.58
Compound (SMILES): N#CCC1(n2ccc(-c3nc(-c4ccc(N)cn4)cc4ncccc34)n2)CC(C#N)C1
InChIKey: BMHGXAASLMLUDN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% 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 JAK 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% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 1312 nM (pChEMBL 5.880000114440918)
Descriptors: MW 406.45, ALogP 3.68, HBA 8, HBD 1, PSA 130.09, QED 0.55
Compound (SMILES): CN1CCC(c2ccc3c(c2)[nH]/c(=N\C(=O)c2ccnc(-c4cccnc4)c2)n3C2CCCC2)CC1
InChIKey: IKUFIGKDUOXFCR-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Biochemical EGFR Inhibition Assays (100 mM ATP): 5 μL of EGFR enzyme form in assay buffer (50 mM HEPES pH 7.3, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween 20, 2 mM DTT) are dispensed into columns 1-23, than 5 μL of ATP and ULight-poly-GT substrate (PerkinElmer; TRF0100-M) mix in assay buffer is added to all wells (final assay concentration of the ULight-poly-GT substrate 200 nM). Each of the different EGFR enzyme form assays is available at high ATP levels (final assay concentration 100 μM). After 90 minutes incubation at room temperature 5 μL EDTA (final assay concentration 50 mM) and LANCE Eu-anti-P-Tyr (PT66) antibody (PerkinElmer, AD0069) (final assay concentration 6 nM) mix are added to stop the reaction and start the binding of the antibody. After additional 60 minutes incubation at room temperature the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the TR-FRET LANCE Ultra specs of PerkinElmer (used wavelengths: excitation 320 nm, emission 1 665 nm, emission 2 615 nm). Each plate contains 16 wells of a negative control (diluted DMSO instead of test compound; w EGFR enzyme form; column 23) and 16 wells of a positive control (diluted DMSO instead of test compound; w/o EGFR enzyme form; column 24). Negative and positive control values are used for normalization and IC50 values are calculated and analysed using a 4 parametric logistic model.
Measurement: IC50 = 27.7 nM (pChEMBL 7.559999942779541)
Descriptors: MW 480.62, ALogP 5.09, HBA 5, HBD 1, PSA 79.17, QED 0.44
Compound (SMILES): COc1ccc(-n2nc(C(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4cc(C(=O)NCCO)ccc4OC)n3)c3ccccc23)cc1
InChIKey: ZGVCPFSIDNPESN-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: c-Src Enzyme Inhibition 1: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The c-Src enzyme (3000 ng/mL, 2.5 μL) is incubated with the test compound (either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL, 2.5 μL each) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solutions (2.5 μL, 800 μM) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 702.77, ALogP 6.86, HBA 11, HBD 5, PSA 173.78, QED 0.09
Compound (SMILES): CCc1c(-c2ccc(OC)c(OC)c2)[nH]c2ccc(C(=O)NCCN3CCCCC3)cc12
InChIKey: YNVMTDIPVPOBCX-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 2409 nM (pChEMBL 5.619999885559082)
Descriptors: MW 435.57, ALogP 4.63, HBA 4, HBD 2, PSA 66.59, QED 0.54
Compound (SMILES): C[C@H](CN(C)C)NC(=O)c1ncc(-c2noc(C(F)(F)F)n2)cc1Cl
InChIKey: GSQJZSWRISFRIE-SSDOTTSWSA-N
Target: Histone deacetylase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P56524
Assay: HDAC4 Assay: Human recombinant HDAC4 was expressed in full length form (aa 2-1084) in Sf9 insect cells (obtained from ATCC) using baculovirus generated with Bac-to-Bac system (Invitrogen). Test compounds were serially diluted to reach final test concentrations from 0.000128 μM to 10 μM. HDAC4 and test compounds were incubated in 25 mM Tris buffer pH 8.0 containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.05% (w/v) bovine serum albumine and 0.005% (v/v) Triton-X-100 for 2 hours at room temperature in presence of 5 μM of acetyl-Gly-Ala-Lys(ε-trifluoroacetyl)-AMC (AMC=7-amino-4-methyl coumarin) in a final volume of 9 μl. Control wells with HDAC4 only (positive control) and without HDAC4 (negative control) were included on the microplate. Bovine trypsin (4.5 μl of a 300 nM solution) was added and the plate incubated for additional 15 minutes at room temperature. The plate was placed in a fluorescence microplate reader, and read at an excitation wavelength of 360 nm and an emission wavelength of 450 nm with a 10 nm bandpath. Fluorescence values for all wells containing HDAC4 (positive control and wells with test compound) were corrected by subtracting negative control fluorescence values, and IC50 values were calculated by fitting the dose-response curves to a 4-parameter logistic function.
