lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(Cn2c(=O)n(CC(C)(C)C(=O)O)c(=O)[nH]/c2=N\c2ccc(Oc3ccc(C)cn3)cc2)cc1
InChIKey: PCHZTGCPGFSPKI-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 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, 0.5% BSA, and 0.04% Pluronic P-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 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 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, 150 nM ATP solution (25 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 4 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.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 515.57, ALogP 3.53, HBA 8, HBD 2, PSA 131.57, QED 0.37
Compound (SMILES): Cc1c(C)c2cc(OCCN3CCOCC3)c(O)c(C=O)c2oc1=O
InChIKey: CJJIDUOGGDSOTE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase/endoribonuclease IRE1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75460
Assay: IRE-1alpha Assay: A fusion protein comprising glutathione S transferase (GST) and human IRE-1α (GST-IRE-1α) obtained from a 500 ml baculovirus-infected insect cell culture can be used to measure IRE-1α activity in vitro. Five μl of a reaction mixture comprising 1× reaction buffer (5× reaction buffer is 100 mM Hepes pH 7.5, 250 mM KOAc, 2.5 mM MgCl2), 3 mM DTT, and 0.4% polyethylene glycol water is added to each well of 384 well plates. Twenty-five nanoliters of a 1 mM test compound solution are added to test wells. Three μl of a 128 ng/ml IRE-1α preparation are added to each test well and to positive control wells (final concentration 5.82 ng/well). Negative control wells contain only reaction mixture and test compound. After spinning the plates at 1200 rpm for 30 seconds, 3 μl of an IRE-1α human mini-XBP-1 mRNA stem-loop substrate 5'-CAGUCCGCAGCACUG-3' (SEQ ID NO:1), labeled with the fluorescent dye Cy5 at the 5' end and Black Hole Quencher 2 (BH2) at the 3' end, are added to each well of a control plate. The plates are again spun at 1200 rpm for 30 seconds. Final concentrations for the assay are: 63 nM IRE-1α substrate, 5.82 ng IRE-1α protein, and 2.5 μM test compound. The plates are covered with lids and incubated for one hour at 30° C. The plates are then transferred to an ACQUEST™ microplate reader. Data is analyzed using data analysis software, and the percent activity of IRE-1α is calculated.
Measurement: EC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 347.37, ALogP 1.64, HBA 7, HBD 1, PSA 89.21, QED 0.65
Compound (SMILES): O=C(CCO)N1CCN(Cc2ccc(-c3cnc4[nH]cc(-c5ccc6[nH]ccc6c5)c4c3)cc2)CC1
InChIKey: NNTDMHUTGDBABF-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16584
Assay: Radiometric Filter Plate MLK3 Assay: 200 ng (130 nM) MLK3 (Dundee, DU8313) was incubated with 1 μM inactive MKK7b (Dundee, DU703) in the presence of 2 μM cold ATP (Km) and 0.5 μCi/assay 33P ATP and appropriate concentrations of compounds. After a twenty minute incubation, the reactions were washed through filter plates and read on a scintillation counter.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 479.58, ALogP 4.4, HBA 4, HBD 3, PSA 88.25, QED 0.34
Compound (SMILES): O=C(O)C[C@H](O)Cn1c(=O)[nH]/c(=N\c2ccc(Oc3ccc(F)cn3)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: WJDWVKBIAGOSQO-IBGZPJMESA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 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, 0.5% BSA, and 0.04% Pluronic P-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 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 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, 150 nM ATP solution (25 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 4 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.
