lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CNC(=O)N1CCC(CS(=O)(=O)NNC(=O)c2cc(-c3cn(C)cn3)cc(Cl)c2F)C1
InChIKey: AJBMMUNILOMGSS-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: KAT-6A Enzyme Assay: KAT-6A. 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-6A enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 1 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 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 472.93, ALogP 1.11, HBA 6, HBD 3, PSA 125.43, QED 0.54
Compound (SMILES): CCc1ccc(C(C)NC(=O)c2cc(=O)[nH]c(C3CC3)n2)cc1
InChIKey: FYIHVNKVBDJMQM-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 311.39, ALogP 2.7, HBA 3, HBD 2, PSA 74.85, QED 0.89
Compound (SMILES): CC1(C)CC([C@H](O)c2c(C3CC3)sc3cncn23)C1
InChIKey: ZZXIKNNANYDHKL-ZDUSSCGKSA-N
Target: Tryptophan 2,3-dioxygenase (SINGLE PROTEIN), Homo sapiens
UniProt: P48775
Assay: Enzymatic Assay: TDO2: Recombinant human TDO comprising amino acids 19-407 with a N-terminal hexahistidine tag expressed in E. coli and purified to homogeneity is incubated at a final concentration of 15 nM in assay buffer consisting of 75 mM phosphate buffer at pH7 supplemented with 100 μM ascorbic acid, 50 U/ml Catalase, 0.01% BSA, and 0.01% Tween 20 (protocol modified from Seegers et al, JBS 2014). Example compounds are serially diluted in DMSO, further diluted in phosphate buffer, and added to the reaction mixture at final concentrations ranging from 10 μM to 0.5 nM. The final DMSO concentration is 0.6%. Following a pre-incubation of 30 minutes at RT, the reaction is started by the addition of L-tryptophan at a final concentration of 200 μM in assay buffer. After 30 minutes of incubation at RT, 3 μL of the reaction mixture are transferred to a 384 deep well plate containing 25 μL of deionized water. 100 μl of 200 nM L-Tryptophan-(indole-d5) in cold 100% methanol are added followed by a 10 minutes centrifugation at 4,000 rpm at 4° C. An additional 75 μL of deionized water are then added and followed by a 10 minutes centrifugation at 4,000 rpm at 4° C. The product of the reaction N′-Formylkynurenine (NFK) is quantified by LCMS and normalized to the L-Tryptophan-(indole-d5) signal. Samples with 0.6% DMSO (0% effect) and a TDO/IDO inhibitor (100% effect) are used as control samples to set the parameters for the non-linear regression necessary for the determination of the half-maximal inhibitory concentration (IC50) for each compound. For each compound concentration the percentage of activity compared to 0% and 100% effect is calculated as average±STDEV (each concentration measured in duplicate). IC50 values and curves are generated with XLfit software (IDBS) using Dose-Response One Site model 203 (four parameter logistic curve model). When compounds are measured multiple times, mean values are given.
