lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@H](Oc1cc(-n2nc3n(c2=O)CCCN3C)c(F)cc1C(=O)Nc1c(F)cccc1F)C(F)(F)F
InChIKey: OVPYJXAGXINBKS-NSHDSACASA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Enzymatic Assay: Assay1: The enzymatic assay couples DHODH activity with bleaching of the dye 2,6-dichlorophenolindophenol (DCIP) (Knecht and Loffler, 1998; Miller et al., 1968). The assay was conducted in buffer containing 50 mM Tris, 0.1% Triton X-100, 150 mM potassium chloride, 2 nM DHODH, 1 mM dihydroorotate, 0.1 mM decylubiquinone, 0.06 mM DCIP, and 2% DMSO at pH 8.0 at 32 degree Celsius. The reaction was initiated by addition of substrates. Enzyme activity was monitored kinetically by the reduction in DCIP absorbance at 600 nm. Purified recombinant human DHODH enzyme was purchased from Novus (cat. no. NBP1-98916). Other chemicals were purchased from Sigma-Aldrich. Absorbance measurements were obtained using a BMG clarion star plate-reading spectrophotometer.
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 515.41, ALogP 3.87, HBA 7, HBD 1, PSA 81.39, QED 0.52
Compound (SMILES): COc1ccccc1C[C@@H](C)Oc1ncc(-c2cc(F)c(C(=O)NS(C)(=O)=O)cc2C)cc1Cl
InChIKey: KBVYWSWLHOYBOF-OAHLLOKOSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav 1.7 In Vitro Patch Express (PX) Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes. Peak sodium current was measured at the end of the compound addition period to determine percent inhibition. Three to five cells were tested per concentration, and IC50 curves were fitted to percent inhibition as a function of concentration.
Measurement: IC50 = 92.9 nM (pChEMBL 7.03000020980835)
Descriptors: MW 506.98, ALogP 4.56, HBA 6, HBD 1, PSA 94.59, QED 0.48
Compound (SMILES): Cc1ccc(-c2cc3c(cc2NC(=O)c2cnc(C)o2)CN(C[C@@H](F)C(C)(C)O)C3=O)nc1
InChIKey: DWZQUHUVXLAKCL-OAQYLSRUSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Transcreener-Fluorecescence Polarization Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 μM ATP; 10 μM MgCl2; 125 μM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 1.5 nM IRAK1; 62.5 μM ATP; 5 μM MgCl2, and 62.5 μM IRAK1 peptide in reaction buffer for 60 min.
Measurement: Ki = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 452.49, ALogP 3.67, HBA 6, HBD 2, PSA 108.56, QED 0.59
Compound (SMILES): Cc1ccc(C(=O)NC2CC2)cc1-c1cnn(-c2cnc(N3CCOCC3)s2)c1
InChIKey: ODEUCTMBTLLYKB-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43353
Assay: V9103X ADP-Glo Kinase Assay: In a 384-well plate, test compound diluted in assay buffer (1% DMSO final) is mixed with 8His-RIPK2 FL enzyme (final concentration of 8 nM). After 15 minutes of pre-incubation at RT, ATP dissolved in assay buffer is added (final concentration 5 μM). The mixture is incubated for 60 minutes at 37° C. in a humidified incubator. Then, ADP Glo Reagent is added, followed by a 40 minute incubation at rt. Finally, Kinase Detection Reagent is added and the entire mixture is incubated for 40 min at RT. The luminescence signal is measured with an Envision reader to determine the amount of ADP produced. Assay buffer: 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid), 0.1% BSA (bovine serum albumin), 10 mM MgCl2, 5 mM MnC12, 50 mM KCl, 0.01% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 10 μM Na3VO4, 1 mM DTT (dithiothreitol), pH 7.5 All plates contain wells with vehicle controls instead of compound (1% DMSO) as reference for the high signal (100% CTL (100% of control), high signal), and wells without enzyme as reference for low signal (0% CTL, low signal). The luminescent signal generated is proportional to the ADP produced and is correlated with enzyme activity. The analysis of the data is performed by the calculation of the percentage of ADP production in the presence of the test compound and RIPK2 as compared to the ADP production in the presence of RIPK2 plus 50 μMGefitinib.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 409.52, ALogP 3.03, HBA 7, HBD 1, PSA 72.28, QED 0.7
Compound (SMILES): COCCOCCOCCOCCOCCOCCOCCn1c(=O)cc(NC2CCN(S(=O)(=O)C(F)(F)F)CC2)c2cc(C(c3ccc(Cl)cc3)c3ccc(Cl)cc3)ccc21
InChIKey: PDZFPTCXJOJGJS-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay : Cells plated the day prior to the assay in clear bottom plates were viewed on an inverse microscope to ensure confluency in the range of 60-75%. The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 μM cAMP Standard (50 μM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses @ ½ dilutions resulting in a dose range of 1 μM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 mL d-H2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat # 62AM4PEB reconstituted with 5 mL d-H2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 μM in Buffer 2.