Measurement: IC50 = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 541.92, ALogP 2.43, HBA 9, HBD 3, PSA 151.8, QED 0.29
Compound (SMILES): CC(=O)N1CCN(c2cccc(-c3cc(-c4cnnn4C(C)C)c4c(N)ncnn34)c2)C(C)(C)C1
InChIKey: RGOPOOXTYGJFHL-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 473.59, ALogP 3.27, HBA 9, HBD 1, PSA 110.47, QED 0.48
Compound (SMILES): Cn1cc(-c2ccc3c(ncn3C)c2Oc2ccc(F)cc2F)c2cc[nH]c2c1=O
InChIKey: PMAYQUBINLZCDL-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech). IC50 determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 5.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 406.39, ALogP 4.49, HBA 5, HBD 1, PSA 64.84, QED 0.48
Compound (SMILES): NC[C@H]1CC[C@H](C(=O)N[C@H]2C/C=C\CCCCc3ccccc3-c3c[nH]c2n3)CC1
InChIKey: DEKJPODSYVPCPW-LYIDWPMZSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: In Vitro Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 6174 nM (pChEMBL 5.210000038146973)
Descriptors: MW 406.57, ALogP 4.67, HBA 3, HBD 3, PSA 83.8, QED 0.65
Compound (SMILES): CCOc1ccc2cc(-c3nn(CCOC)c(N)c3C(=O)NCCOC)cnc2c1
InChIKey: BBTFPYWNKYLKIS-UHFFFAOYSA-N
Target: Calmodulin-domain protein kinase 1 (SINGLE PROTEIN), Toxoplasma gondii
UniProt: Q9BJF5
Assay: Luminescent Kinase Assay: Inhibition of TgCDPK1 and CpCDPK1 was determined using a luminescent kinase assay which measures ATP depletion in the presence of the Syntide 2 peptide substrate (KinaseGlo). For description of the methods and assays, see WO 2011/094628, incorporated by reference herein. Similar to TgCDPK1, exogenous calcium was necessary for CpCDPK1 to possess maximum catalytic activity (data not shown). Notably, both kinases were tested at the same ATP concentration which allows direct comparison of inhibitor potencies due to these enzymes possessing similar Kms for this cofactor.
Measurement: IC50 = 321 nM (pChEMBL 6.489999771118164)
Descriptors: MW 413.48, ALogP 2.1, HBA 8, HBD 2, PSA 113.52, QED 0.49
Compound (SMILES): O=C(O)c1ccc(NC(=O)[C@@H](CC2CC2)c2ccc(-c3c(OC(F)F)ccc(Cl)c3F)c[n+]2[O-])cc1
InChIKey: KRBJCJHQECPFLG-KRWDZBQOSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Factor XIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIa can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at room temperature or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the half-maximal inhibitory concentrations (IC50), or the inhibitory constant, Ki.Compounds were pre-incubated for 30 minutes at 25° C. with human (0.04 nM) Factor XIa in 50 mM HEPES buffer with 150 mM sodium chloride, 5 mM calcium chloride, 0.1% PEG 8000, pH 7.4. Factor XIa enzymatic activity was determined by addition of the substrate glycine-proline-arginine-7-amido-4-trifluoromethylcoumarin (GPR-AFC) and measurement of the fluorescence at 400/505 nm after a 60 minute incubation at 25° C. The % inhibition for each data point was calculated from the data and analyzed using the log (inhibitor) vs. response four parameters equation to determine the half-maximal inhibitory concentrations (IC50). The IC50 were converted to equilibrium inhibitory constants (Ki) using the Cheng-Prusoff equation.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 520.89, ALogP 5.6, HBA 4, HBD 2, PSA 102.57, QED 0.28
Compound (SMILES): CCn1nc(C(=O)NC[C@]2(CC)CC[C@H](S(C)(=O)=O)CC2)c(Cl)c1-c1ccc(CC(C)(C)C(F)(F)F)cc1OC(F)F
InChIKey: QGUWIYPCVCYMGW-LIZUVYQKSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Development, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature.
