lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=c1cc(-c2c[nH]c3ncc(N4CCOCC4)cc23)ccn1C(CO)c1cccc(Cl)c1
InChIKey: RFHBLSVKYMKZKM-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 Enzymatic Assay: To assess compounds capacity to inhibit ERK2 enzymatic activity, Z′-Lyte biochemical assay from Life technologies was used according to manufacturer's instructions. Briefly, black 384-well plates containing 100 nl of 100× compound solution in 100% DMSO, 2.4 μl kinase buffer, 5 μl 2×MAPK1 (ERK2)/Ser/Thr 03 mixture and 2.5 μl 4×ATP solution were used. Plates were shaken for 30 seconds and incubated for 60 minutes at room temperature. Then, 5 μl of a 1:1024 dilution of Development Reagent A was added. Plates were shaken for 30 seconds and incubated for 60 minutes at room temperature. A plate reader was used to read fluorescence. In this assay, ERK2 enzyme was used at a concentration of 0.4 μg/ml (5.74 nM) at ATP Km (100 μM). Kinase buffer consisted of 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. Compounds IC50 were determined with a 3-fold serial dilution (10 point titrations in duplicate).
Measurement: IC50 < 150 nM (pChEMBL 6.823999881744385)
Descriptors: MW 450.93, ALogP 3.46, HBA 6, HBD 2, PSA 83.38, QED 0.49
Compound (SMILES): CCN(C)c1cc(O)c(=O)[nH]n1
InChIKey: DCJSGIACBCVQMB-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P14920
Assay: Enzyme Assay: The functional activity of compounds inhibiting the DAAO enzyme was determined by utilizing the co-product of the catalysis of D-Serine, H2O2, which can be quantitatively measured using the Amplex (trade mark) Red (Invitrogen) detection. Amplex Red reagent is a colorless substrate that reacts with hydrogen peroxide (H2O2) with a 1:1 stoichiometry to produce highly fluorescent resorufin (excitation/emission maxima=570/585 nm). The changes in fluorescence were monitored by a fluorescence plate reader, Envision (Perkin Elmer) and increases in DAAO activity were readily detected upon addition of D-Serine and suppression of this response observed with the application of test compounds.Human DAAO enzyme was supplied by the Takeda Pharmaceutical Company (Osaka) and each batch was tested and used at concentrations giving comparable levels of activity. The Km of D-Serine was measured for each enzyme batch to maintain consistency; this Km was used in subsequent assays.On the day of the assay compounds were serially diluted in DMSO before being diluted 1:20 with assay buffer (20 mM Tris ph 7.4). A 5 μl portion of assay buffer was added to the wells of a 384 clear base black-walled plate (Corning), 5 μl of diluted compound was then added via automated plate-to-plate transfer using the Bravo liquid handler (Agilent technologies) followed by 5 μl of human DAAO enzyme, and then 5 μl D-Serine 50 mM was added to all but the negative control wells (final concentration of 10 mM). Finally 5 μl Amplex red reagent (Invitrogen) was added to all wells as per manufacturer's protocol. The plate was incubated for 60 minutes in the dark at 25° C. and the fluorescence in each well was measured in the Envision plate reader.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 169.18, ALogP -0.07, HBA 4, HBD 2, PSA 69.22, QED 0.65
Compound (SMILES): CC(=O)N1CCN(c2ccc(NC(=O)Cc3cnc(-c4ccnc(C)c4)c(C)c3)cn2)C[C@@H]1C
InChIKey: BWMCXCGQZHGQHP-IBGZPJMESA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: Wnt-Luc Reporter Assay: Human embryonic kidney 293 cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are cultured in DMEM medium (Gibco/Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS (Gibco/Invitrogen, Carlsbad, Calif.), 50 unit/mL penicillin and 50 μg/mL of streptomycin (Gibco/Invitrogen, Carlsbad, Calif.) at 37° C. with 5% CO2 in air atmosphere. 293 cells in a 10 cm dish are co-transfected with 8 μg of STF-reporter plasmid containing a luciferase gene driven by Wnt-responsive elements and 2 μg of pcDNA3.1-Neo (Gibco/Invitrogen, Carlsbad, Calif.) with 30 μL of FuGENE6 (Roche Diagnostics, Indianapolis, Ind.) following the manufacturer's protocol. Stable cell lines (293 Wnt-Luc) were selected with 400 μg/mL of G418 (Gibco/Invitrogen, Carlsbad, Calif.). The 293 Wnt-Luc cells and L-cell Wnt3a cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are trypsinized and co-cultured into a 384-well plate with DMEM medium supplemented with 2% FBS, and treated with different concentrations of a compound of the invention. After 24 hours, the firefly luciferase activities are assayed with the Bright-Glo Luciferase Assay System (Promega, Madison, Wis.). The IC50 is measured when the effect of the compound reduces the luminescence signal by 50%.