Measurement: EC50 = 4910 nM (pChEMBL 5.309999942779541)
Descriptors: MW 932.88, ALogP 6.96, HBA 12, HBD 1, PSA 136.02, QED 0.05
Compound (SMILES): COc1cccc(CCC2CCc3cc([C@H]4CC[C@](N)(COP(=O)(O)O)C4)ccc3C2)c1
InChIKey: OPWNVYPWPMIBGP-MAYCQTMOSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08530
Assay: S1P1 Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1×109 cells/pellet) were suspended in buffer containing 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 2 mM EDTA (Ethylenediaminetetraacetic acid) and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at −80° C. after protein concentration determination.Membranes (2 μg/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, Perkin elmer or American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA (bovine serum albumin), 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 l/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes onto 384-well Millipore FB filter plates, and radioactivity was measured by TOPCOUNT®. The competition data of the test compounds over a range of concentrations was plotted as percentage inhibition of radioligand specific binding. The IC50 is defined as the concentration of competing ligand needed to reduce specific binding by 50%.
Measurement: IC50 = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 459.52, ALogP 4.51, HBA 4, HBD 3, PSA 102.01, QED 0.5
Compound (SMILES): CN1CCC2(CN(Cc3cccc(F)c3Cc3nc4c(N)nc(-c5cccc(C#N)c5F)cn4n3)C2)C1=O
InChIKey: YMNGTFKKNDUJJX-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: HTRF Assay: Assays were conducted in black low volume 384-well polystyrene plates (Greiner 784076-25) in a final volume of 10 μL. Test compounds were first serially diluted in DMSO and 100 nl added to the plate wells before the addition of other reaction components. The final concentration of DMSO was 1%. Tag-Lite® Adenosine A2A labeled cells (CisBio C1TT1A2A) were diluted 1:5 into Tag-lite buffer (CisBio LABMED) and spun 1200 g for 5 mins. The pellet was resuspended at a volume 10.4× the initial cell suspension volume in Tag-lite buffer, and Adenosine A2A Receptor Red antagonist fluorescent ligand (CisBio L0058RED) added at 12.5 nM final concentration. 10 ul of the cell and ligand mix was added to the assay wells and incubated at room temperature for 45 minutes before reading on a PHERAstar FS plate reader (BMG Labtech) with HTRF 337/620/665 optical module. Percent binding of the fluorescent ligand was calculated; where 100 nM of A2A antagonist control ZM 241385 (Tocris 1036) displaces the ligand 100% and 1% DMSO has 0% displacement. The % binding data versus the log of the inhibitor concentration was fitted to a one-site competitive binding model (GraphPad Prism version 7.02) where the ligand constant=12.5 nM and the ligand Kd=1.85 nM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 514.54, ALogP 2.78, HBA 8, HBD 1, PSA 116.44, QED 0.44
Compound (SMILES): CCOC(=O)Nc1coc(-c2cncc([C@@]34CC[C@@H](c5cc(-c6c(F)cccc6F)nnc53)C4(C)C)n2)n1
InChIKey: BGWGSHJQHFNGJQ-QZXCRCNTSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Ligand Binding Assay: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present. Assay plates were 96-well polypropylene V-bottom plates. 10 uL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 uL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 uL of 5 uM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 uL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 uL of 1.5 ug/mL RORc receptor was added to wells (or 40 uL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25° C. Filtration
Measurement: IC50 = 8.1 nM (pChEMBL 8.09000015258789)
Descriptors: MW 518.52, ALogP 5.64, HBA 8, HBD 1, PSA 115.92, QED 0.36
Compound (SMILES): Cn1nc(C(C)(C)C)cc1C(=O)NC[C@@H]1C[C@H]2CC[C@@H](C1)N2CC(=O)NC(C)(C)C