Measurement: Kd = 10 nM (pChEMBL 8)
Descriptors: MW 642.13, ALogP 7.07, HBA 6, HBD 1, PSA 90.29, QED 0.27
Compound (SMILES): CC1(Oc2cc3c(-c4cc(N5CCc6nccnc6C5)ncn4)n[nH]c3cc2F)CC1
InChIKey: VQUUMQFYGQCGHD-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Km ATP LanthaScreen Assay: a) 400 nl of a 1:2.15 serial dilution of test compound (98 uM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 ul of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1x assay buffer (50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 ul of 800 nM fluorescein labeled LRRKtide peptide substrate and 186 uM ATP solution in 1x assay buffer to all wells. d) After a 60 minute room temperature incubation, 20 ul of TR-FRET Dilution Buffer (Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision multi-mode plate reader with Lantha Screen settings.
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 417.45, ALogP 3.44, HBA 7, HBD 1, PSA 92.71, QED 0.54
Compound (SMILES): COc1cn(Cc2cc(F)cc(F)c2)c(Nc2cc(S(C)(=O)=O)cc(C)c2C)nc1=O
InChIKey: DFTGRAIGNZTGFT-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
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. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 449.48, ALogP 3.34, HBA 7, HBD 1, PSA 90.29, QED 0.62
Compound (SMILES): COc1ccc2[nH]c(NC(=O)c3cccc(Cn4nc(-c5cccnc5)ccc4=O)c3)nc2c1
InChIKey: PETOGDHWSNAURO-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P51617
Assay: IRAK1 Enzymatic Assay: IRAK1 is a human purified recombinant enzyme (His-TEV-IRAK1 (194-712)) In this assay, IRAK-1 hydrolyses ATP and autophosphorylates.Measurement of IRAK-1 inhibition is performed in streptavidin coated 384 well FlashPlate (PerkinElmer #SMP410A).His-TEV-IRAK-1 (15 ng/well), ATP (1 μM, [33P]ATP 0.25 μCi/well) and compounds in DMSO (range of concentrations from 20 μM to 1 nM) or controls (2% DMSO) are incubated for 3 hours at 30° C. in assay buffer: Hepes pH7.0 50 mM, Fatty acid-free BSA 0.1%, Dithiothreitol DTT 2 mM, MgCl2 10 mM, EGTA 0.5 mM, Triton-X-100 0.01%. Kinase reaction is stopped by addition of EDTA. Supernatant is discarded, plates are washed three times with 150 mM NaCl and radioactivity is then measured in a Microbeta Trilux reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 452.47, ALogP 3.49, HBA 7, HBD 2, PSA 114.79, QED 0.41
Compound (SMILES): Cc1nn2c3c(nc2s1)CCN(C(=O)COc1ccc(C#N)cc1Cl)C3c1ccc(Cl)nc1F
InChIKey: STJMGYOHQCYQOG-UHFFFAOYSA-N
Target: Tryptophan 5-hydroxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17752
Assay: Biochemical In Vitro Assay: To generate the enzyme, full length human TPH1 is cloned into the plasmid pET20b(+) (Novagen) and expressed in E. coli. The bacterial cells are ruptured by sonication on ice and the lysate is cleared by centrifugation. The resulting protein in the pellet is re-extracted and TPH1 is purified from the obtained lysate by affinity chromatography using a pterin cosubstrate analog immobilized to the resin of the column. The protein is further purified by size exclusion chromatography to remove protein aggregates. The activity of TPH1 is determined by using a fluorescence assay. The enzyme activity assay is carried out at 15° C. with atmosphere oxygen for the duration of 60 minutes in a volume of 64 μl. The reaction is carried out in a 0.1 M Tris-HCl buffer, adjusted to pH 7.6, containing 1 mM DTT, 0.2 mg/mL catalase, 100 μM (±)-6-methyl-5,6,7,8-tetrahydropterine dihydrochloride, 40 μM L-tryptophan, and 40-80 nM of TPH1. The reaction is started by bringing together L-tryptophan with all the other reaction substituents and stopped by quenching with perchloric acid (HClO4). The amount of 5-hydroxy-L-tryptophan produced during the enzymatic reaction is determined by fluorescence readout. Fluorescence, as determined at 540 nm when excited at 300 nm wavelength, increases proportionally to the 5-hydroxy-L-tryptophan formed. Compounds 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 the assay plate. Fluorescence is measured for each well and the fluorescence at 540 nm wavelength is compared to the fluorescence of the vehicle in place of compound. Inhibitory activities of example compounds with respect to the TPH1 protein are determined by calculating the IC50 value (the concentration of compound needed to inhibit 50% of the enzyme activity).
