lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCCN1CCC(N2Cc3c(cnn3C(C)C)-c3n[nH]c4ccnc2c34)CC1
InChIKey: UOQZUVUTYBJAMY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 Assay: JAK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% 0-mercaptoethanol, 1 mg/ml BSA) is assayed against PDKtide (KTFCGTPEYLAPEVRREPRILSEEEQ-EMFRDFDYIADWC) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% 0-mercaptoethanol, 100 μM substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μl of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.
Measurement: IC50 = 9900 nM (pChEMBL 5)
Descriptors: MW 395.51, ALogP 2.83, HBA 7, HBD 1, PSA 75.1, QED 0.72
Compound (SMILES): Cc1ccccc1C(=O)c1c[nH]c2ncnc(N[C@H]3CC[C@@H](NC(=O)CN4CCN(C)CC4)CC3)c12
InChIKey: ZAIXEVZXCATZAQ-BGYRXZFFSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Mobility Shift Assay (MSA) Type A: Full length unphosphorylated form of BTK expressed in Sf9 cells was employed to test inhibitory activity in the inactive BTK assay. The assay was measured in buffer solution containing 100 mM HEPES pH7.5, 0.01% Triton X-100, 0.1% BSA, 5 mM MgCl2, 1 mM DTT. The enzyme and increasing concentrations of inhibitor was incubated at room temperature for 30 minutes and the kinase reaction was initiated by the addition an activation mixture diluted in assay buffer containing Srctide peptide substrate, DOPS/DOPC, PtdIns(3,4,5)P3, and ATP for final concentrations of 1 μM Srctide, 5.5 μM DOPS, 5.5 μM DOPC, 0.5 μM PtdIns(3,4,5)P3, and 16 μM ATP. The plates were incubated for 60 minutes at room temperature, and then the reaction stopped with 100 mM HEPES buffer containing 0.01% Triton and 40 mM EDTA and read on Caliper Life Sciences Labchip EZ Reader II instrument.
Measurement: IC50 = 8.3 nM (pChEMBL 8.079999923706055)
Descriptors: MW 489.62, ALogP 2.58, HBA 7, HBD 3, PSA 106.25, QED 0.44
Compound (SMILES): Cn1cnc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)CC3CCOC3)c2)c1
InChIKey: UPSFLEQIRCEVCX-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92993
Assay: KAT-5 Enzyme Assay: Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-5 enzyme (9 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 0.5 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 416.86, ALogP 1.48, HBA 6, HBD 2, PSA 102.32, QED 0.69
Compound (SMILES): Nc1nc2c(ncn2[C@@H]2O[C@@H]3COP(=O)(O)OC4[C@@H](COP(=O)(O)O[C@H]2C3O)O[C@@H](n2ccc3c(N)ncnc32)[C@H]4O)c(=O)[nH]1
InChIKey: UTYHBKZTJXGVRM-AINHVTRJSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: 3H-cGAMP Filtration Binding Assay (HAQ STING): The compounds were serially titrated by the Hamilton STARPlus CORE in a 96-well plate (Greiner, #651201) using a 1:3 ten-point dose response format. After compound preparation, a 2.2 ug/ml working concentration of STING membrane (SEQ. ID. No. 2) was prepared by diluting concentrated membrane into assay buffer (1×PBS; Invitrogen #SH30028.02) and douncing 7× using a manual tissue homogenizer (Wheaton, #357546). 148 uL of prepared membrane was then manually added to each well of a 96-well deep-well polypropylene plate (Fisher Scientific, #12-566-121). Following membrane addition, 2 uL of either titrated test compound, DMSO control (Sigma #276855), or cold cGAMP control (prepared in-house) was added to the appropriate wells using a Biomek FX. Compound and membrane then preincubated for 60 min at RT to allow compound binding to equilibrate. Following equilibration, 8 nM of [3H] c-GAMP ligand was prepared by diluting into assay buffer, and 50 uL of this working stock was then manually added to each well of the assay plate. Plates were then incubated at RT for 60 min, and the contents of each assay plate were then filtered through a 96-well GF/B filter plate (PerkinElmer, #6005250) using a TomTec MachIII Cell Harvester equipped with 20 mM HEPES buffer (Fisher Scientific, #BP299500). The filter plates were then dried at 55° C. for 30 min using a pressurized VWR oven before 30 uL of Ultima GoldF scintillate was added to each well. Tritium levels for each reaction well were then measured using a PerkinElmer TopCount plate reader.