InChIKey: IVSKYWUZWNEKJL-OSYLJGHBSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Activity Assay: The HEK293 cells having human Cav3.2 stably expressed therein were cultured at 37° C. in Alpha-MEM, to which 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 μg/mL), and G418 (250 g/mL) had been added. The cells were suspended in the culture liquid and were inoculated onto a 96-well plate. Subsequently, the cells were cultured for 48 hours. The culture liquid was removed, and the liquid was changed to S-MEM, to which 5% (v/v) FBS, calcium chloride (0.5 mmol/L), L-glutamine (2 mmol/L), L-alanine (8.9 ng/mL), L-asparagine (13.2 ng/mL), L-aspartic acid (1.33 ng/mL), L-glutamic acid (14.7 ng/mL), glycine (7.5 ng/mL), L-proline (11.5 ng/mL, L-serine (10.5 ng/mL), penicillin (100 U/mL), and streptomycin (100 μg/mL) had been added. The cells were cultured for another 24 hours. The culture liquid was removed again, and the cells were washed with an assay buffer (140 mmol/L sodium chloride, 5 mmol/L potassium chloride, 0.5 mmol/L magnesium chloride, 0.5 mmol/L calcium chloride, 10 mmol/L glucose, 0.4 mmol/L magnesium sulfate, 10 mmol/L HEPES, and 250 mol/L sulfinpyrazone, pH 7.4) that had been kept warm at 37° C. Subsequently, an assay buffer prepared by dissolving Fura2-AM, which is a fluorescent Ca2+ indicator, to a concentration of 5 μM, was added to the cells, and the system was incubated for 30 minutes at 37° C. The assay buffer having Fura2 dissolved therein was removed, and the cells were washed with the assay buffer. Subsequently, an assay buffer prepared by adding a test compound was added to the cells, and the system was incubated for 15 minutes. The plate was mounted on a fluorescence analyzer (FLEX STATION II, Molecular Devices, LLC), and the baseline was measured for 20 seconds. Subsequently, any change in the intracellular calcium concentration that was induced when an assay buffer prepared by adding 100 mmol/L calcium chloride was added to the cells, was measured (excited at 340 nm or 380 nm, and detected at 510 nm).
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 417.6, ALogP 2.61, HBA 5, HBD 2, PSA 79.26, QED 0.77
Compound (SMILES): CCn1c(=O)[nH]/c(=N\c2cccc(Oc3nccs3)c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: XHIBJCIYXAUMDD-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 = 177 nM (pChEMBL 6.75)
Descriptors: MW 455.93, ALogP 3.54, HBA 8, HBD 1, PSA 94.27, QED 0.48
Compound (SMILES): CCS(=O)(=O)Nc1ccc(-c2cc(NC(=O)C3CC3)nc3c2ccn3C)cc1F
InChIKey: BHWNREXFSITKHU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 > 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 416.48, ALogP 3.49, HBA 5, HBD 2, PSA 93.09, QED 0.64
Compound (SMILES): C=CC(=O)Nc1cccc(-c2cnc3[nH]cc(-c4cccc5occc45)c3c2)c1
InChIKey: DWBOJPBWGKDVQR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: ITK Inhibitor Binding Kinetics: This assay is based on the binding of a proprietary, Alexa Fluor 647-labeled, ATP-competitive kinase inhibitor (kinase tracer-236) to the ITK expression construct in the presence of a Europium-conjugated antibody, resulting in a FRET (fluorescence resonance energy transfer) signal. Displacement of the kinase tracer by compound results in a lower emission ratio upon excitation of the Europium chelate. Candidate compounds are designed as potential irreversible inhibitors of ITK, capable of ligating to an active site cysteine residue resulting in time dependent covalent binding. The time dependent nature of irreversible inhibition is investigated by performing the binding assay with and without a pre-incubation of compound and ITK.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 379.42, ALogP 5.77, HBA 3, HBD 2, PSA 70.92, QED 0.39
Compound (SMILES): Cn1nccc1Nc1nccc(-c2cc3n4c(nnc4c2)C(Oc2ccc(F)c(F)c2)CCO3)n1
InChIKey: OBYRTZDXYUNRII-UHFFFAOYSA-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 < 0.02 nM (pChEMBL 10.699000358581543)
Descriptors: MW 476.45, ALogP 3.84, HBA 10, HBD 1, PSA 104.28, QED 0.41
Compound (SMILES): O=C(Nc1ccc(Oc2ccnc3cc(OCCCO)c4c(c23)OCCO4)cc1Cl)NC1CC1