Measurement: IC50 = 3600 nM (pChEMBL 5.440000057220459)
Descriptors: MW 377.75, ALogP 2.48, HBA 6, HBD 1, PSA 84.15, QED 0.86
Compound (SMILES): Cc1cnc2cc(-n3nc(-c4nc(-c5cnn(C6CCNCC6)c5)cnc4N)ccc3=O)ccc2c1
InChIKey: LHHDRTUNEIVZGK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Enzyme Assay: FGFR1, 2 and 3 kinase activity was measured by the Invitrogen LanthaScreen Assay technology which directly measures the amount of substrate phosphorylation by TR-FRET using a fluorescein-labeled peptide and Europium-labeled antibody.To measure FGFR1 kinase activity, 200 pM His-tagged recombinant human FGFR1 catalytic domain (amino acids 308-731) (Life Technologies Cat. No. PR4660A) was incubated with 100 nM Alexa Fluor® 647-Poly-GT Peptide Substrate (Life Technologies Cat. No. PV5836) and ATP in the presence of Mg++, along with test compound in a buffer consisting of 250 mM HEPES, 25 mM MgCl2, 0.05% TritonX-100, pH 7.5, and 2% DMSO. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After 20 minutes incubation at 22° C., an equal volume of 2 nM LanthaScreen® Eu-PY20 Antibody (Life Technologies Cat. No. PV5691) and EDTA were added to quench the kinase reaction and start the detection reaction. After an additional 60 minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compound and 0 POC is determined using no enzyme. The POC values were fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.To measure FGFR2 kinase activity: 200 pM His-tagged recombinant human FGFR2 cytoplasmic domain (amino acids 403-822), (Life Technologies Cat. No. PR5332A); 20 minutes incubation at 22° C., 60 minute detection incubation at 22° C.To measure FGFR3 kinase activity: 750 pM N-terminal GST-HIS6 fusion protein with a 3C cleavage site recombinant human FGFR3 (amino acids R397-T806) (ProQinase Cat. No. 1068-0000-1); 10 minutes incubation at 22° C., 60 minute detection incubation at 22° C.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 479.55, ALogP 2.92, HBA 10, HBD 2, PSA 129.43, QED 0.4
Compound (SMILES): COC(=O)c1c(OC)cccc1O[C@@H]1CC[C@@H](C)N(C(=O)c2ccccc2-c2ncccn2)C1
InChIKey: YWOYASQIABQMBZ-QZTJIDSGSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: FLIPR Ca2+ Flux Assay: In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 461.52, ALogP 4.01, HBA 7, HBD 0, PSA 90.85, QED 0.51
Compound (SMILES): Cc1cc(F)ccc1-c1cc(N2CCC(CC(=O)O)CC2)nc(C#N)c1N(C)C(=O)C(C)(C)c1cc(C(F)(F)F)cc(C(F)(F)F)c1
InChIKey: XAMIKPXOHVPWBZ-UHFFFAOYSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibitory Effect on CYP3A4: A dimethyl sulfoxide (DMSO) solution of a test compound with a concentration 1000 times higher than the evaluation concentration was prepared, and a reaction solution was prepared by diluting the solution. Enzyme reaction was performed by incubating in a potassium phosphate buffer solution (pH 7.4) containing 1 nM to 20 μM test compound, 3.2 mM magnesium chloride, 0.2 pmol human CYP3A4 (BD Biosciences), 0.5 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 3 μM Luciferin-IPA (Promega) at 37° C. for 10 minutes. The volume of the reaction solution was 50 μL/well. The 30-minute pre-incubation group was incubated at 37° C. for 30 minutes before adding the substrate, the Luciferin-IPA solution (12.5 μL/well). At the end of the enzyme reaction, 50 μL/well of a Luciferin detection reagent (Promega) was added to the wells, and the plate was left at room temperature for 20 minutes. Then, the emission intensities were measured with Infinite M1000 (TECAN). The enzyme activities (%) relative to the value of the group to which the test compound was not added were calculated. A dose-response curve was drawn using analysis software, GraphPad Prism (GraphPad Software), and the concentration of each compound that exhibited 50% inhibition, IC50, was calculated. As a comparative example, aprepitant, which is an NK1 receptor antagonist, was tested in the same manner.