Measurement: IC50 = 102 nM (pChEMBL 6.989999771118164)
Descriptors: MW 458.57, ALogP 3.39, HBA 6, HBD 1, PSA 91.32, QED 0.63
Compound (SMILES): CCCS(=N)(=O)c1cnc(N2CCN(C(C)c3ccc4c(c3)OCC4)CC2)nc1
InChIKey: OWAFXLFISYMNSK-UHFFFAOYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Human O-GlcNAcase enzyme inhibition assay: 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λexc 485 nm; (Δemm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 415.56, ALogP 3.11, HBA 7, HBD 1, PSA 82.41, QED 0.78
Compound (SMILES): CCCCc1nc(=O)c(S(=O)(=O)c2ccc(-c3ccccc3F)cc2)c(O)n1-c1c(OC)cccc1OC
InChIKey: CVCYVZXZOVJFKI-UHFFFAOYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyrl)apelin-13) peptide, which was set to 100%.
Measurement: EC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 538.6, ALogP 4.94, HBA 8, HBD 1, PSA 107.72, QED 0.32
Compound (SMILES): CCn1cc(C(F)(F)F)nc1-c1ccc(Cn2ncc3cnc(-c4c(C5CC5)cnn4CC)nc32)cc1
InChIKey: FWVBEBBERVBFHM-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: Inhibitor Assay: Deubiquitinase activity was measured using ubiquitin-rhodamine 110 as a substrate. Cleavage of the amide bond between rhodamine and the c-terminal glycine of ubiquitin yields an increase in fluorescence signal. The assay was conducted in 20 ul total volume of assay buffer (50 mM Tris-HCl, pH 7.8, 0.5 mM EDTA, 0.01% Bovine Serum Albumin, 1 mM DTT, 0.01% Tween-20), and 0.05 nM USP1/UAF1 enzyme. Reaction was initiated by addition of 150 nM Ubiquitin-rhodamine (Boston Biochem) substrate.Compounds, dissolved in DMSO were tested in dose response format, beginning at 10 uM.Compounds depicted below were added to enzyme/assay buffer mix and incubated 10 min. Substrate mix was added, and reaction mix was read in kinetic mode for 30 min at Ex480/Em540 and IC50 response curves were plotted.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 506.54, ALogP 5.54, HBA 8, HBD 0, PSA 79.24, QED 0.29
Compound (SMILES): N#Cc1ccc(NC2CC3CCC2N3C(=O)c2cccc(F)c2-n2nccn2)nc1
InChIKey: JNECNNFBFDAMJO-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Rat and Human Orexin 1 Receptor Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat #SH30022), 10% FBS, 1× Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat #SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −800 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 403.42, ALogP 2.53, HBA 7, HBD 1, PSA 99.73, QED 0.72
Compound (SMILES): COc1ccc(-c2c(-c3ccc(C#N)cc3)nc(N3CCC(N)CC3)n(C)c2=O)cn1
InChIKey: MJQRQURVBRMRKC-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: In Vitro Enzyme Inhibition Assay: The enzymatic assay of LSD1 activity is based on Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The inhibitory properties of compounds to LSD1 were determined in 384-well plate format under the following reaction conditions: 0.1-0.5 nM LSD1, 50 nM H3K4mel-biotin labeled peptide (Anaspec cat # 64355), 2 μM FAD in assay buffer of 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-unmodified histone H3 lysine 4 (H3K4) antibody (PerkinElmer) in the presence of LSD1 inhibitor such as 1.8 mM of Tranylcypromine hydrochloride (2-PCPA) in LANCE detection buffer (PerkinElmer) to final concentration of 12.5 nM and 0.25 nM respectively.The assay reaction was performed according to the following procedure: 2 μL of the mixture of 150 nM H3K4mel-biotin labeled peptide with 2 μL of 11-point serial diluted test compound in 3% DMSO were added to each well of plate, followed by the addition of 2 μL of 0.3 nM LSD1 and 6 μM of FAD to initiate the reaction. The reaction mixture was then incubated at room temperature for one hour, and terminated by the addition of 6 μL of 1.8 mM 2-PCPA in LANCE detection buffer containing 25 nM Phycolink Streptavidin-allophycocyanin and 0.5 nM Europium-anti-unmodified H3K4 antibody. Enzymatic reaction is terminated within 15 minutes if 0.5 LSD1 enzyme is used in the plate.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 416.49, ALogP 2.32, HBA 8, HBD 1, PSA 110.06, QED 0.69
Compound (SMILES): COc1ccc(-c2nc3c(n2C(C)C)C(c2ccc(Cl)cc2)N(c2cc(C)c4nnn(C)c4c2)C3=O)cn1
InChIKey: UVCPBOQPPSLUPY-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In-Vitro Binding Assay: All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 ul per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5ul per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 ul per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (IC50 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 528.02, ALogP 5.53, HBA 8, HBD 0, PSA 90.96, QED 0.3
Compound (SMILES): CC(C)Oc1ccc(/N=c2\[nH]c(=O)n(CCc3ncn[nH]3)c(=O)n2Cc2ccc(Cl)cc2)cc1F
InChIKey: GXAFLNJRFCAUCP-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 499.93, ALogP 2.56, HBA 8, HBD 2, PSA 122.95, QED 0.39
Compound (SMILES): COC1CC(CC#N)(n2cc(-c3nc(-c4cc(CO)n[nH]4)cc4ncccc34)cn2)C1
InChIKey: QOUIXZBLWSFJLI-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
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 = 63 nM (pChEMBL 7.199999809265137)
Descriptors: MW 415.46, ALogP 2.79, HBA 8, HBD 2, PSA 125.53, QED 0.5
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCN(C(=O)OCCCN)CC4)cc3c2C(C)C)cn2ncnc2c1C
InChIKey: QWHYPUONSUBBFN-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: 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 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 488.64, ALogP 5.28, HBA 6, HBD 2, PSA 101.54, QED 0.36
Compound (SMILES): CCS(=O)(=O)N[C@H]1C[C@@H](C)N(C(=O)OC)[C@H]1CO[C@@H]1CC[C@]2(c3ncc(F)cn3)C[C@@H]2C1
InChIKey: ZFJVRNBKXJAGJA-GMBQTQCJSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: IP-One assay kit: IP-One, Cisbio, cat #621PAPEC, is a commercially available assay.
Measurement: IC50 = 13.8 nM (pChEMBL 7.860000133514404)
Descriptors: MW 470.57, ALogP 1.98, HBA 7, HBD 1, PSA 110.72, QED 0.65
Compound (SMILES): CC1(C)CN(c2ccccc2NC(=O)NC2CCN(CC(F)(F)F)CC2)c2c(O)ccc(C(F)(F)F)c21
InChIKey: YKSIIZROYXPAPI-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 μL containing 15 fmol of P2Y1 receptor (1.7 μg of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 μM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 μg of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 μL of the aqueous solution was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKER (Amersham) Image Reader.