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 517.37, ALogP 4.36, HBA 8, HBD 0, PSA 96.41, QED 0.37
Compound (SMILES): CC(C)CN(C)C1CCOCC1
InChIKey: FJZCSTFWLIEROM-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 171.28, ALogP 1.75, HBA 2, HBD 0, PSA 12.47, QED 0.64
Compound (SMILES): CNCCC(Oc1cccc2cnccc12)c1cncc(Cl)c1
InChIKey: FAIQDJPZKHQIMM-UHFFFAOYSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay: Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. The binding of the test compound was measured at five different concentrations. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4. Filter plates were dried at 60° C. for 1 h and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 327.82, ALogP 4.01, HBA 4, HBD 1, PSA 47.04, QED 0.74
Compound (SMILES): O=C(O)[C@@H]1C[C@H]1CCc1ccc(N2CCCC(Oc3ccccc3)CC2)cc1
InChIKey: NQJRASXYQXCMOE-QQHBYLCDSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: GPR120 pERK AlphaScreen SureFire Assay: The human and mouse GPR120-mediated intracellular phosphorylated ERK assays were also established using CHO-K1 cells stably transfected with the short form of human or mouse GPR120 receptor. Cells were cultured in growth medium consisting of F-12 media (Invitrogen Cat. #11765) with 5% Charcoal/Dextran FBS (Invitrogen Cat. #12676-029) and 500 μg/mL GENETICIN® (Life Technologies Cat. #10131-027). Cells were cryo preserved at a concentration of 3×106 cells/mL, in 70% F-12, 20% Charcoal/Dextran FBS and 10% DMSO, and frozen in liquid nitrogen at a low passage number.For the pERK assay, 3×106 cells/mL cryopreserved human and mouse cells were thawed rapidly in a 37° C. water bath and added to a T-225 flask containing 50 mL growth medium. The flasks were placed in a tissue culture incubator overnight (37° C., 5% CO2). The next day, cells were harvested with trypsin (Gibco Cat. #25300-054), resuspended in serum-containing growth medium and counted using a Cellometer and volume adjusted to a concentration of 0.5×106 cells/mL. Cells were plated into 384-well clear bottom tissue culture plates (BD Cat. #353962) at 50 μL/well, for a density of 25,000 cells/well using a MULTIDROP® and incubated for 16-18 hours (overnight) at 37° C. with 5% CO2. The next day, cells were washed once with 50 μL of PBS without Ca++/Mg++ (Gibco Cat. #14190-036) and serum starved in 25 μL of F-12 media without any serum or antibiotics for 2 hours at 37° C.