Measurement: IC50 = 246 nM (pChEMBL 6.610000133514404)
Descriptors: MW 276.41, ALogP 3.74, HBA 4, HBD 1, PSA 37.53, QED 0.93
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C(C)(C)C(=O)NC[C@H]4CC[C@H](N)CC4)cc3c2C(C)C)ccn1
InChIKey: LINLYQDATVBZNX-XYWHTSSQSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 325 nM (pChEMBL 6.489999771118164)
Descriptors: MW 446.64, ALogP 5.57, HBA 3, HBD 3, PSA 83.8, QED 0.46
Compound (SMILES): Cc1nc(C(=O)NC(c2ccc(S(F)(F)(F)(F)F)cc2F)C(C)(C)C)cc(=O)[nH]1
InChIKey: OJRISADALOEALA-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 1217 nM (pChEMBL 5.920000076293945)
Descriptors: MW 443.41, ALogP 5.39, HBA 3, HBD 2, PSA 74.85, QED 0.62
Compound (SMILES): CCn1cc(CN2C(=O)N(c3c(F)c(OC)cc(OC)c3F)Cc3cnc(-c4cn(C)nc4C)cc32)cn1
InChIKey: HQOORGXQDKMTBO-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 523.54, ALogP 4.45, HBA 8, HBD 0, PSA 90.54, QED 0.36
Compound (SMILES): [C-]#[N+]CN1C2CCC1CC(c1ccc3[nH]c(-c4cn5ncnc5c(C)c4C)c(C(C)C)c3c1)C2
InChIKey: SHSLULYZPWJOFX-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 452.61, ALogP 6.21, HBA 4, HBD 1, PSA 53.58, QED 0.37
Compound (SMILES): CC1NC(=O)c2c(Cl)cc(-n3ccc4cncnc43)cc21
InChIKey: DIBVCVHQDKYHEP-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: Biochemical Enzymatic Assay: Compounds are screened for MNK inhibition using the ADP-Glo kinase assay kit (Promega, catalogue No. V9101). All kinase reactions are performed in Reaction Buffer E (15 mM HEPES pH7.4, 20 mM NaCl, 1 mM EGTA, 10 mM MgCl2, 0.1 mg/ml BGG, and 0.02% Tween-20). Final MNK 1 reactions contained 10 nM recombinant MNK1 (Life Technologies, PR9138A), 100 μM MNK substrate peptide Ac-TATKSGSTTKNR-NH2 (American Peptide Company), 300 μM ATP, and varying concentrations of the inhibitory compound of interest. Final MNK2 reactions contained 3 nM recombinant MNK2 (Life Technologies, PV5607), 50 μM MNK substrate peptide Ac-TATKSGSTTKNR-NH2 (American Peptide Company), 10 μM ATP, and varying concentrations of the inhibitory compound of interest. Final DMSO concentration in each reaction is 1%.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 298.73, ALogP 2.88, HBA 4, HBD 1, PSA 59.81, QED 0.75
Compound (SMILES): CCn1c(-c2cccnc2C(C)OC)c2c3cc(ccc31)-c1cccc(c1)C[C@H](NC(=O)[C@H](C(C)C)N(C)C(=O)[C@H]1CCN(C(=O)C#CC3CCO3)C1)C(=O)N1CCC[C@H](N1)C(=O)OCC(C)(C)C2
InChIKey: OSPHUDXDINZSCP-FUTRASTKSA-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 942.17, ALogP 5.87, HBA 11, HBD 2, PSA 164.64, QED 0.15
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C5CCCC5(C)C#N)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: LRGXVIPVHAWSGO-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Biological Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 16.4 nM (pChEMBL 7.789999961853027)
Descriptors: MW 480.66, ALogP 6.57, HBA 5, HBD 1, PSA 73.01, QED 0.36
Compound (SMILES): FC(F)(F)c1cccc(-c2ccc([C@H]3C[C@@H]3NCC3CCNCC3)cc2)c1
InChIKey: GJXFBKDQIXYBIK-RTWAWAEBSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Biological Assay: Human recombinant monoamine oxidase proteins MAO-A and MAO-B were purchased from Sigma Aldrich. MAOs catalyze the oxidative deamination of primary, secondary and tertiary amines. In order to monitor MAO enzymatic activities and/or their inhibition rate by inhibitor(s) of interest, a fluorescence-based (inhibitor)-screening assay was set up. 3-(2-Aminophenyl)-3-oxopropanamine (kynuramine dihydrobromide, Sigma Aldrich), a non fluorescent compound was chosen as a substrate. Kynuramine is a non-specific substrate for both MAO-A and MAO-B activities. While undergoing oxidative deamination by MAO activities, kynuramine is converted into 4-hydroxyquinoline (4-HQ), a resulting fluorescent product.The monoamine oxidase activity was estimated by measuring the conversion of kynuramine into 4-hydroxyquinoline. Assays were conducted in 96-well black plates with clear bottom (Corning) in a final volume of 100 uL. The assay buffer was 100 mM HEPES, pH 7.5. Each experiment was performed in duplicate within the same experiment. Briefly, a fixed amount of MAO (0.25 ug for MAO-A and 0.5 ug for MAO-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of at least eight 3-fold serial dilutions each. Clorgyline and Deprenyl (Sigma Aldrich) was used as a control for specific inhibition of MAO-A and MAO-B respectively. After leaving the enzyme(s) interacting with the inhibitor, KM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. In the dark. The oxidative deamination of the substrate was stopped by adding 50 uL of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure levels of fluorescence produced in the absence and/or in the presence of inhibitor. The maximum of oxidative deamination activity was obtained by measuring the amount of 4-hydroxyquinoline formed from kynuramine deamination in the absence of inhibitor and corrected for background fluorescence in the absence of MAO enzymes. The IC50 values of each inhibitor were calculated with GraphPad Prism Software.