Measurement: EC50 = 7.2 nM (pChEMBL 8.140000343322754)
Descriptors: MW 673.43, ALogP -1.74, HBA 19, HBD 7, PSA 316.76, QED 0.12
Compound (SMILES): CN(C)CCNC(=O)c1n[nH]c2ccc(-c3cccnc3)cc12
InChIKey: CYPMWQXBYDLBDW-UHFFFAOYSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: Wnt activity assay: Reporter cell lines were generated by stably transducing cells of cancer cell lines (e.g., colon cancer) with a lentiviral construct that include a Wnt-responsive promoter driving expression of the firefly luciferase gene.Lentiviral constructs were made in which the SP5 promoter, a promoter having eight TCF/LEF binding sites derived from the SP5 promoter, was linked upstream of the firefly luciferase gene. The lentiviral constructs included a hygromycin resistance gene as a selectable marker. The SP5 promoter construct was used to transduce SW480 cells, a colon cancer cell line having a mutated APC gene that generates a truncated APC protein, leading to de-regulated accumulation of β-catenin. A control cell line was generated using another lentiviral construct containing the luciferase gene under the control of the SV40 promoter which does not require β-catenin for activation.Cultured SW480 cells bearing a reporter construct were distributed at approximately 10,000 cells per well into 384 well multiwell plates. Compounds were then added to the wells in half-log dilutions using a three micromolar top concentration. A series of control wells for each cell type received only buffer and compound solvent. Twenty-four hours after the addition of compound, reporter activity for luciferases was assayed, for example, by addition of the BrightGlo luminescence reagent (Promega) and the Victor3 plate reader (Perkin Elmer). Readings were normalized to DMSO only treated cells, and normalized activities were then used for the IC50 calculations.
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 309.37, ALogP 1.92, HBA 4, HBD 2, PSA 73.91, QED 0.75
Compound (SMILES): CC(C)C[C@](C)(O)CN1CCN(c2cccc3c2cnn3-c2ccccc2F)C1=O
InChIKey: JHCGKDLPHAAUPR-QHCPKHFHSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 μg/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 μL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 μM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 μL assay buffer containing 2.5 μM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR®TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics.
Measurement: IC50 = 816 nM (pChEMBL 6.090000152587891)
Descriptors: MW 410.49, ALogP 4.2, HBA 4, HBD 1, PSA 61.6, QED 0.66
Compound (SMILES): CC(C)(O)[C@]1(C)Cc2cc(NC(=O)c3cnn4cccnc34)c(N3CCOCC3)nc2O1
InChIKey: BMCKJFJQDZNUAK-QFIPXVFZSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Enzymatic 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 uM ATP; 10 uM MgC2; 125 uM 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 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.
Measurement: Ki = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 438.49, ALogP 1.68, HBA 9, HBD 2, PSA 114.11, QED 0.63
Compound (SMILES): C[C@H]1OCCn2c1nc1c(N3CC4CC3C4)nc(-c3cnc(N)nc3)nc12
InChIKey: RWQUHTBMJUYUKO-GRZMOONWSA-N
Target: Mitogen-activated protein kinase kinase kinase 12 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12852
Assay: DLK TR-FRET Inhibition Assay: DLK kinase reactions (20 μL) containing 5 nM N-terminally GST-tagged DLK (catalytic domain amino acid 1-520) (Carna Bioscience), 40 nM N-terminally HIS tagged MKK4 K131M substrate, and 30 μM ATP in kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Triton X-100, 0.01% Bovine γ-Globulins, 2 mM DTT, 10 mM MgCl2 and 1 mM EGTA), and testing compound 1:3 serial diluted starting at 20 uM were incubated at ambient temperature for 60 minutes in 384 well OptiPlate (Perkin Elmer). To quench kinase reactions and detect phosphorylated MKK4, 15 μL of TR-FRET antibody mixture containing 2 nM anti-phosphorylated MKK4 labeled with Europium cryptate (Cisbio) and 23 nM anti-HIS labeled with D2 (Cisbio) in detection buffer (25 mM Tris pH 7.5, 100 mM NaCl, 100 mM EDTA, 0.01% Tween-20, and 200 mM KF) was added to the reaction mixture. The detection mixture was incubated for 3 hours at ambient temperature and the TR-FRET was detected with an EnVision multilabel plate reader (Perkin-Elmer) using the LANCE/DELFIA Dual Enh label from Perkin-Elmer (excitation filter: UV2 (TRF) 320 and emission filters: APC 665 and Europium 615).