InChIKey: DRWNHOJYURSOFF-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Assay of Small Molecular Compounds for Inhibiting the Activity of RET Kinase: The assay is based on the LANCE TR-FRET technology of Perkin Elmer Inc., and the assay method is as follows:1. Dilution of compounds: a total of 11 concentrations were obtained using a 3-fold gradient dilution from the highest concentration of 2500 nM (the maximum final concentration of the drug used in this assay was 2500 nM, and the minimum final concentration was 0.042 nM).2. 2.5 μL of the gradient-diluted compounds were taken with a transfer pipette to a 384-well plate.3. Addition of enzyme: 5 μL of 2× RET kinase solution (concentration was 0.8 nM) was taken with a transfer pipette to the corresponding reaction well of the 384-well plate, mixed well and pre-reacted at room temperature for 30 minutes.4. 2.5 μL 4× Ultra ULight-JAK-1 (Tyr1023) Peptide (concentration was 200 nM)/ATP (concentration was 40 μM) mixture was taken with a transfer pipette to the corresponding reaction well of the 384-well plate.5. Negative control: 2.5 μL/well 4× substrate/ATP mixture and 7.5 μL 1× Kinase Assay Buffer were added to the wells of the 384-well plate. Positive control: 2.5 μL/well 4× substrate/ATP mixture, 2.5 μL/well 1× Kinase Assay Buffer containing 16% DMSO, and 5 μL/well 2× RET kinase solution were added to the 384-well plate. The final concentration of DMSO in the reaction system was 4%.6. The mixture was mixed well and then centrifuged and reacted at room temperature in dark for 60 min.7. Termination of the enzymatic reaction: 5 μL of 4× stop solution was taken with a transfer pipette to the wells of the 384-well plate, mixed and then centrifuged, and reacted at room temperature for 5 min.8. Development of the reaction: 5 μL of 4× detection solution was taken with a transfer pipette to the wells of the 384-well plate for color development, and the mixture was mixed and then centrifuged and reacted at room temperature for 60 min.9. The 384-well plate was placed into the Envision plate reader and the signal was detected using the appropriate program.10. Analysis and processing of the raw data:The drug concentrations and the corresponding inhibition rates were input into GraphPad Prism5 for calculation, and the inhibition rate of the compounds were calculated as follows: inhibition rate (%)=(reading of positive well−reading of experimental well)/(reading of positive control well−reading of negative control well)×100%. Processing with GraphPad Prism5 software yielded the corresponding IC50 values (the concentration of the compound at which 50% of the highest inhibition of the enzyme is achieved).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 485.92, ALogP 4.5, HBA 7, HBD 3, PSA 111.17, QED 0.4
Compound (SMILES): CCOc1ncc(-c2cnc(NCc3c(F)ccc4c3CCO4)n3cnnc23)c(C)n1
InChIKey: LXAVJONJUUHXQC-UHFFFAOYSA-N
Target: Polycomb protein EED (SINGLE PROTEIN), Homo sapiens
UniProt: O75530
Assay: EED-H3K27Me3 Peptide Competition Binding Assay by AlphaScreen: To assess the compounds potency in the EED-H3K27Me3 competition binding assay, compounds were serially diluted 3-fold in DMSO to obtain a total of twelve concentrations. Then compounds at each concentration (75 nL of each) were transferred by Mosquito into a 384-well Perkin Elmer ProxiPlate 384 plus plates. 8 uL of solutions containing 30 nM EED (1-441)-His protein and 15 nM biotin-H3K27Me3 (19-33) peptide in the buffer (25 mM HEPES, pH 8, 0.02% Tween-20, 0.5% BSA) were added to the wells and then incubated with compound for 20 min. AlphaScreen detection beads mix was prepared immediately before use by mixing nickel chelate acceptor beads and streptavidin donor beads in a 1:1 ratio (Perkin Elmer, Product No. 6760619C/M/R) into the buffer described above. Then 4 μL of detection beads mix was added to the plate and incubate in the dark at the rt for 1 h. The final concentration of donor and acceptor beads was 10 μg/mL for each. Plates were read on EnVision (PerkinElmer) using the AlphaScreen setting adapted for optimal signal detection with a 615 nm filter, after