Measurement: IC50 = 2600 nM (pChEMBL 5.579999923706055)
Descriptors: MW 664.62, ALogP 7.74, HBA 5, HBD 1, PSA 97.53, QED 0.26
Compound (SMILES): Cc1cc(CC(=O)N[C@H]2CC[C@H](CCN3C[C@@H]4C[C@]4(c4cc(C(C)(F)F)on4)C3)CC2)on1
InChIKey: RLCITMZJUDPBHU-KOAQGZTJSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Binding Inhibition for Dopamine D2 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl2.6H2O (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl2 (067-31, NAKARAI CHEMICALS, LTD.)Radioligand: (final concentration) 1.2 nM [3H]-Methylspiperone ([3HN-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 μM Butaclamol [(+)-Butaclamol Hydrochloride, D033; Sigma]SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well)Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 462.54, ALogP 3.96, HBA 6, HBD 1, PSA 84.4, QED 0.64
Compound (SMILES): O=C(C1CCS(=O)(=O)C1)N(Cc1ccc(-c2noc(C(F)(F)F)n2)cn1)c1cccc(F)c1
InChIKey: PKCJSFYDCXQFAP-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzymatic Assay: HDAC1 and 6 reagents: FLAG-tagged HDACs 1 and 6 were prepared in-house by protein expression in HEK293F cells followed by anti-FLAG affinity purification. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 137 mM NaCl [Sigma, catalog #S5150, 5M stock], 2.7 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], and 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock]. In addition to the above buffer ingredients, TCEP [CalBiochem, catalog #580561, 500 mM stock] was added at a final concentration of 0.5 mM to the buffer for the HDAC6 assays. HDAC1, 2, 3, and 6 enzymes were run at the final concentrations of 0.3 nM, 1.5 nM, 0.3 nM, and 1.333 nM, respectively. Fluor-de-Lys substrate [BioMol Research Laboratories, catalog #KI-104], used to evaluate enzyme activity, was added at the final concentrations of 20 uM, 40 uM, 20 uM, and 2.5 uM for HDACs 1, 2, 3, and 6. To enable detection of the signal, Developer [BioMol Research Laboratories, catalog #KI-105] was added at a 1:250 dilution to the stop solution, which also included 10 uM SAHA [Sigma, catalog #SML0061] to ensure complete termination of the reaction.
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 484.43, ALogP 3.26, HBA 7, HBD 0, PSA 106.26, QED 0.51
Compound (SMILES): COc1cncc(OC[C@@H](C)CN)c1-c1cc(Nc2cnc(C#N)cn2)n[nH]1
InChIKey: YRHQDDRNFFJEKC-NSHDSACASA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: hERG Assay: The test compounds were added to cultured hERG (human Ether-a-go-go Related Gene) gene stably expressing CHO cell line cells to achieve 0.27 to 100 μM. The hERG current under electric potential stimulation was measured using Qube384 (Sophion Bioscience) to calculate the concentration at which each test compound suppresses 50% hERG current (IC50 value; μM).
Measurement: IC50 = 13900 nM (pChEMBL 4.860000133514404)
Descriptors: MW 380.41, ALogP 1.86, HBA 9, HBD 3, PSA 147.65, QED 0.53
Compound (SMILES): CN(C)CCN(C)C(=O)c1cccc(-c2cnc(N)c(C(=O)Nc3ccccc3)n2)c1
InChIKey: HQVIRTZEPFATMS-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 (3 mCi 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).