Measurement: Ki = 239 nM (pChEMBL 6.619999885559082)
Descriptors: MW 364.41, ALogP 1.56, HBA 9, HBD 1, PSA 107.87, QED 0.73
Compound (SMILES): COc1cc(N2CCc3nc(-c4ncccn4)ncc3C2)cc(F)n1
InChIKey: BBIHQKJIKXNHTK-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), GB virus-B
UniProt: Q69422
Assay: HBsAg Assay: HepG2.2.15 cells were seeded in duplicate into white, 96-well plates at 1.5×104 cells/well. The cells were treated with a three-fold serial dilution series of the compounds in DMSO. The final DMSO concentration in all wells was 1% and DMSO was used as no drug control.The HBsAg chemiluminescence immunoassay (CLIA) kit (Autobio Diagnostics Co., Zhengzhou, China, Catalog number: CL0310-2) was used to measure the levels of secreted HBV antigens semi-quantitatively. For the detection 50 μL/well culture supernatant was used and HBsAg was quantified using HBsAg chemiluminescence immunoassay (CLIA) kit (Autobio Diagnostics Co., Zhengzhou, China, Catalog number: CL0310-2), 50 μL of the supernatant was transferred to the CLIA assay plate and 50 μL of enzyme conjugate reagent was added into each well. The plates were sealed and gently agitated for 1 hour at room temperature. The supernatant-enzyme-mixture was discarded and wells were washed 6 times with 300 μL of PBS. The residual liquid was removed by plating the CLIA plate right side down on absorbent tissue paper. 25 μL of substrates A and B were added to each well. Luminance was measured using a luminometer (Mithras LB 940 Multimode Microplate Reader) after 10 minutes incubation. Dose-response curves were generated and the IC50 value was extrapolated by using the E-WorkBook Suite (ID Business Solutions Ltd., Guildford, UK). The IC50 was defined as the compound concentration (or conditioned media log dilution) at which HBsAg secretion was reduced by 50% compared to the no drug control.
Measurement: IC50 = 553 nM (pChEMBL 6.260000228881836)
Descriptors: MW 338.35, ALogP 2.04, HBA 7, HBD 0, PSA 76.92, QED 0.68
Compound (SMILES): Oc1ccc(C(O)CN2C[C@@H]3C[C@](F)(Cc4ccccc4)C[C@@H]3C2)cc1
InChIKey: BWLCVUVDSLQCFZ-NGJZOURWSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: FDSS Assay: To each well of the plate, 10 μL test compound, control (MK801) or HHnoCa buffer was added to a final concentration of 10 μM with a final concentration of DMSO of 0.1%. Following 10 minutes pre-incubation in the dark, plates are loaded onto the Hamamatsu FDSS 6000. After collecting baseline fluorescence images, 3 μM glutamate, 3 glycine, and 1 mM Ca2+ in HHnoCa buffer is added to each well, and Ca2+ is recorded for 3 minutes. Data were processed by computing ratio of fluorescence at the end of data collection to baseline fluorescence to assess degree of Ca′ influx inhibition relative to that observed in MK801.
Measurement: IC50 = 448 nM (pChEMBL 6.349999904632568)
Descriptors: MW 355.45, ALogP 3.72, HBA 3, HBD 2, PSA 43.7, QED 0.86
Compound (SMILES): O=C(Nc1cn2nc(-c3c(-c4ccc(F)cc4)nc4ccccn34)ccc2n1)c1cc(Cl)ncc1C(F)(F)F
InChIKey: TXZLIURWTIIEJG-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 16.3 nM (pChEMBL 7.789999961853027)
Descriptors: MW 551.89, ALogP 6.17, HBA 7, HBD 1, PSA 89.48, QED 0.21
Compound (SMILES): FC(F)(F)c1cnc(N2CCCC2)nc1Nc1ccc2c(c1)OCO2
InChIKey: RDZCZCDOBVQANL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ULK1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75385
Assay: ULK1 inhibition assay: Gamma-32P assays to measure ULK1 kinase activity were performed as previously described. Briefly, Flag ULK1 was transfected into HEK293T cells and 20 hours later treated as indicated. The immmunoprecipitate was washed in IP buffer 3 times, and washed in kinase buffer (25 mM MOPS, pH 7.5, 1 mM EGTA, 0.1 mM Na3VO4, 15 mM MgCl2,). Hot and cold ATP were added at a 100 μM final concentration. As substrates, GST or the recombinant protein GST-Atg101 purified from E. coli were used at 1 μg for each reaction. Reactions were boiled, run out on SDS page gel. The gel was dried, and imaged using PhosphoImager software. For cold assays to asses ULK1, Flag ULK1 which was transiently overexpressed and immunoprecipited from HEK293T cells. Reactions were then run out on SDS page gel, transferred to PVDF membrane and blotted for total levelsFluorescence MicroscopyVps34flox/flox MEFs were reconstituted with Flag-VPS34 and either p40FX or GFP-DFCP1. 48 hours post infection with adenovirus expressing Cre recombinase (MOI of 100), cells were plated on glass coverslips at a density of 3×105 cells per well in 6-well tissue culture plates. 18 h later, cells were fixed in 4% PFA in PBS for 10 minutes and permeabilized in 0.2% Triton in PBS for 10 minutes. The following primary antibodies were used: mouse anti-Myc epitope and LC3B XP antibody (2276 and 3868 respectively, Cell Signaling Technologies). Secondary antibodies were anti-rabbit Alexa488 and anti-mouse Alexa594 (Molecular Probes, 1:1000. Cells were then fixed and counter stained with DAPI. Coverslips were mounted in FluoromountG (SouthernBiothech). Images were acquired on a Zeiss Axioplan2 epifluorescence microscope coupled to the Openlab software. Confocal images of mitotracker were taken on Zeiss LSM 710 laser scanning confocal microscope. 10 random fields per condition were acquired using the 100× objective and representative images shown. Glass coverslips were mounted directly on plate with FluoromountG and images taken on Zeiss Axioplan2 epifluorescence microscope.