sample excitation at 680 nm. The emission signal at 615 nm was used to quantify compounds inhibition. AlphaScreen signals were normalized based on the reading coming from the positive (maximum signal control) and negative controls (minimum signal control) to give percentage of activities left. The data were then fit to a dose response equation using the program Helios (Novartis) to get the IC50 values. Helios is a Novartis in-house assay data analysis software using the methods described by Normolle, D. P., Statistics in Medicine, 12:2025-2042 (1993); Formenko, I. et al, Computer Methods and Programs in Biomedicine, 82, 31-37 (2006); Sebaugh, J. L., Pharmaceutical Statistics, 10:128-134 (2011); Kelly, C. et al., Biometrics, 46(4):1071-1085 (1990); and Kahm, M. et al., Journal of Statistical Software, 33(7): (2010) (grofit: Fitting Biological Growth Curves with R, pages 1-21, available at http://www.jstatsoft.org/).Each compound was counterscreened to determine if it interfered with the AlphaScreen beads. Compounds were diluted as described in the preceding section, and the assay was performed by adding 12 μL of 10 nM biotin-miniPEG-His6 peptide in the above buffer and incubating for 20 min at rt prior to addition of the beads to 10 μg/mL each. The plates were then incubated for 1 h at rt in dark before being read on EnVison.
Measurement: IC50 = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 421.44, ALogP 2.97, HBA 9, HBD 1, PSA 99.35, QED 0.51
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3=CCCN(S(C)(=O)=O)CC3)cc12
InChIKey: LFCHDVBWVGMFRB-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 415.49, ALogP 3.82, HBA 4, HBD 1, PSA 75.29, QED 0.7
Compound (SMILES): COC(=O)N1[C@H](C)C[C@H](NS(=O)(=O)N(C)C)[C@@H]1CO[C@H]1CC[C@@H](c2ccc(C#N)c(C)c2)CC1
InChIKey: CALXAGUURZEISL-JGOTUQMDSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Receptor Activity Assay: In a typical experiment, the OX1 and OX2 receptor agonist activity is determined in accordance with the following general experimental method. Chinese hamster ovary (CHO) cells expressing human OX1R and/or the human OX2R were grown in Iscove's modified DMEM containing glutaMAX®, 1% G418, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat-inactivated qualified fetal bovine serum (FBS). The OX2R cells were seeded at 10,000 cells/well/50 L and the OX1R cells were seeded at 20,000 cells/well/50 μL into 384-well white tissue culture plates (Greiner; cat #781080). All cell/media reagents were from GIBCO-Invitrogen Corp. The seeded cell plate(s) were incubated at 37° C. with 5% CO2 and 85% humidity for 20-24 hours. On the day of the assay, assay-ready compound plates were prepared using an acoustic liquid handler (ECHO; Labcyte), which dispensed sufficient volume of test compound stock (10 mM in DMSO) or 100% DMSO to prepare 10 point, ½-log dilutions in a final volume of 202.5 nL/well in all test wells of a 384-well diamond plate (Labcyte). Following completion of assay-ready plates, importantly, the next three steps were performed with minimal delay: 1) 20 μl of 1× stimulation buffer was added to the compound plate using a Multidrop Combi (small cassette, Thermo Fisher Scientific cat #24073290); 2) culture medium was removed from the cell plate using the Bluewasher plate washer (gentle spin; BlueCatBio); 3) 14 μl of compound/stimulation buffer mixture was added to the cell plate using a Bravo liquid handler (Agilent) prior to incubating cell plates at 37° C. with 5% CO2 and 85% humidity for 1 or 2 hours (OX1R and OX2R, respectively). During this incubation, IP-one detection reagents were prepared (38:1:1 lysis buffer:D2:AB-cryptate reagents). Six L of mixed detection reagents were added to the cell plate using a Multidrop Combi (small cassette, Thermo Fisher Scientific cat #24073290) and incubated 60 minutes at room temperature in the dark. Fluorescence signal was detected using an Envision plate reader (Perkin Elmer) [LANCE/DELFIA Dual Enh (Em: APC 665; Ex: Cy5 620)].