Measurement: IC50 = 1020 nM (pChEMBL 5.989999771118164)
Descriptors: MW 374.45, ALogP 4.82, HBA 2, HBD 2, PSA 24.06, QED 0.78
Compound (SMILES): CCOc1ncccc1-c1noc([C@@H]2CCCN2C(=O)c2ccccc2-n2nccn2)n1
InChIKey: QTNHBXHXZBQJHF-SFHVURJKSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
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 = 99 nM (pChEMBL 7)
Descriptors: MW 431.46, ALogP 3.09, HBA 9, HBD 0, PSA 112.06, QED 0.46
Compound (SMILES): CNCC(c1ccc(Br)c(Cl)c1)[C@H](O)c1ccccc1
InChIKey: YYXLUZNBTYTCJI-FQNRMIAFSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Uptake Assay Protocol: DAT: This protocol was designed to measure inhibition of uptake by the human dopamine transporter. The reagents were human DAT (HEK293F) cells, GBR 12909 (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 pt polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1×HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human dopamine transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.12 μL of test compound (10 mM) in DMSO was added to the wells, and nomifensine was used as a control. GBR 12909 (final assay concentration of 10 μM) was used for background signal. The neurotransmitter transporter dye was prepared in AB prior to use.
Measurement: Ki = 6.6 nM (pChEMBL 8.180000305175781)
Descriptors: MW 354.68, ALogP 4.14, HBA 2, HBD 2, PSA 32.26, QED 0.85
Compound (SMILES): CCCOC1(c2ccccc2C)CN(C(=O)[C@@H](Cc2ccc(OC)cc2)NC(=O)CCc2c[nH]cn2)C1
InChIKey: FOLVJMUNZVVKBU-AREMUKBSSA-N
Target: Melanocortin receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P32245
Assay: Transactivation Assay: In the presence of an activator (agonist), the melanocortin receptor will activate the cAMP pathway which, via the CRE-Luc vector, will result in the synthesis of luciferase. After the addition of a lysis buffer containing a luminescent substrate for luciferase, it will be possible to measure the luminescence proportional to the degree of activation or of inhibition of the receptor. The products are solubilized at 10 mM in DMSO. They are tested in the form of dose-response at a final DMSO concentration of 0.1%. The range comprising 10 points and one zero begins at 10 μM with 4-fold dilutions. For testing agonists, the products are tested alone. For determining the behaviour of antagonists, the products of interest are tested in the presence of 1 nM NDP-MSH (reference agonist). The cells are seeded at a rate of 5000 cells per well (384 well plate) in serum-free DMEM medium, and incubated overnight at 37° C., 5% CO2.The products and the reference ligand (NDP-MSH) are added the following day, and the plates are again incubated for 6 h at 37° C., 5% CO2. After the addition of lysis buffer containing luciferin, the plates are read on a Top-Count instrument. The results are standardized as % activity using the 100% (cells+NDP-MSH at 10 nM) and 0% (cells alone) controls. An EC50 is calculated for each product using the XLFit software. The results are given in nM.