Measurement: IC50 > 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 352.32, ALogP 3.57, HBA 6, HBD 1, PSA 59.51, QED 0.91
Compound (SMILES): CC(CCCc1ccccc1)c1cc(C(=O)N2CCC(NC(=O)C3CC3)C2)n[nH]1
InChIKey: XRCCYPSLTYKAIY-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: KDM5 Enzyme Assay: Full length KDM5A enzyme was expressed and purified inhouse. Biotin-H3K4me3 peptide was purchased from New England Biolabs. HTRF reagents (containing Eu-labeled H3K4me1-2 antibody, and streptavidin-XL665) were purchased from Cis-Bio International. Plates were read on an Envision multi-label plate reader (Perkin Elmer).The HTRF assay mixture contained 2 nM full length KDM5A enzyme, 100 nM H3K4Me3 peptide substrate, 1 uM 2-OG, 100 uM Fe2+, 500 uM ascorbate. 50 mM HEPES pH 7.0 buffer, 0.01% Triton-X 100, 2 mM DTT, 0.25% DMSO at a final volume of 10 uL.The enzyme reaction was carried out at room temperature in black Proxiplate 384-Plus plate (Corning. Costar) within 30 minutes, in the presence of varying concentration of a test compound. At the end of enzyme reaction, 5 uL of 1 mM EDTA were added to quench the reaction and then the detection reagents (5 uL) were added to give final concentrations of 0.5 nM Eu-labeled H3K4me1-2 antibody, and 50 nM streptavidin-XL665. The plates were incubated at room temperature for 60 minutes and then read in the Envision plate reader. The readouts were transformed into % inhibition, and IC50 value of a test compounds was generated by using four parameters curve fitting (Model 205 in XLFIT5, iDBS).
Measurement: IC50 = 0.12 nM (pChEMBL 9.920000076293945)
Descriptors: MW 394.52, ALogP 3.28, HBA 3, HBD 2, PSA 78.09, QED 0.72
Compound (SMILES): CC[C@@H](NC(=O)[C@@H](CO)N1Cc2ccc(-c3nc(NC4CCOCC4)ncc3Cl)cc2C1=O)c1cc(OC)ccc1F
InChIKey: UCTJOZVXCNGCND-CLJLJLNGSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 598.08, ALogP 4.12, HBA 8, HBD 3, PSA 125.91, QED 0.32
Compound (SMILES): O=C(Nc1cn2cc(-c3ccc4sc(=O)[nH]c4c3)ccc2n1)[C@@H]1C[C@@H]1F
InChIKey: JWOJVNKGVGVDHF-NEPJUHHUSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Kinase Assay: c-abl: ADP-Glo assay kit was purchased from Promega. Magnesium chloride (MgCl2), bovine serum albumin (BSA), ethylene glycol-bis(p-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), triton X-100, 1,4-dithiothreitol (DTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. HEPES buffer was purchased from Gibco. ABL1 kinase and Abltide were purchased from Signalchem.c-Abl kinase activity was measured by Promega's ADP-Glo Assay. In this assay, His-tagged recombinant human ABL1 (0.25 ng/μl) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of Abltide (0.01 μg/μl) and 5 μL of ATP (25 μM) in buffer (50 mM HEPES, 7.5; 10 mM MgCl2; 1 mM EGTA; 0.05% BSA; 0.01% Triton X-100; 2 mM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30° C. for 30-min. After the incubation, 25 μL ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 μL per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software and Sigma Plot 13.0.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 368.39, ALogP 3.2, HBA 5, HBD 2, PSA 79.26, QED 0.58
Compound (SMILES): CC1(C)CC=C(c2nc(C3CC(C)(C)OC(C)(C(=O)O)C3)ccc2NC(=O)c2nc(C#N)c[nH]2)CC1
InChIKey: MZZYKYGSHDFBTD-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: Kinase Assay: c-fms(h) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 250 μM KKKSPGEYVNIEFG, 10 mM MgAcetate and [γ-33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The reaction was initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of 3% phosphoric acid solution. 10 μL of the reaction was then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 491.59, ALogP 5.04, HBA 6, HBD 3, PSA 140.99, QED 0.53
Compound (SMILES): Cc1cccc([C@@]2(N)CCCN(c3cnc(-c4ccc(F)cc4)cc3Cn3cnc4c(N)nccc43)C2)n1
InChIKey: ZEIPMJNFQMCDSC-GDLZYMKVSA-N
Target: Histone-lysine N-methyltransferase NSD2 (SINGLE PROTEIN), Homo sapiens
UniProt: O96028