Measurement: Ki = 2550 nM (pChEMBL 5.590000152587891)
Descriptors: MW 418.5, ALogP 2.61, HBA 6, HBD 2, PSA 104.45, QED 0.61
Compound (SMILES): CCN1CCN(c2ccc(NC(=O)c3ccc(C)c(NC(=O)c4cncc(-c5cccs5)c4)c3)cc2C(F)(F)F)CC1
InChIKey: KAEHLZHGEOJDKT-UHFFFAOYSA-N
Target: Nuclear receptor subfamily 2 group C member 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P49116
Assay: Inhibition Assay: The in vitro activity of the compounds described herein in inhibiting TAK1, HCK, and other kinases were obtained using an Invitrogen Select Screening assay as known in the art.
Measurement: IC50 = 117 nM (pChEMBL 6.929999828338623)
Descriptors: MW 593.68, ALogP 6.78, HBA 6, HBD 2, PSA 77.57, QED 0.25
Compound (SMILES): C[C@@H](O)COc1cc(-c2ccc(N3CCN(Cc4ccccn4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: ZVGAVNAMWIVXCT-LJQANCHMSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype 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; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog 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 = 1076 nM (pChEMBL 5.96999979019165)
Descriptors: MW 469.55, ALogP 2.74, HBA 9, HBD 1, PSA 102.81, QED 0.44
Compound (SMILES): O=c1n(Cc2ccc(Cl)cc2F)c2sc3c(c2c2ncnn12)CCOC3
InChIKey: WWBSJDAJGFOJCZ-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: PDE1B Inhibitory Assay: PDE1B inhibition was determined by an IMAP TR-FRET assay. The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, Calmodulin, cAMP or cGMP substrate, DMSO tolerance, and incubation time. Into each well of a solid white 1536 well plate (Corning) was dispensed 250 pg full-length recombinant NH-terminal GST tagged human PDE1B enzyme (BPS Bioscience Cat #60011, San Diego, Calif.) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 10 U/ml Calmodulin and 2.5 mM CaCl2 (Sigma Aldrich.) After a brief centrifugation, 30 nl compound was added by transfer from 1 mM stock in DMSO using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 533 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 200 nM. After a brief centrifugation, the plates were incubated for 30 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer). The instrument was set to excite using the DUG11 filter and measure using 490/10 nm and 520/10 nm filters. Ratios of acceptor and donor were then calculated.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 390.83, ALogP 3.02, HBA 7, HBD 0, PSA 61.42, QED 0.53
Compound (SMILES): CCN(CC(F)(F)F)C(=O)c1ccc(Nc2nn([C@@H](CC#N)C3CC3)c3cc[nH]c(=O)c23)cc1C
InChIKey: UYBJTXWMDFRTGQ-SFHVURJKSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Jak Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 810 nM (pChEMBL 6.090000152587891)
Descriptors: MW 486.5, ALogP 4.67, HBA 6, HBD 2, PSA 106.81, QED 0.48
Compound (SMILES): CN1CCN(S(=O)(=O)N(Cc2ccc(-c3nnc(C(F)F)o3)cc2)c2ccc(F)c(Cl)c2)CC1
InChIKey: XPMZKRDZZWCBDE-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzyme Activity Inhibition Assay: HDAC enzyme activity was measured using a HDAC Fluorimetric Drug Discovery Kit (BML-AK511, 516, Enzo Life Science). For the HDAC1 enzyme activity test, human recombinant HDAC1 (BML-SE456) was used as an enzyme source, and Fluor de Lys-SIRT1 (BNL-KI177) was used as a substrate. A 5-fold dilution of the compound was seeded into a 96-well plate, and then 0.3 μg of the enzyme and 10 μM of the substrate were added to each well of the plate and allowed to react at 30 for 60 minutes. Then, Fluor de LysDeveloper II (BML-KI176) was added thereto and allowed to react for 30 minutes, after which the fluorescence value (Ex 360, Em 460) was measured using a multi-plate reader (Flexstation 3, Molecular Device). The HDAC6 enzyme was tested using human recombinant HDAC6 (382180) (Calbiochem) according to the same protocol as the HDAC1 enzyme activity test method.