Measurement: EC50 = 10300 nM (pChEMBL 4.989999771118164)
Descriptors: MW 379.5, ALogP 4.78, HBA 3, HBD 1, PSA 49.77, QED 0.75
Compound (SMILES): CC(F)Cn1cc(Nc2nc(Oc3cc(NC(=O)CCl)ccc3F)c3cccn3n2)cn1
InChIKey: KEUCNFKDCJEWJU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Inhibitory Activity Assay: Specifically, the inhibitory activities of JAK3 and BTK kinase were measured using a JAK3 kinase assay kit (Promega, V9441) and a BTK kinase assay kit (Promega, V9071) which were purchased from Promega. Recombinant purified human JAK3 and BTK were diluted with 1× kinase reaction buffer (JAK3: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 uM DTT/BTK: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM MnCl2 and 50 uM DTT) and added to a 96 well plate (JAK3: final concentration of 4 ng per reaction/BTK: final concentration of 8 ng per reaction). The compounds were treated so as to be finally a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (JAK3: final concentration of 5 uM/BTK: final concentration of 10 uM) and 0.2 ug/uL of Poly(Glu4, Tyr1)peptide (JAK3 and BTK final concentration) in the total 25 uL reactants was added to a 96-well plate to initiate enzymatic reaction. After incubation (30° C.) for 1 hour, equivalent volume (25 uL per reaction) of ADP Glo was added and incubated (30° C.) for 40 minutes at room temperature. Then, a kinase detection reagent (50 uL per reaction) was added and incubated (30° C.) for 30 minutes at room temperature.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 461.86, ALogP 4.14, HBA 8, HBD 2, PSA 98.37, QED 0.38
Compound (SMILES): COc1cc(C(=O)Nc2nnc(-c3ccncc3)s2)ccc1OCc1cscn1
InChIKey: VJIZYYJAKBONGL-UHFFFAOYSA-N
Target: NADPH oxidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5S8
Assay: ROS Production Measurement on hNOX1 Membranes: All solutions were placed on ice and protected from light. The final concentration in the 1x hNOX1 membrane fluorescent assay buffer was PBS pH7, 6 μM FAD, 15 μM PA, 1 mM MgCl2, 12.5 μM AR, 0.02 u/ml, 125 ng membranes, 1.5 μg of cofactors and 30 μM NADPH.The NADPH was dissolved in water at a concentration of 12 mM and was transferred in a metal transfer plate kept at 4° C. The NADPH was added to the assay plate to initiate the reaction just before the measurement.
Measurement: Ki = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 425.5, ALogP 3.9, HBA 9, HBD 1, PSA 99.12, QED 0.48
Compound (SMILES): Cc1ccc(C(=O)Nc2ccc(CN3CCN(C)CC3)c(C(F)(F)F)c2)cc1C#Cc1nn(C(=O)OC(C)(C)C)c2ncnc(N)c12
InChIKey: VREZKFPACUPTJS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Yes (SINGLE PROTEIN), Homo sapiens
UniProt: P07947
Assay: Kinase Inhibition Assays: The aim of this experiment is to detect the inhibitory activity of the compounds of the present invention against in vitro protein kinases using isotope labeling (labeled gamma phosphate groups on ATP). Kinase inhibition profiles were determined using KinaseProfiler services provided by Euro fins, and ATP concentrations used are the Km of corresponding kinases. In this study, we examined Abl (T315I) (h), ALK (h), ARK5 (h), Axl (h), Blk (h), Bmx (h), BTK (h), B-Raf (H), ckit (h), cSRC (h), CDK7, CHK1 (h), c-RAF (h), DDR2 (h), EGFR (h), EphA1 (h), EphA2 (h), EphA8 (h (H), FGF (h), Ft (h), Ft (h), F (h), F (h), Ft (h), Hb (h), ErbB2 (h), FAK (h) (H), JK3 (h), IKKalpha (h), IKKbeta (h), Itk (h), JAK3 (h), JNK11 (h), KDR (h), Lyn (h), MAPK1 (h), MEK1 (h (H), PKA (h), PKB [alpha](h), PKB [beta] (h), PKC [alpha] (h), Ret (H), RIPK2 (h), Src (1-530) (h), TAK1 (h), TBK1 (h), Tec (h) activated, Tie2 (h), TrkA (h), ULK3 (h) Yes (h), PI3 Kinase a (h) and other kinase in vitro inhibitory activity. The kinase inhibitory activity of the test compound is expressed as IC50 (half inhibitory concentration) or the inhibitory rate of the test compound at a concentration of 10 ??M for kinase activity. IC50 values can be obtained by calculating the inhibition rate of the kinase activity by the test compound at a series of different