Assay: LC-MS/MS-Based NSD2 Enzymatic Assay: This assay employed LC-MS/MS technology to monitor the SAH production from the NSD2 enzymatic reaction. The enzymatic reaction was performed in white proxiplate plus 384-well microplate (Perkin Elmer). The reaction mixture (10 μL) were composed of 8 nM NSD2 [1-1365], 1 M SAM (USB, 10601) and 400 nM nucleosome (purified from mouse liver) in reaction buffer (20 mM Tris-HCl, pH at 8.0, 0.01% Tween 20, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT, 1 mM TCEP (pH7.5)). After 90 min incubation at room temperature, 3 μL quenching solution containing 2.5% TFA with 320 nM d4-SAH was added to stop the reaction.For the inhibition assay, compound solutions were transferred into the wells by Mosquito (TTP LabTech). The inhibition assays were carried out by preincubating various concentrations of the inhibitor with 5 uL reaction mixture containing 16 nM NSD2 [1-1365] and 2 uM SAM (USB, 10601) in reaction buffer. After 20 min preincubation, 5 uL solution containing 800 nM nucleosome (purified from mouse liver) in reaction buffer was added to initiate the reaction. The reaction was stopped by 3 μL quenching solution containing 2.5% TFA with 320 nM d4-SAH after 90 min.The SAH production from the enzymatic assays were monitored by LC-MS/MS on an API 4000 triple quadrupole mass spec with Turbolon Spray (Applied Biosystem) coupled with Prominenece UFLC(Shimazu). Liquid chromatography was performed on a Chromolith FastGradient HPLC column (RP-18e, 25-2 mm, from Merck) at a flow rate of 0.8 ml/min. The column was connected directly to the turbo ion electrospray operating in the positive-ion mode. Mobile phase A is 0.1% FA and 2% methanol in water and mobile phase B is 0.1% FA in ACN. Injection volume was 3 μl and the autosampler was kept at 4 degree. SAH and d4-SAH were simultaneously monitored. Data were acquired and processed by Analyst software.To quantify the formed SAH, d4-SAH was added as internal standard (IS). A series of SAH (Sigma, A9384) solution at varying concentration (1-500 nM in reaction buffer) was mixed with quenching solution mentioned above. Then LC-MS/MS was carried out on API 4000 LC/MS/MS system to detect both SAH and d4-SAH. The plot of SAH peak area/IS peak area vs SAH concentration was used to generate the normalization factor of SAH. The SAH production from real enzymatic reaction was derived from the standard curve of SAH. The down-limit of our system for the detection of SAH is around 1-2 nM, and the linear range can reach up to 500 nM.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 508.61, ALogP 4.42, HBA 8, HBD 2, PSA 111.77, QED 0.36
Compound (SMILES): N[C@H]1CCC[C@H]1NC(=O)c1sc2nccc3c2c1NC(=O)N3c1cccc(-c2ccccn2)c1
InChIKey: SZEQFTVUBDADNL-FUHWJXTLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 470.56, ALogP 4.65, HBA 6, HBD 3, PSA 113.24, QED 0.4
Compound (SMILES): Cn1nc2c(c1-c1cc(F)c(F)c(F)c1)C[C@@H]1CCC[C@H]2N1C(=O)c1ccc2cccnc2c1
InChIKey: QCPJUIZYOCJCJU-HTAPYJJXSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Activity Assay: The assay used to measure the in vitro activity of MGL is adapted from the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson S J, Lovenberg T W, Barbier A J. Anal Biochem. 2003 Jul. 15; 318(2):270-5). The assay consists of combining endogenously expressed MGL from HeLa cells with test compounds, adding [glycerol-1,3-3H]-oleoyl glycerol, incubating for one hour, and then measuring the amount of cleaved [1,3-3H]-glycerol that passes through an activated carbon filter. The amount of cleaved, tritiated glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme in a particular well/test condition. Standard conditions for this assay combine 300 nM [Glycerol-1,3-3H]-oleoyl glycerol with human MGL from HeLa cells and test compounds for one hour, after which the reaction is filtered through activated carbon and tritium is measured in the flow through. The test compound concentration in screening mode is 10 uM, while the highest compound concentration in IC50 assays is determined empirically.