Measurement: Ki = 4.9 nM (pChEMBL 8.3100004196167)
Descriptors: MW 530.51, ALogP 5.99, HBA 4, HBD 3, PSA 67.84, QED 0.43
Compound (SMILES): Cn1cc(-c2cccc(C(=O)NC(CCOCCCCc3ccc4c(n3)NCCC4)C(=O)O)c2)cn1
InChIKey: CDHBGORYZXVXCW-UHFFFAOYSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Binding Assay: The biochemical potency of compounds was determined using a proximity-based assay (ALPHASCREEN®, Perkin Elmer, Waltham, Mass.) as described previously (Ullman E F et al., Luminescent oxygen channeling immunoassay: Measurement of particle binding kinetics by chemiluminescence. Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 5426-5430, June 1994). To gauge the potency of inhibitors of binding to human integrin αvβ6, inhibitor compounds and integrin were incubated together with TGF-b1 LAP and biotinylated anti-LAP antibody plus acceptor and donor beads, following the manufacture's recommendations. The donor beads were coated with streptavidin. The acceptor beads had a nitrilotriacetic acid Ni chelator, for binding to a 6×His Tag on human integrin αvβ6. All incubations occurred at room temperatures in 50 mM Tris-HCl, pH 7.5, 0.1% BSA supplemented with 1 mM each CaCl2 and MgCl2.The order of reagent addition was as follows:1. Alpha-v-beta-6 integrin, test inhibitor compound, LAP, biotinylated anti-LAP antibody and acceptor beads were all added together.2. After 2 hours, donor beads were added. After another 30 minute incubation, samples were then read.Integrin binding was evaluated by exciting donor beads at 680 nm, and measuring the fluorescent signal produced, between 520-620 nm, using a Biotek Instruments (Winooski, Vt., USA) SynergyNeo2 multimode plate reader.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 491.59, ALogP 3.45, HBA 7, HBD 3, PSA 118.37, QED 0.33
Compound (SMILES): COc1ccc(Nc2nc(=O)n(C)c3ncn(Cc4ccccc4)c23)c(C)c1
InChIKey: MEXXRTSEWPWMKF-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Inhibition Assay: Evaluation of a human P2X7 receptor inhibitory activity Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1× HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 375.43, ALogP 3.24, HBA 7, HBD 1, PSA 73.97, QED 0.58
Compound (SMILES): C=CC(=O)Nc1ccc(F)c(Oc2nc(Nc3cccnc3)nn3cccc23)c1
InChIKey: LCNHWLNGKLWZQJ-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 = 15.5 nM (pChEMBL 7.809999942779541)
Descriptors: MW 390.38, ALogP 3.92, HBA 7, HBD 2, PSA 93.44, QED 0.48
Compound (SMILES): C[C@@H](Nc1nc(C(=O)O)nc2nc(-c3cc(C(F)(F)F)ccn3)n(Cc3ccc(C(F)(F)F)cc3)c12)C1CCC1
InChIKey: JDLIPAZRZYPFIF-CYBMUJFWSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 15.18 nM (pChEMBL 7.820000171661377)
Descriptors: MW 564.49, ALogP 6.27, HBA 7, HBD 2, PSA 105.82, QED 0.25
Compound (SMILES): CC(=O)NC[C@H]1C[C@]2(CN(C(C)C)C(=O)c3c(O)c(=O)c(C(=O)NCc4ccc(F)cc4F)cn32)C1
InChIKey: OQPQSUBMOCYLSD-AHPHSXTOSA-N
Target: Protein Nef (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: P03406
Assay: Measurement of Antiviral (HIV-1) Activity: The medium (40 μL), a test substance (10 μL) diluted with the medium, and a 1×105 cells/mL MT-4 cell suspension (50 μL) wherein HIV-1 NL4-3 strain was infected with MOI (infection multiplicity) 0.05 were added to each well of a 96-well black plate (manufactured by Corning Incorporated), and the mixture was cultured at 37° C. for 5 days.medium composition: RPMI1640, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin.Then, Cell Titer-Glo (manufactured by Promega Corporation, 100 μL) was added to each well, and the mixture was stood at room temperature for 10 min, and the luminescence intensity was measured.
Measurement: EC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 502.52, ALogP 1.87, HBA 6, HBD 3, PSA 120.74, QED 0.56
Compound (SMILES): O=C(N[C@H](C(=O)N1C[C@H](Cl)[C@H]2OCC(=O)[C@H]21)C1CCCCC1)c1cc(F)c(-c2cccnn2)s1
InChIKey: KIGJQHZQBSCHKN-YGHPHNMRSA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Enzyme Assay: Recombinant human cathepsins (CatS, CatK, CatL) were purchased from a Enzo Life Sciences. All assays were carried out in 96-well format using a buffer of 50 mM KH2PO4, 50 mM NaCl, 2 mM EDTA, 0.5 mM DTT and 1% Triton-X-100, pH 6.5 for Cathepsin S and a buffer of 50 mM NaOAc, 10 mM EDTA, 1 mM DTT and 0.01% Triton-X-100, pH 5.5 for CatK/L/B. For CatS, the enzyme (0.0007 mU/well) was incubated with fluorogeninc substrate (Z-VVR-AMC, 5 μM) at RT for 10 min. For CatK the enzyme (0.00175 mU/well) was incubated with fluorogeninc substrate (Z-FR-AMC, 40 μM) at RT for 10 min. For CatL, the enzyme (0.000874 mU/well) was incubated with fluorogeninc substrate (Z-VVR-AMC, 40 μM) at RT for 10 min. Flourogenic substrate turnover was detected using a microplate reader (Synergy H4, BioTek). Ki values were calculated using the Cheng Prusoff equation (Cheng & Prosoff 1973).