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 492.64, ALogP 2.9, HBA 6, HBD 1, PSA 111.97, QED 0.63
Compound (SMILES): COc1cc(Cl)c(-c2noc(C3CC3)c2CO[C@@H]2C[C@@H]3C[C@H]2CN3c2nc3c(F)cc(C(=O)O)cc3s2)c(Cl)c1
InChIKey: JAGZHTKTLRGDEF-VFCRVFHLSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: Binding Assay: The affinity of FXR ligands for the ligand binding domain of FXR was determined using a commercially available human FXR ligand binding assay (LanthaScreen, Thermofisher Cat # PV4833). The purified ligand binding domain of human FXR tagged with GST (glutathiones-S-transferase) is incubated with a terbium labelled anti-GLT antibody and a fluorescein-labelled SRC2-2 peptide (LKEKHKILHRLLQDSSSPV (SEQ ID NO.: 1)). Binding of FXR ligands to the FXR ligand binding domain promotes binding of the fluorescein-labelled SRC2-2 peptide. This causes a FRET signal between the terbium-labelled anti-GST antibody and the fluorescein-labelled SRC peptide which are both bound to the FXR ligand binding domain.Test compounds are dissolved in DMSO and a 3-fold serial dilution series is generated, then further diluted into assay buffer. The compounds are mixed with 5 nM GST-tagged FXR ligand binding domain, 5 nM Tb-labelled anti-GST antibody and 500 nM fluorescein-labelled SRC2-2 peptide in a pH7.4 buffer. The reaction is incubated at room temperature for 1 hour, then the FRET signal is measured as the ratio of the 520 nm/495 nm emission following excitation at 340 nm. The change in FRET signal is plotted against the test article concentration and fit to a 3-parameter logistical equation.
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 604.49, ALogP 7.17, HBA 8, HBD 1, PSA 97.92, QED 0.23
Compound (SMILES): COc1cc2ncnc(Sc3cccc(NC(=O)Nc4cc(C(C)(C)C)nn4CC(C)C)c3)c2cc1OC
InChIKey: UGGGYSTZAPTBNH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005310). For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in polypropylene 384-well plates in a final volume of 34 μL, while Kd determinations were performed in polystyrene 96-well plates in a final volume of 135 μL. The assay plates were incubated at room temperature with shaking for 1 hour, long enough for binding reactions to reach equilibrium, and the affinity beads were washed extensively with wash buffer (1×PBS, 0.05% Tween 20) to remove unbound protein. The beads were then resuspended in elution buffer (1×PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound. KdS were determined using eleven serial threefold dilutions.