Measurement: IC50 = 3.09 nM (pChEMBL 8.510000228881836)
Descriptors: MW 462.48, ALogP 5.34, HBA 4, HBD 0, PSA 51.02, QED 0.38
Compound (SMILES): Cn1cc(Nc2ncc3cc(-c4cc(C(=O)Nc5cccc(C(F)(F)F)c5)ccc4C4CC4)ccc3n2)cn1
InChIKey: XEEYQEHUTWMPDJ-UHFFFAOYSA-N
Target: Discoidin domain-containing receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16832
Assay: Enzyme-linked immunosorbent assay (ELISA): Enzyme reaction substrate Poly(Glu, Tyr) 4:1 was diluted to 20 μg/mL with potassium-free PBS (10 mM sodium phosphate buffer, 150 mM NaCl, pH 7.2-7.4), and the microtiter plate was coated with 125 μL/well and placed at 37° C. for 12-16 hours of reaction. The liquid in the well was discard. The plate was washed three times with T-PBS (potassium-free PBS containing 0.1% Tween-20, 200 μL/well), 5 minutes each time. The ELISA plate was dried in an oven at 37° C. for 1 to 2 hours.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 528.54, ALogP 6.92, HBA 6, HBD 2, PSA 84.73, QED 0.25
Compound (SMILES): O=C(O)c1ccncc1Nc1nn(C2CCOC2)c2c1CCCC2
InChIKey: JZTNSFWOTHOOMW-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: Enzymatic Assay: The enzymatic assay of Jarid1A activity is based upon Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The ability of test compounds to inhibit the activity of Jarid1A was determined in 384-well plate format under the following reaction conditions: 1 nM Jarid1A, 300 nM H3K4me3-biotin labeled peptide (Anaspec cat #64357), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-mono- or di-methylated histone H3 lysine 4 (H3K4me1-2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 25 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K4me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO was added to each well of plate, followed by the addition of 2 μl of 3 nM Jarid1A to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 50 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K4me1-2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 328.37, ALogP 2.56, HBA 6, HBD 2, PSA 89.27, QED 0.9
Compound (SMILES): c1ccc(C2CN(c3nccc(Nc4ccc(-c5ccncc5)cc4)n3)CCCN2)cc1
InChIKey: VTVOOOAULZKYMR-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Determination of Compound's ROCK Inhibitory Activity In Vitro (Z'Lyte Assay): Recombinant ROCK1 (amino acids 1-535) and ROCK2 (amino acids 1-552) proteins were purchased from ThermoFisher Scientific. Compound's activities were measured by Z′-lyte kinase kit (ThermoFisher Scientific) and IC50 was calculated
Measurement: IC50 = 206 nM (pChEMBL 6.690000057220459)
Descriptors: MW 422.54, ALogP 4.82, HBA 6, HBD 2, PSA 65.97, QED 0.48
Compound (SMILES): N#Cc1cncc(COc2cc(O[C@H]3CCc4c(-c5ccc6c(c5F)OCCO6)cccc43)c(Cl)cc2CN2CC(O)C2)c1
InChIKey: LGKWJNPRZZRKDL-LJAQVGFWSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayâELISA: Plates were coated with 1 Î¼g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4Â° C. The wells were then blocked with PBS containing 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37Â° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the samples were diluted to 1:5 in dilution medium in the ELISA plates. Human PD-1 and biotin 0.3 Î¼g/mL (ACRO Biosystems) were added and incubated for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was added with PBS containing 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37Â° C. and was washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37Â° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 614.07, ALogP 6.01, HBA 8, HBD 1, PSA 97.07, QED 0.26
Compound (SMILES): C=C1C[C@@H](c2nc3cc(C)c(C)cc3[nH]2)N(C(=O)c2ccccc2-n2nccn2)C1
InChIKey: DLVLLKYWAYBERE-NRFANRHFSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 398.47, ALogP 3.9, HBA 5, HBD 1, PSA 79.7, QED 0.53
Compound (SMILES): N#Cc1ccc2ncnc(N[C@H]3CC[C@H](N4CCN(S(=O)(=O)CCOCCOCCOc5ccc6c(c5)C(=O)N(C5CCC(=O)NC5=O)C6=O)CC4)CC3)c2c1