concentrations. The assay was as follows: In a reaction tube, the buffer (8 mM MOPS, pH 7.0, 0.2 mM EDTA, 10 mM MnCl2), the kinase to be tested (5-10 mU), the substrate to be tested kinase, 10 mM acetic acid Magnesium and gamma 33P-ATP solutions, as well as different concentrations of the test compound. MgATP was then added to the reaction to initiate the enzymatic reaction and incubated at room temperature for 40 minutes. The reaction was finally quenched with 5 ul of 3% phosphate buffer and 10 uL of the reaction solution was titrated onto a filtermat A membrane, washed three times with 75 mM phosphate solution for 5 minutes each, and washed again with methanol. Finally, the filtermat A film is dried and scintigraphized, and the size of the scintillation count reflects the degree to which the substrate is phosphorylated, thereby demonstrating that the kinase activity is inhibited. Among them, the percentage of the remaining active protein=the active protein of the experimental group the active protein of the control group 100%.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 648.69, ALogP 4.92, HBA 10, HBD 2, PSA 131.5, QED 0.3
Compound (SMILES): CCc1nc(C(=O)OCCCc2nn(CC)c3c2CC2(CCOCC2)CNC3=O)cs1
InChIKey: YSCRIWNUKLERET-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: PDE4 Assay: The human PDE4D catalytic domain (UniProt no. Q08499 [S380-L740]) was incubated with a mixture of non-labelled cAMP (cyclic adenosine monophosphate) and fluorescein amidite (FAM) conjugated cAMP and titrated test or reference Compound. Following brief incubation the enzymatic reaction was stopped by addition of binding buffer containing nanoparticles with immobilized trivalent metal ions capable of binding 1) AMP phospho groups and 2) terbium (Tb) donor fluorophores. Subsequent excitation of the Tb donor triggers time-resolved FRET to adjacent FAM acceptor molecules resulting in light emission. In the presence of a PDE4 inhibitor, AMP generation was reduced resulting in a lower fluorescence signal. The cAMP phosphodiester is not bound by the detection system.The results were calculated as the molar concentrations resulting in 50% inhibition of the substrate cleavage compared to controls samples, and are expressed as a range of IC50 (nM).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 446.57, ALogP 2.79, HBA 8, HBD 1, PSA 95.34, QED 0.52
Compound (SMILES): Cc1nnc(-c2cccc(-n3ccc(N(C(=O)c4cccs4)C4CCN(Cc5ccccn5)CC4)n3)c2)o1
InChIKey: DMGZJIZQYPBJRJ-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Human mu-Opioid Receptor Radioligand Assay: To investigate binding properties of test compounds to human μ-opioid receptor, transfected CHO-K1 cell membranes and [3H]-DAMGO (Perkin Elmer, ES-542-C), as the radioligand, were used. The assay was carried out with 20 μg of membrane suspension, 1 nM of [3H]-DAMGO in either absence or presence of either buffer or 10 μM Naloxone for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM, MgCl2 5 mM at pH 7.4. Plates were incubated at 27° C. for 60 minutes. After the incubation period, the reaction mix was then transferred to MultiScreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH 7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail.
Measurement: Ki > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 525.64, ALogP 5, HBA 9, HBD 0, PSA 93.18, QED 0.3
Compound (SMILES): CN1C(=O)C[C@@H](CC(F)(F)F)c2nnc3cc(-c4ccnc(Nc5ccnn5C)c4)cc1n23
InChIKey: FKAMZHMNVQECDN-AWEZNQCLSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 2 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 456.43, ALogP 3.67, HBA 8, HBD 1, PSA 93.24, QED 0.5
Compound (SMILES): COc1ccccc1C(=O)N[C@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(=N)N)C(=O)CF)C(C)C
InChIKey: LMAULNZAVHAIDN-WFXMLNOXSA-N
Target: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UDY8
Assay: Biological Assay: For exemplary compounds of the disclosure, Ki for inhibition of MALT1 was measured (Table E11). A concentration of 100 nM MALT1 was used for the assay.