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 515.95, ALogP 3.97, HBA 6, HBD 0, PSA 82.78, QED 0.47
Compound (SMILES): O=C(Nc1cccc(-c2nnc(Nc3ccc4[nH]ncc4c3Cl)s2)c1)C1CCNCC1
InChIKey: GFWJDNLJIOICHS-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Biological Assays for Inhibition of Rho-Associated Protein Kinase: Assays for ROCK inhibition were performed using the following protein constructs: glutathione S-transferase (GST)-tagged human ROCK1 catalytic domain 1-477 from Carna Biosciences (cat #01-109; apparent Km value for ATP is 10 μM) and GST-tagged human ROCK2 catalytic domain 1-553 from Carna Biosciences (Cat#01-110; apparent Km value for ATP is 15 μM). Protein constructs were purified from a baculovirus expression system. The peptide substrate was fluorescent LANCE® Ultra ULight-CREBtide: CKRREILSRRPSYRK (PerkinElmer, # TRF0107-D). Kinase reactions were carried out in in a 10 μL volume in 384-well plates: 50 nM ULight-CREBtide substrate, 2 nM constitutively active ROCK1 or ROCK2 kinase, and test compound in DMSO (or DMSO only for controls) were diluted into assay buffer containing 50 mM Tris-HCl (pH=7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, and 2 mM DTT such that the final concentration of DMSO was 0.5%. After a 90 minute incubation at room temperature on a shaker table, the kinase reaction was stopped by addition of 10 mM EDTA, and phosphorylation of the substrate was detected by adding 1 nM LANCE® Ultra Europium-anti-phospho-CREB (ser133) antibody (PerkinElmer, # TRF0200-D) and incubating for 60 minutes on a shaker at room temperature. The flurorescence resonance energy transfer (FRET) signals were read and analyzed on an Envision¿ 2103 Multilabel Reader (Perkin Elmer). The concentration of test compound required to inhibit substrate phosphorylation by 50% (the IC50) was calculated by non-linear regression using GraphPad PRIZM.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 453.96, ALogP 4.42, HBA 7, HBD 4, PSA 107.62, QED 0.36
Compound (SMILES): C[C@H](Nc1nccc(N2C(=O)OCC2[C@@H](C)C(F)(F)F)n1)c1nc(-c2ccc(Cl)cc2)no1
InChIKey: VWTGBFKWGWEASC-ZAZPPBBNSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 = 2085 nM (pChEMBL 5.679999828338623)
Descriptors: MW 482.85, ALogP 4.88, HBA 8, HBD 1, PSA 106.27, QED 0.53
Compound (SMILES): CO[C@H]1CCN(Cc2cc3c(nc2C=O)N(C(=O)Nc2cc(NC4COC4)c(C#N)cn2)CCC3)C1=O
InChIKey: IJIXBFOMNRPTTQ-NRFANRHFSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymologic Experiment of FGFR1: In this experiment, the inhibitory effect of the compounds on FGFR1 kinase activity was tested by a fluorescence resonance energy transfer (TR-FRET) method, and the half maximal inhibitory concentration (IC50) of the compounds on the FGFR1 kinase activity was determined. 1) 15 μL of FGFR1 enzymatic solution was added to a 384-well plate, and the final concentration of the enzyme was 0.15 nM. 2) 15 μL of diluted solution in gradient of the compound was added.3) 15 μL of a substrate mixture containing substrate polypeptide with a final concentration of 550 nM and ATP with a final concentration of 10200 μM was added. 4) The mixture was incubated for 0.5-3 hours at room temperature. 5) 10 μL of EDTA and a test solution comprising labeled antibody were added, and the plate was incubated for 1 hour at room temperature. 6) The fluorescence signal values of each plate were determined by a microplate reader at 665 nm. 7) The inhibition rates were calculated according to the fluorescence signal values.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 505.54, ALogP 1.7, HBA 9, HBD 2, PSA 149.78, QED 0.54