InChIKey: PFZFDVVJTFBIPW-HSFMFCDSSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: A Z′-Lyte assay based on a fluorescence resonance energy transfer (FRET) readout was developed for measuring IRAK4-dependent phosphorylation of a FRET-peptide substrate as described by Thermo Fisher Scientific (Grand Island, N.Y.). At a starting concentration of 1.5 mM, the identified compounds were serially diluted with 100% dimethyl sulfoxide (DMSO) in 3-fold increments, into Greiner bio-one 96-well plates (cat. #: 650201, Greiner bio-one, Monroe, N.C.). Compounds were then transferred to intermediate Greiner bio-one 96-well plates and diluted 10-fold with Kinase assay buffer (25 mM HEPES, pH 7.5, 10 mM MgCl2, 10 mM MnCl2, 1 mM EGTA, and 0.01% Brij-35, 2 mM DTT). Three μL of the serially diluted compounds were then transferred in duplicate to low-volume 384-well black proxiplates (cat. #: 6008269; Perkin Elmer, Akron, Ohio), to give duplicate twelve-point concentration curves. Six μl of a 2.5× solution of IRAK4 enzyme (cat #: 40064, BPS Bioscience, San Diego, Calif.) in kinase buffer was added to each well, followed by a 10 minutes pre-incubation step. Reactions were initiated by adding 6 μl of a 2.5× substrate mix of ATP and Z′-Lyte Ser/Thr 7 peptide (cat. #: PV3180, Thermo Fisher Scientific, Grand Island, N.Y.) and proceeded at room temperature for 1 hour. The final concentration of key reagents in the 15 uL kinase reactions were 2 μM substrate, 2 nM enzyme, and 1 mM ATP, with dose responses starting at 30 μM compound. At the end of the kinase reactions, 5 μl of developing solution (as instructed in for cat. #: PV3180, Thermo Fisher Scientific, Grand Island, N.Y.) was added to each well and incubated at room temperature for 1 hour. All wells were read on an Envision 2105 Multilabel fluorescence plate reader (Perkin Elmer, Waltham, Mass.) at 400 nm excitation and 460 nm/530 nm emission. Plus and minus 100% inhibition controls were used to calculate percent inhibition and IC50 curves were generated using Graphpad Prism (La Jolla, Calif.).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 788.88, ALogP 1.69, HBA 14, HBD 2, PSA 213.46, QED 0.17
Compound (SMILES): Cc1ccc(S(=O)(=O)N2CCN3C(=O)OC[C@H]3C2)cc1-c1cnc2c(N)ncnn12
InChIKey: IFJARHWHSGCNSB-GFCCVEGCSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay: PI3K-γ:[γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 429.46, ALogP 0.51, HBA 9, HBD 1, PSA 136.02, QED 0.64
Compound (SMILES): Nc1nc2c(-c3ccc(S(=O)(=O)NC[C@@H]4CCCN4)c(S(N)(=O)=O)c3-c3nn[nH]n3)cccc2[nH]1
InChIKey: JSSNYCYCQLWUEO-JTQLQIEISA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: BIOLOGICAL ASSAYS: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1; for a review, see: Meine, M.-R.; Llarrull, L. I.; Vila, A. J. Antibiotics, 2014, 3, 285-316) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 μM to 0.00063 μM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 0.077 nM (pChEMBL 10.109999656677246)
Descriptors: MW 518.59, ALogP -0.33, HBA 10, HBD 6, PSA 227.52, QED 0.19
Compound (SMILES): O=C1CCc2cc(/C=C/C(=O)N3CC4CC(n5nccn5)CC4C3)cnc2N1
InChIKey: BJZMKFZXAANTDL-DAFODLJHSA-N
Target: Enoyl-[acyl-carrier-protein] reductase [NADPH] FabI (SINGLE PROTEIN), Staphylococcus aureus
UniProt: Q6GI75
Assay: Enzyme Inhibition Assay: FabI enzyme inhibition assays were carried out in half-area, 384-well microtitre plates. Compounds were evaluated in 40-μl assay mixtures containing 100 mM NaADA, pH 6.5 (ADA=N-[2-acetamido]-2iminodiacetic acid), 250 μM crotonoyl-CoA, 625 μM NADH and 50 μg/ml S. aureus ATCC 29213 FabI. Inhibitors were typically varied over the range of 50 to 0.39 μM. The reaction mixtures were incubated for 30 minutes at room temperature and the reaction was stopped by adding 200 mM Tris buffer (pH 9.0) to create a pH-shift. The consumption of NADH was monitored by measuring the change in absorbance at 340. By comparing sample readings to those of negative (absence of compound) and positive (absence of enzyme) controls, the percent inhibition of enzymatic activity of the compounds was determined. A best-fit curve is fitted by a minimum of squares method.