Measurement: Ki = 109 nM (pChEMBL 6.960000038146973)
Descriptors: MW 520.61, ALogP 0.73, HBA 6, HBD 5, PSA 166.71, QED 0.15
Compound (SMILES): CC(=O)NC[C@@H](NC(=O)c1cccn(Cc2ccc(F)c(F)c2)c1=O)c1nc(-c2c[nH]c3ncccc23)cs1
InChIKey: JDMDIKDJSUKFPE-JOCHJYFZSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: Kinase Assay: A PDK1 kinase assay was performed as follows. PDK1 (amino acids 51-360) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to greater than 90% homogeneity. PDK1 protein was divided into two fractions, one of which was subsequently dephosphorylated. To generate dephosphorylated PDK1, the PDK1 was reacted with GST-tagged lambda-phosphatase in vitro. GST was subsequently cleaved proteolytically for both phosphorylated and dephosphorylated PDK1. Protein preparations were run on glutathione Sepharose columns to remove GST and GST-tagged lambda-phosphatase, if present. Phosphorylated PDK1 and dephosphorylated PDK1 were verified by mass-spectrometry. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay using either phosphorylated or unphosphorylated PDK1 and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 3-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide. (final concentrations: 25 nM un-phosphorylated PDK1 or 0.5 nM phosphorylated PDK1, 30 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 24 μM for both forms of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 548.58, ALogP 3.78, HBA 7, HBD 3, PSA 121.77, QED 0.27
Compound (SMILES): CCc1cc(O)c(F)cc1-c1ccc2c(-c3nc4c([nH]3)CN(C)[C@H](C(=O)N3CC(C)(N(C)C)C3)C4)n[nH]c2c1
InChIKey: CVHSFUPKHBNGLI-DEOSSOPVSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical Kinase Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.
Measurement: Ki = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 531.64, ALogP 3.55, HBA 6, HBD 3, PSA 104.38, QED 0.36
Compound (SMILES): Cc1nc(Nc2ccc(N3C[C@@H](C)O[C@@H](C)C3)cc2)nc(OCC2CCC(O)CC2)c1F
InChIKey: FKJZXMKOHOOGNE-QIMBPPIPSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Enzymatic Assay: LRRKtide substrate (peptide sequence RLGRDKYKTLRQIRQ, derived from human ezrin [amino acids 561-573], moesin [amino acids 539-553] and radixin [amino acids 558-570], obtained from SignalChem, catalogue #L10-58, reconstituted in 20 mM Tris-HCl at pH 7.5 to a final concentration of 1 mg/mL, assay concentration 20 μM) and recombinant human LRRK2 (catalytic domain only [amino acids 970-2527], GST-tagged, expressed in insect cells, obtained from ThermoFisher Scientific, catalogue #PV4874, 0.35 mg/mL, assay concentration 30 nM) were mixed in assay buffer (20 mM Hepes pH 7.5, 10 mM MgCl, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM NaVO, 2 mM DTT, 1% DMSO). Compounds of interest (in DMSO, serial 3-fold dilution from 10 μM to 0.5 nM) or control (1% DMSO) were dispensed into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range). After incubation at room temperature for 20 minutes, the kinase reaction was initiated by addition of [P]-ATP (Specific activity 10 μCi/μl) and the mixture was incubated at room temperature for 2 hours. The reaction was then stopped by spotting the reaction mixture on strips of phosphocellulose P81 paper. Following washing, the radioactivity of the P81 paper was measured and kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions.