Measurement: Ki = 528 nM (pChEMBL 6.28000020980835)
Descriptors: MW 506.99, ALogP 2.81, HBA 7, HBD 1, PSA 101.49, QED 0.63
Compound (SMILES): CC[C@H](NC(=O)C1(OC)CN(c2ncnn3cc(-c4cnn(C)c4)cc23)C1)c1ccc(Cl)cc1
InChIKey: YUQWIBUHWLRUIH-FQEVSTJZSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit Wild Type Assay: In each well of a 384-well plate, 0.2 ng/ul final (2 nM) of wild type Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 479.97, ALogP 3.26, HBA 8, HBD 1, PSA 89.58, QED 0.44
Compound (SMILES): CC(C)c1c(-c2ccnc3[nH]ncc23)[nH]c2ccc(-c3ccc(C(C)N4CCOCC4)cc3)cc12
InChIKey: VCJAQZAPMLSJCU-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
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-κc Band 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 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-Bue 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 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 465.6, ALogP 6.29, HBA 4, HBD 2, PSA 69.83, QED 0.32
Compound (SMILES): [C-]#[N+]c1ccc(-c2cc3sc(C(C(=O)NCC(=O)NC4CC4)S(=O)(=O)C(C)C)nc3cc2F)cn1
InChIKey: BFYDLOFTYHCHDA-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Biological Activity: Endothelial lipase (EL) and hepatic lipase (HL) activities were measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 571 μL of 29 mM DMPG in a 1:1 mixture of MeOH and CHCl3 with 2000 μL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen in multiple vials then resuspended in 20 mL total volume of 50 mM HEPES pH 8.0 buffer containing 50 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at room temperature for 15 min and then was sonicated 3×4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.11 for the FRET substrate.The enzymatic assay was measured using 384-well white Optiplates. Each well contained 20 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 0.25 μL of a DMSO solution containing a compound of interest. EL or HL (10 μL) was added and allowed to incubate with the compound for 30 min at 37° C. The source of EL was conditioned media obtained from HT-1080 cells that were transformed using RAGE technology (Athersys) to overexpress endogenous EL, and HL was partially purified from conditioned media obtained from COS cells overexpressing HL. The reaction was started by the addition of 10 μL of a 1:10 dilution of vesicles. The final total reaction volume was 20.25 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Readings were taken over a period of 60 minutes, and the slope between 300 and 900 secs of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 515.59, ALogP 3.31, HBA 7, HBD 2, PSA 122.48, QED 0.44
Compound (SMILES): CN1CCN(CCOc2ccc(-c3cnc4c(-c5cccc6ncccc56)cnn4c3)nc2)CC1
InChIKey: LCNMZQCSKWYNQF-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: ALK1-6 enzymatic assays: Specifically, compounds were assayed using LANCE® Ultra ULight¿ technology (Perkin Elmer) against human ALK1-6 enzymes (ALK1: Life Technologies, ALK2-6: Carna Biosciences). Briefly, ALK enzyme (10 nM) was prepared in kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 3 mM MgCl2, 0.005% Tween-20 and 2 mM DTT) and dispensed at 2.5 μL/well into a 1536 well, white, solid bottom, microtiter plate (Greiner, 789175-F). Negative controls for the assay were generated by adding one column containing kinase buffer only. Compounds in DMSO solution were transferred to the assay plates at 23 nL/well via a NX-TR pin tool workstation (WAKO, San Diego, Calif.) and incubated with enzyme for 10 minutes at ambient temperature. ULight Topo IIa Substrate (50 nM) was prepared in kinase buffer containing either 10 μM or 1000 μM ATP and dispensed at 2.5 μL/well into the assay plate. Following a 1 hour incubation at ambient temperature, Europium anti-phospho DNA Topoisomerase 2-alpha antibody (4 nM) was prepared in 1× LANCE detection buffer containing 12 mM EDTA and dispensed at 5 μL/well into the assay plate. Plates were measured using the EnVision plate reader (Perkin Elmer), with excitation 320 nm and emissions of 615 nm and 665 nm.