Measurement: Kd < 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 534.69, ALogP 6.59, HBA 8, HBD 2, PSA 103.19, QED 0.25
Compound (SMILES): CCC[C@H](NC(=O)[C@H](C)NC)C(=O)N1CCC[C@H]1Cc1c(-c2nc3cc(F)ccc3n2C[C@@H]2CCCN2C(=O)[C@@H](NC(=O)[C@H](C)NC)C(C)C)[nH]c2cc(F)ccc12
InChIKey: AHDSDZLGMYADMK-NCIVLJBASA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF-alpha inducing NF-kappaB reporter: First day:Dilution of Compounds:  1) tested compounds were added into DMSO to give parent liquor (10 mM);  2) reference compound Birinapant (32 μL, 10 mM) was added into DMSO (128 μL) to give a solution (2 mM);  3) 0.5 μL reference compound Birinapant (2 mM) and tested compounds (10 mM) were taken by Echo and added into Greiner 96 well black cell culture plate respectively. The initial concentration of the reference compound and tested compound was 10 μM and 50 μM, respectively. Then, the reference compound and tested compounds were diluted at 9 points in series by 5-fold; and every plate was repeated for 3 times.  4) the reference compound WXFL2012A001 (Birinapant) (final concentration: 10 μM) as HPE and DMSO (final concentration: 0.5%) as ZPE.Second DayTNF-α Inducing1) TNF-α (100 μg/mL) was diluted into 20 ng/mL with 0.1% FBS medium and transferredto the third plate with 100 μL per well2) Removing medium of the first 96 well black cell culture plate (compound activity testing plate) after 24 h, replacing with fresh medium containing the compound and 20 ng/mL TNF-α in the third plate3) Placing cell culture plate into cell incubator (37° C., 5% CO2) and incubatingfor 6 h4) after 6 h, compound's activity in the first plate was tested according to Bright-Glo (Promega) manual method, luciferase signal in each well was tested by Envision plate reader5) compound's cytotoxicity in the second plate was tested according to ATPlite 1Step (Perkin Elmer) manual method, luciferase signal in each well was tested by Envision plate reader6) analyzing with software to obtain EC50 of compound.
Measurement: EC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 804, ALogP 4.63, HBA 8, HBD 5, PSA 156.49, QED 0.11
Compound (SMILES): CC(C)Oc1ccc(S(=O)(=O)N2C[C@H](n3nc(CC(=O)O)c4ccc(F)cc43)C[C@@H]2COc2cccc(F)c2)cc1
InChIKey: IJOZDHYYXGZQSP-FGZHOGPDSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Receptor Binding Inhibition Assay: Cell membrane was prepared from CRTH2-expressing K562 cell. The cell membrane (20 μg/well) was mixed with reaction buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2). Then, [3H]-PGD2 was added to the mixture and incubated for 60 min at room temperature. The concentration of [3H]-PGD2 was determined as equal to Kd calculated from Scatchard plot. Then, the mixture was filtrated with glass paper immediately. The filter paper was washed several times and radio activity on the filter was measured. Specific binding was defined as difference between total and non-specific binding (radio activity with 10 μM PGD2). Ki values for each compound were calculated from displacement curve.
Measurement: Ki = 2505 nM (pChEMBL 5.599999904632568)
Descriptors: MW 585.63, ALogP 4.81, HBA 7, HBD 1, PSA 110.96, QED 0.28
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CCC[C@H]2c2noc(-c3cc(C)ccc3C)n2)c1
InChIKey: HXLWTZGQBQSCHZ-NRFANRHFSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 428.5, ALogP 4.22, HBA 7, HBD 0, PSA 89.94, QED 0.48
Compound (SMILES): COC1CN(C(=O)C2CCc3c(-c4ccc(C(=O)O)cc4F)nn(C(=O)c4c(Cl)cccc4C4(C(F)F)CC4)c3C2)C1
InChIKey: XPDGSFBLRUKGJV-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 mg/ml bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per ml in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 ml SF9 lysate per 15 ml of diluted RORγ-LBD.Compounds to be tested were injected to the 384-well assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, CA).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-SPSSHSSLTERHKILHRLLQEGSP) (SEQ ID NO:1) and APC-conjugated streptavidin (final concentrations 100 nM and 8 nM respectively) were also added to each well.