Measurement: IC50 > 400 nM (pChEMBL 6.3979997634887695)
Descriptors: MW 444.55, ALogP 4.21, HBA 7, HBD 2, PSA 79.74, QED 0.69
Compound (SMILES): CS(=O)(=O)c1ccc(-c2cccc([C@@H](Cc3ccccc3)c3ccc(-c4cc(Cl)ccc4-n4cnnn4)c[n+]3[O-])c2)cc1
InChIKey: UYKJEZDHLCBKRB-SSEXGKCCSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 40 pM (Sekisui Diagnostics) and the synthetic substrate, Z-Gly-Pro-Arg-AFC, TFA salt (Sigma #C0980) at a concentration of 100 μM.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 608.12, ALogP 6.06, HBA 7, HBD 0, PSA 104.68, QED 0.15
Compound (SMILES): CCOc1nnccc1-c1cc(NCc2cnn(C)c2)c2c(n1)c(C)nn2C(C)CC
InChIKey: UXVRWXLBBBUCGV-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P54750
Assay: Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays were performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 hr at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 hr in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 420.52, ALogP 3.91, HBA 9, HBD 1, PSA 95.57, QED 0.46
Compound (SMILES): CC1(NC(=O)O[C@@H]2CC[C@H](c3cc(NC(=O)Cc4cc(C(F)F)ccn4)n[nH]3)C2)CC1
InChIKey: KUKODEROBKWGOL-SWLSCSKDSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Biochemical Assay: The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:1). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Matl enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA.
Measurement: Ki = 0.36 nM (pChEMBL 9.4399995803833)
Descriptors: MW 433.46, ALogP 3.84, HBA 5, HBD 3, PSA 109, QED 0.61
Compound (SMILES): CCCNC(=O)c1ccc(-n2ncc(-c3ccc(C#N)cc3C)c2O)nc1
InChIKey: MVUHNPANESKMDK-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition Assay: The IC50 values for the PHD2 enzyme (residues 181-417) were determined by mixing increasing amounts of inhibitor with a fixed amount of enzyme (5 nM, final concentration) and Biotin labeled peptide (Biotin-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu, 1 uM final concentration) and 2-Oxyglutarate (2 uM final concentration) in 50 mM HEPES, 50 mM KCl, 0.5 mM TCEP, 2 uM FeCl2, 0.1 mg/ml BSA, at pH 7.3. The reaction was conducted by pre-incubating the enzyme in the presence of inhibitor for 60 min at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-Oxoglutarate (see above for final concentrations), and Ascorbic Acid (1 mM final concentration). The enzymatic activity was quenched after 60 min by adding an excess of a tight binding inhibitor to the assay mixture. The amount of product released was measured by using a LC/MS system (Agilent HPLC with Applied Biosystems API3000 Mass Spectrometer).
Measurement: IC50 = 72.4 nM (pChEMBL 7.139999866485596)
Descriptors: MW 361.41, ALogP 2.96, HBA 6, HBD 2, PSA 103.83, QED 0.73
Compound (SMILES): Cc1cc(Br)cc2c1cc(Cc1c(Cl)ccc(C(=O)N3CCOCC3)c1Cl)n2C
InChIKey: IKNSPBUPYSEYDB-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: transient transactivation assay: The primary screen was performed by transient transactivation assays. These cell-based assays were carried out using Cos-7 cells transfected with a chimeric human RORγ-Gal4 receptor expression plasmid and a 5Gal4 pGL3 TK Luc reporter plasmid. Transfections were performed by a chemical agent (Jet PEI). Transfected cells were distributed in 384-wells plates and were allowed to recover for 24 h. The culture medium was then removed and fresh medium containing the compounds to be tested was added (final concentration ranging from 10−4 M to 3 10−10 M). After an overnight incubation, luciferase expression was measured by adding SteadyGlo according to the manufacturer's instructions (Promega). T0901317 at 10−5 M was used as reference. Results were expressed as fold induction compared to basal level or as percentage activity compared to references taken as 100% (basal) or 0% (T0901317). Dose-effect curves and IC50 were calculated using the software Assay Explorer (MDL).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 496.23, ALogP 5.62, HBA 3, HBD 0, PSA 34.47, QED 0.47