Compound (SMILES): CN1CCC(c2ccc3c(c2)[nH]/c(=N\C(=O)c2ccnc(-c4cnn5cccnc45)c2)n3CC(C)(C)O)CC1
InChIKey: AEVLWBNAYZFMNY-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Biochemical EGFR Inhibition Assays (100 mM ATP): 5 μL of EGFR enzyme form in assay buffer (50 mM HEPES pH 7.3, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween 20, 2 mM DTT) are dispensed into columns 1-23, than 5 μL of ATP and ULight-poly-GT substrate (PerkinElmer; TRF0100-M) mix in assay buffer is added to all wells (final assay concentration of the ULight-poly-GT substrate 200 nM). Each of the different EGFR enzyme form assays is available at high ATP levels (final assay concentration 100 μM). After 90 minutes incubation at room temperature 5 μL EDTA (final assay concentration 50 mM) and LANCE Eu-anti-P-Tyr (PT66) antibody (PerkinElmer, AD0069) (final assay concentration 6 nM) mix are added to stop the reaction and start the binding of the antibody. After additional 60 minutes incubation at room temperature the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the TR-FRET LANCE Ultra specs of PerkinElmer (used wavelengths: excitation 320 nm, emission 1 665 nm, emission 2 615 nm). Each plate contains 16 wells of a negative control (diluted DMSO instead of test compound; w EGFR enzyme form; column 23) and 16 wells of a positive control (diluted DMSO instead of test compound; w/o EGFR enzyme form; column 24). Negative and positive control values are used for normalization and IC50 values are calculated and analysed using a 4 parametric logistic model.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 524.63, ALogP 3.4, HBA 8, HBD 2, PSA 116.7, QED 0.36
Compound (SMILES): COc1ccc2nc([C@@H]3CCCN3C(=O)c3nc(C)sc3-c3cccc(Cl)c3Cl)[nH]c2c1
InChIKey: NSTXIYYXGQYSCJ-SFHVURJKSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 76 nM (pChEMBL 7.119999885559082)
Descriptors: MW 487.41, ALogP 6.29, HBA 5, HBD 1, PSA 71.11, QED 0.37
Compound (SMILES): CCc1nn(-c2cc(OC(C)C(CC)CC)c(C(=O)NC(CC)CC)cc2F)c(=O)n1C
InChIKey: PTLNNNORUJAFFA-UHFFFAOYSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Enzymatic Assay: Assay 2: The enzymatic assay couples DHODH activity with bleaching of the dye 2,6-Dichlorophenolindophenol (DCIP) (Knecht and Loffler, 1998; Miller et al., 1968). The assay was conducted in aqueous buffer containing 50 mM Tris, 0.1% Triton X-100, 150 mM potassium chloride, 0.4 μg/mL DHODH, 1 mM dihydroorotate, 0.1 mM decylubiquinone, 0.06 mM DCIP, and 0.17% DMSO at pH 8.0 at room temperature. Compounds were added via pin transfer or via D300 digital dispenser, and the reaction was initiated by addition of substrates. Enzyme activity was monitored kinetically by the reduction in DCIP absorbance at 600 nm. Purified recombinant human DHODH (full-length, C-terminal MYC/DDK-tag) enzyme was purchased from Origene (cat. no. TP039034). Other chemicals, including leflunomide and teriflunomide, were purchased from Sigma-Aldrich. Absorbance measurements were obtained using a Molecular Devices Spectramax M5 plate-reading spectrophotometer.