Measurement: IC50 = 151 nM (pChEMBL 6.820000171661377)
Descriptors: MW 465.56, ALogP 3.63, HBA 8, HBD 0, PSA 71.68, QED 0.38
Compound (SMILES): C[C@H]1Cc2c(oc3ccccc23)[C@H](c2c(F)cc(/C=C/C(=O)O)cc2F)N1CC(C)(C)F
InChIKey: DMJJJIYHTDJNMV-CZHFBYBJSA-N
Target: G-protein coupled estrogen receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99527
Assay: Biological Assay: The compounds of the present invention have an inhibition effect on the binding of E (estrogen) to ER (estrogen receptor), thereby blocking the binding of a complex of E and ER to ERE (estrogen responsive element), and subsequently blocking the expression of downstream luciferase protein.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 443.47, ALogP 5.89, HBA 3, HBD 1, PSA 53.68, QED 0.5
Compound (SMILES): Cc1cc(CC(=O)N[C@H]2CC[C@H](CCN3CCc4nc(OCC(F)(F)F)sc4CC3)OC2)on1
InChIKey: LJTCNKUXICKSFP-JKSUJKDBSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Binding Assay: 225 nL of the solutions of the non-specific ligand or the compounds of the present invention at each concentration (in case of vehicle, final concentration 0.3% DMSO) were added in each well of a 384-well white/clear bottom microplate (3706, Corning). Jump-In HEK Cell membranes (final reaction amount: 2 μg protein/well), SPA beads (final reaction amount: 0.2 mg/well) and the buffer were mixed and the mixed solution was left still for more than or equal to 1 h at 4° C. Then, 50 μL of the mixture was added to each well of the plate. In addition, 25 μL of 3.6 nM [3H]-Metylspiperone (final concentration: 1.2 nM) was added to each well. The plate was sealed by putting TopSeal-A 96/384 well (6050185, PerkinElmer) on the top of the plate, mixed using stirring deaerator (Welltornado, FK-62, Sakaki) and incubated for 120 min at 25° C. After incubation, the radioactivity of [3H]-Metylspiperone which was binded to D2 receptor was determined by liquid scintillation counter (1450 Microbeta, PerkinElmer) in each well. Non-specific binding was calculated based on the radioactivity of [3H]-Metylspiperone in the presence of 10 μM non-labeled Butaclamol. The total binding was calculated using the radioactivity of [3H]-Metylspiperone in the absence of the compounds of the present invention (Vehicle). The Ki values were calculated from dose-response curves.
Measurement: Ki > 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 502.56, ALogP 3.08, HBA 8, HBD 1, PSA 89.72, QED 0.59
Compound (SMILES): C[C@@H]1CN(c2ccc(Nc3ncc4ccc(=O)n(CCC5CN(C(=O)/C=C/CN(C)C)C5)c4n3)cc2)CCN1C
InChIKey: FFHPKCFQQZTEHD-IOUFHMPJSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical JAK and Tyk2 Kinase Assays: 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 or discretely 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.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.
Measurement: Ki > 100 nM (pChEMBL 7)
Descriptors: MW 544.7, ALogP 2.64, HBA 9, HBD 1, PSA 89.84, QED 0.41
Compound (SMILES): O=C(c1nc2n(n1)[C@@H](c1ccccc1)C[C@H]2Cl)C1CC1
InChIKey: QVMPFORRCHVDES-VXGBXAGGSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Enzyme Assay: The ability of the receptor interacting protein kinase (RIPK1) to catalyze the hydrolysis of adenosine-5′-triphosphate (ATP) is monitored using the Transcreener ADP (adenosine-5′-diphosphate) assay (BellBrook Labs). Purified human RIP1 kinase domain (2-375) (50 nM) derived from a baculovirus-infected insect cell expression system is incubated with test compounds for 2 hours in 50 mM Hepes buffer (pH 7.5) containing 30 mM MgCl2, 1 mM dithiothreitol, 50 μM ATP, 0.002% Brij-35, and 0.5% dimethyl sulfoxide (DMSO). Reactions are quenched by the addition of 1× Bell Brooks Stop buffer B (20 mM Hepes (ph 7.5), 40 mM ethylenediaminetetraacetic acid and 0.02% Brij-35) containing an additional 12 mM EDTA and 55 ug/mL ADP2 antibody and 4 nM ADP-AlexaFluor® 633 tracer. The tracer bound to the antibody is displaced by the ADP generated during the RIP1K reaction, which causes a decrease in fluorescence polarization that is measured by laser excitation at 633 nm with a FP microplate reader M1000.