Measurement: IC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 602.01, ALogP 4.99, HBA 6, HBD 1, PSA 101.73, QED 0.41
Compound (SMILES): Nc1ncnc2ccc(-c3cc(F)cc(CN4CCC[C@@H](O)C4)c3)nc12
InChIKey: BFVBNWMXJGBWIB-OAHLLOKOSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O95819
Assay: Inhibition Assay: The kinase activity of purified human MAP4K4 kinase domain was measured by monitoring the phosphorylation of a peptide substrate derived from moesin protein (Leu-Gly-Arg-Asp-Lys-Tyr-Lys-Thr-Leu-Arg-Gln-Ile-Arg-Gln) fluorescently labeled on the N-terminus with 5-carboxyfluorescein using the Caliper LabChip technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (IC50), compounds were serially diluted in DMSO and added to 10 uL kinase reactions containing 1 nM purified MAP4K4 enzyme, 1 uM peptide substrate, 10 uM ATP, 10 mM MgCl2, 1 mM EGTA, 50 mM Hepes pH 7.2, 1 mM DTT, 0.01% Triton X-100, and 2% DMSO. Reactions were incubated at room temperature in Perkin Elmer Proxiplates for 45 minutes and stopped by the addition of 10 uL of an EDTA-containing solution (50 mM Hepes pH 7.2, 40 mM EDTA, 0.02% Triton X-100). The fraction of phosphorylated peptide was determined as a fraction of total peptide substrate using the Caliper Lab Chip 3000 according to the manufacturer's instructions. IC50 values were determined using the four-parameter non-linear fit model.
Measurement: IC50 = 571 nM (pChEMBL 6.239999771118164)
Descriptors: MW 353.4, ALogP 2.37, HBA 6, HBD 2, PSA 88.16, QED 0.75
Compound (SMILES): CC1(C)Oc2cc(Cl)ccc2C2OCC3(CCN(CCn4ccnc4CO)CC3)CC21
InChIKey: HTHOAOYVNGKOOM-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Test for Evaluating hERG Inhibition: To a cultured CHO cell strain which stably expresses hERG was added each test compound to adjust the final concentration of DMSO to 0.0135 to 0.5%. The hERG current was measured with QPatch HT (Sophion Inc.), and the concentration at which 50% of the hERG current was inhibited by each test compound (IC50 value; μM) was calculated.
Measurement: IC50 = 1400 nM (pChEMBL 5.849999904632568)
Descriptors: MW 445.99, ALogP 4.06, HBA 6, HBD 1, PSA 59.75, QED 0.77
Compound (SMILES): Cn1ncc2c(C(=O)NC(CCOCCCCc3ccc4c(n3)NCCC4)C(=O)O)cccc21
InChIKey: DCCYSDVCRVEPKV-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 > 1000 nM (pChEMBL 6)
Descriptors: MW 465.55, ALogP 2.94, HBA 7, HBD 3, PSA 118.37, QED 0.37
Compound (SMILES): Cc1ccccc1CNC(=O)c1ccc(NC(=O)NCc2ccncc2)cc1
InChIKey: XSXPTHMNEFVJRV-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Enzyme Assay and NMN Detection: NAMPT reaction was performed in 50 mM HEPES, pH 7.5, containing 5 mM MgCl2, 0.1% Prionex, 0.005% Tween 20, and 1 mM TCEP at room temperature in Greiner 384-well black polypropylene plates. The concentration of substrates and enzymes in the final assay were as follows: ATP (120 uM), nicotinamide (5 uM), phosphoribosylpyrophosphate (6.25 uM), NAMPT enzyme (16 nM), pyrophosphatase (0.04 U/ml) in the final reaction volume 6 uL. Following 2 h incubation the reaction was stopped by addition 2.5 ul 20% acetophenone in dimethyl sulfoxide (DMSO) and 2.5 ul 2 MKOH. Plates were centrifuges and added with 10.5 ul 88% formic acid. The fluorescence (ex380/em460) was measured following another short incubation and 30 min incubation. The increased concentration of NMN was determined from a calibration curve obtained at the time of experiment.
Measurement: EC50 = 1050 nM (pChEMBL 5.980000019073486)
Descriptors: MW 374.44, ALogP 3.64, HBA 3, HBD 3, PSA 83.12, QED 0.62
Compound (SMILES): Cn1cc(Nc2nccc(N3C[C@H]4CC[C@@H](C3)N4C(=O)[C@H]3CC3(F)F)n2)cn1
InChIKey: BUWBRTXGQRBBHG-HZSPNIEDSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper Enzyme Assay: 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 = 702 nM (pChEMBL 6.150000095367432)
Descriptors: MW 389.41, ALogP 1.79, HBA 7, HBD 1, PSA 79.18, QED 0.86