Measurement: IC50 = 246 nM (pChEMBL 6.610000133514404)
Descriptors: MW 448.58, ALogP 4.39, HBA 6, HBD 1, PSA 78.15, QED 0.55
Compound (SMILES): N[C@@H](Cc1ccccc1)C(=O)Nc1cccc(-c2nnc(Nc3ccc4[nH]ncc4c3Cl)s2)c1
InChIKey: WCXUGSIOXZKHMD-SFHVURJKSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Biological Assays for Inhibition of Rho-Associated Protein Kinase: Assays for ROCK inhibition were performed using the following protein constructs: glutathione S-transferase (GST)-tagged human ROCK1 catalytic domain 1-477 from Carna Biosciences (cat #01-109; apparent Km value for ATP is 10 μM) and GST-tagged human ROCK2 catalytic domain 1-553 from Carna Biosciences (Cat#01-110; apparent Km value for ATP is 15 μM). Protein constructs were purified from a baculovirus expression system. The peptide substrate was fluorescent LANCE® Ultra ULight-CREBtide: CKRREILSRRPSYRK (PerkinElmer, # TRF0107-D). Kinase reactions were carried out in in a 10 μL volume in 384-well plates: 50 nM ULight-CREBtide substrate, 2 nM constitutively active ROCK1 or ROCK2 kinase, and test compound in DMSO (or DMSO only for controls) were diluted into assay buffer containing 50 mM Tris-HCl (pH=7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, and 2 mM DTT such that the final concentration of DMSO was 0.5%. After a 90 minute incubation at room temperature on a shaker table, the kinase reaction was stopped by addition of 10 mM EDTA, and phosphorylation of the substrate was detected by adding 1 nM LANCE® Ultra Europium-anti-phospho-CREB (ser133) antibody (PerkinElmer, # TRF0200-D) and incubating for 60 minutes on a shaker at room temperature. The flurorescence resonance energy transfer (FRET) signals were read and analyzed on an Envision¿ 2103 Multilabel Reader (Perkin Elmer). The concentration of test compound required to inhibit substrate phosphorylation by 50% (the IC50) was calculated by non-linear regression using GraphPad PRIZM.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 489.99, ALogP 4.99, HBA 7, HBD 4, PSA 121.61, QED 0.26
Compound (SMILES): Cc1cc(C#N)cnc1C(=O)Nc1ccc2[nH]nc(-c3cccc(N(C)C)c3)c2c1
InChIKey: MKJCCXWLMISQRX-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen Kinase Activity Assay: The assay was carried out under the following protocol conditions: 1 mM compound in DMSO was serially diluted 1:3, 11 points in DMSO with a Biomek FX and 0.1 μL of the diluted compound was subsequently stamped into the assay plate (384-well format Lumitrac 200, Greiner, 781075) with an Echo Labcyte such that the final compound concentration in the assay was 10 μM to 169 μM. Subsequently, 5 μL of 2× kinase solution (2.9 nM final concentration) was added to the assay plate in assay buffer composed of 50 mM Tris pH 8.5 (Sigma, T6791), 5 mM MgCl2 (Fluka, 63020), 1 mM EGTA (Sigma, E3889), 0.01% BRIJ-35 (Sigma, P1254) and 2 mM DTT. The reaction was started by addition of 2×ATP/LRRKtide solution in assay buffer such that the final concentration was 400 nM LRRKtide and 25 μM ATP. After 60 min incubation at room temperature, the reaction was stopped by addition of 10 μL of 2× stop solution containing a final concentration of 2 nM anti-pERM antibody and 10 mM EDTA. After a 30 min incubation at RT, the TR-FRET signal was measured on a Wallac 2104 EnVision multilabel reader at an excitation wavelength of 340 nm and reading emission at 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 396.45, ALogP 4.12, HBA 5, HBD 2, PSA 97.7, QED 0.54
Compound (SMILES): N#CCC1CCN(c2nc(C=N)c(C(N)=O)c(Nc3ccc(N4CCC(CO)CC4)cc3)n2)CC1
InChIKey: JZEQJJYKVZQHLI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of Enzymatic JAK2 Kinase Activity: Two μL of 10% DMSO in blank, substrate control and positive control wells and 2 μL of test compound in test wells was added. Thirteen μL of assay buffer in blank and substrate control wells and 13 μL of Enzyme buffer mix in positive and test wells was added. The reaction mixture was incubated for 30 minutes at RT on a plate shaker. Ultra Light-pGT substrate (5 μL) [poly Glu-Tyr (4:1) labeled with U Light TM dye, a tyrosine kinase substrate] and ATP mix was added to all wells. The reaction plate was incubated for 60 minutes at RT on a plate shaker. The reaction was stopped by adding 40 mM EDTA (10 μL) in buffer. Ten μL of antibody was added to all the wells. The plate was read in a Wallac 1420 Multilabel Counter Victor 3 instrument (Ex: 340 nm Em: 615 & 665 nm).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 476.59, ALogP 2.66, HBA 9, HBD 4, PSA 155.25, QED 0.42