Measurement: Ki = 790 nM (pChEMBL 6.099999904632568)
Descriptors: MW 287.75, ALogP 3.14, HBA 4, HBD 0, PSA 47.78, QED 0.64
Compound (SMILES): CC(OC(=O)C(c1ccccc1)c1ccccc1)C(=O)Nc1ccc(F)cc1
InChIKey: DXTIKOVWSAQFPL-UHFFFAOYSA-N
Target: Kallikrein-7 (SINGLE PROTEIN), Homo sapiens
UniProt: P49862
Assay: Inhibition Assay: Substrate:(MeO-Suc-Arg-Pro-Tyr-pNA), acetate salt, Peptide international, lot #906841, previously named S-2586Enzyme: Active human KLK7 (SCCE) 0.62 mg/ml in 0.3 M NaCl, 10 mM NaAc, pH 4Buffer: 10 mM NaPhospate, 0.5 M NaCl, pH 7.2150 μl of Substrate (2.5 mM) in Buffer was added to 96 wells plate (F8 Polysorp Unfra, Nunc cat. no. 469957). 10 μl of DMSO was added to the blank and control wells.10 μl of substance in DMSO was added to wells giving final concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, and 0.15625 μM.40 μl of active KLK7 (SCCE) (diluted to 12.5 μg/ml in activity buffer) was added all wells except blank to which activity buffer was added.Immediately after addition of SCCE the plate was transferred to a Spectramax 250 Microplate Reader (Molecular Devices) and enzyme activity (V) was measured as release of pNa measuring absorbance at 405 nm in 37° C. for 15 min with reading every 30 s.The mean value V of each sample was calculated (n=2 for the substances and n=4 for the control). From the values of V the % of total activity was calculated as (Vinhibitor/Vcontrol (no inhibitor))×100.
Measurement: IC50 = 1620 nM (pChEMBL 5.789999961853027)
Descriptors: MW 377.42, ALogP 4.53, HBA 3, HBD 1, PSA 55.4, QED 0.64
Compound (SMILES): Cc1ccncc1-c1cc(N)c2cnc(NC(=O)[C@@H]3[C@@H](C)[C@H]3c3cnn(C)c3)cc2c1
InChIKey: ZVYLZWVVRXQXTJ-AETJINEWSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK Lantha Binding Assay: For the binding assay, 4 ul 2×HPK1 and Eu-anti-GST antibody were added to each well of the assay plate using a Multidrop reagent dispenser. The solutions were incubated in a 23 C incubator for 1 h. To each well of the assay plate was added 4 ul 2× Tracer-222 using a Multidrop reagent dispenser. The solutions were again incubated in a 23 C incubator for 1 h. The results of the assay were read using an Envision plate reader with the following parameters:TR_FRET, 340ex/615 and 665em; 100 usec Delay; and 200 usec integration.
Measurement: Ki = 0.056 nM (pChEMBL 10.25)
Descriptors: MW 412.5, ALogP 3.91, HBA 6, HBD 2, PSA 98.72, QED 0.5
Compound (SMILES): Cc1c(F)cccc1[C@]1(C(=O)NO)CC[C@@H](c2nc3ccccc3s2)C1
InChIKey: BBHYQXANTPSPQY-XCLFUZPHSA-N
Target: Histone deacetylase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P56524
Assay: Inhibition Assay: The potency of Class IIa Histone Deacetylase (HDAC) inhibitors is quantified by measuring the Histone Deacetylase 4 (HDAC4) catalytic domain enzymatic activity using the fluorogenic substrate, Boc-Lys(Tfa)-AMC. The substrate is deacetylated to Boc-Lys-AMC by HDAC4. Cleavage by trypsin results in the release of the fluorophore AMC from the deacetylated substrate. The fluorescence of the sample is directly related to the histone deacetylase activity in the sample.Serially dilute HDAC inhibitor compounds. Serial dilutions of the HDAC inhibitors and control reference compound (1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone) are made by first resuspending the lyophilized compound to a final concentration of 10 mM in 100% dimethyl sulfoxide (DMSO). Stocks of 6 μl aliquots of the 10 mM compound in DMSO are prepared and stored at −2° C.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 370.45, ALogP 4.45, HBA 4, HBD 2, PSA 62.22, QED 0.53