Measurement: IC50 = 1470 nM (pChEMBL 5.829999923706055)
Descriptors: MW 378.44, ALogP 1.68, HBA 6, HBD 1, PSA 93.01, QED 0.82
Compound (SMILES): CCOc1ncccc1O[C@H](CCNC)c1ccccc1
InChIKey: QEODUGXTASMZPY-OAHLLOKOSA-N
Target: Sodium-dependent noradrenaline transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P23975
Assay: Norepinephrine, Transporter NET (h): Assay Type: BindingSpecies: HumanOrigin: Recombinant/CHO cellsLigand: [3H]-NisoxetineLigand [M]: 1.00E-09Kd (Binding Affinity): 3.00E-09Bmax: 10 pmol/mg proteinMethod: RadioactivityMeasurement: DPM
Measurement: Ki = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 286.38, ALogP 3.21, HBA 4, HBD 1, PSA 43.38, QED 0.81
Compound (SMILES): C=CC(=O)N1CCO[C@@H]2CN(C(=O)N(C)[C@H](C(=O)N[C@H]3Cc4nc(cs4)-c4ccc5c(c4)c(c(-c4cccnc4[C@H](C)OC)n5CC)CC(C)(C)COC(=O)[C@@H]4CCCN(N4)C3=O)C(C)C)C[C@H]21
InChIKey: SWQZFWJGYOPHFX-MTTNLAOXSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 952.19, ALogP 5.38, HBA 13, HBD 2, PSA 180.77, QED 0.16
Compound (SMILES): O=C(c1nsc2cc(Nc3n[nH]c4nccnc34)ccc12)N1CCC(F)(F)CC1
InChIKey: RXUKBPGCTYRGBK-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Metabotropic Glutamate Receptor Activity: In the first mode, a range of concentrations of the disclosed compounds are added to cells, followed by a single fixed concentration of agonist. If the compound acts as a potentiatior, an EC50 value for potentiation and a maximum extent of potentiation by the compound at this concentration of agonist is determined by non-linear curve fitting. If the compound acts as a noncompetitive antagonist, an IC50 value is determined by non-linear curve fitting. In the second mode, several fixed concentrations of the disclosed compounds are added to various wells on a plate, followed by a range in concentrations of agonist for each concentration of disclosed compound. The EC50 values for the agonist at each concentration of compound are determined by non-linear curve fitting. A decrease in the EC50 value of the agonist with increasing concentrations of the sample compound (a leftward shift of the agonist concentration-response curve) is an indication of the degree of mGluR4 potentiation at a given concentration of the sample compound. A decrease in the maximal response of the agonist with increasing concentrations of the sample compounds, with or without a rightward shift in agonist potency, is an indication of the degree of noncompetitive antagonism at mGluR4. The second mode also indicates whether the sample compounds also affect the maximum response to mGluR4 to agonists.
Measurement: EC50 = 827 nM (pChEMBL 6.079999923706055)
Descriptors: MW 415.43, ALogP 3.58, HBA 7, HBD 2, PSA 99.69, QED 0.53
Compound (SMILES): Cc1ccccc1[C@H]1C[C@@H](N(C)C(=O)[C@H]2C[C@]3(COC(=O)N3)C2)C1
InChIKey: KGPYFQMXMFZALW-ZZVSYSDVSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Activity Assay: The assay used to measure the in vitro activity of MGL is adapted from the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson S J, Lovenberg T W, Barbier A J. Anal Biochem. 2003 Jul. 15; 318(2):270-5.). The assay consists of combining endogenously expressed MGL from HeLa cells with test compounds, adding [glycerol-1,3-3H]-oleoyl glycerol, incubating for one hour, and then measuring the amount of cleaved [1,3-3H]-glycerol that passes through an activated carbon filter. The amount of cleaved, tritiated glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme in a particular well/test condition.
Measurement: IC50 = 74 nM (pChEMBL 7.130000114440918)
Descriptors: MW 328.41, ALogP 2.59, HBA 3, HBD 1, PSA 58.64, QED 0.93