Measurement: EC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 504.63, ALogP 3.55, HBA 5, HBD 2, PSA 96.55, QED 0.39
Compound (SMILES): Cc1ccc(NC(=O)c2ccnc(C(C)(C)O)c2)cc1-c1cc(N2CCOCC2)c(=O)n(C)c1
InChIKey: RCWKIQOTRSKJIJ-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity: Raf and biotinylated Mek, kinase dead, were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 462.55, ALogP 3.07, HBA 7, HBD 2, PSA 96.69, QED 0.61
Compound (SMILES): C=CC(=O)NC1COCC1Nc1ncc2c(n1)N(Cc1cn[nH]c1)C(=O)N(c1c(Cl)c(OC)cc(OC)c1Cl)C2
InChIKey: BBMUWVXGFMBUDK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Omnia Assay Protocol for Potency Assessment Against FGFR 4 Enzyme: A 10× stock solution of FGFR4-WT (PR4380C or P3054), (from Invitrogen, Carlsbad, Calif.) corresponding to method a in Table 10, was prepared as described below. Alternatively, a 10× stock solution of FGFR4-WT (F01-11G), (SignalChem, Richmond, BC) corresponding to method b in Table 10, was prepared as described below. A solution of 1.4×ATP (AS001A) and 5× Tyr-Sox conjugated peptide substrate (KNZ3101) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM P3-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of FGFR4 was pipetted into a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.), containing a 0.5 μL volume of 100% DMSO. The serially diluted compounds were prepared on a Tecan EVO100. A second addition of 10 μl of Tyr-Sox FGFR4 substrate was added to each well and the kinase reactions were started with the addition of 35 μL of 1.4×ATP. The reactions were monitored every 71 seconds for 240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to −60 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (seconds) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response (Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.)).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 589.44, ALogP 3.15, HBA 9, HBD 3, PSA 146.83, QED 0.32
Compound (SMILES): CCc1cc(CN2CCCC23CCN(C(=O)OC(C(F)(F)F)C(F)(F)F)CC3)ccc1F
InChIKey: VWUFTHCPWDGASQ-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08914
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (mouse brain membrane fraction or cell lysates) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP¿Rh (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (50 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL, ABHD6 and FAAH using ImageJ 1.43u software. Data from this assay is shown in Table 2 (% inhibition at 1 μM).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 470.43, ALogP 5.45, HBA 3, HBD 0, PSA 32.78, QED 0.55
Compound (SMILES): C=CC(=O)Nc1cc(Nc2ncc3ccc(C4=CCCCC4)n3n2)c(OC)cc1N(C)CCN(C)C
InChIKey: POZRCONTKQCSTI-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: ssay of Inhibitory Activity on Wild Type EGFR and Mutant EGFR Kinase: All reagents used in the following z′-lyte assay were purchased from Invitrogen.The inhibitory activity on T790M/L858R double mutant EGFR kinase (Invitrogen, PV4879) and wild-type EGFR kinase (Invitrogen, PV3872) were determined by the z′-lyte assay.The working concentrations of each component in 10 μL T790M/L858R kinase reaction system were: 25 μM ATP, 0.1 ng/μL T790M/L858R kinase, 2 μM Tyr04 substrate (Invitrogen, PV3193). The concentration of DMSO after addition of the compound prepared in the above examples of the present invention (i.e., the compound to be tested) was 2 vol %.The working concentrations of each component in 10 μL wild-type EGFR kinase reaction system were: 10 μM ATP, 0.8 ng/μL wild-type EGFR kinase, 2 μM Tyr04 substrate (Invitrogen, PV3193). The concentration of DMSO after addition of the compound to be tested was 2 vol %.10 mM drug stock solution was dissolved at room temperature and gradiently diluted to a final concentration of 4-0.002 μM with 8 vol % DMSO solution. 2.5 μL of the solution of the compound to be tested and 5 μL of the mixture of T790M/L858R kinase (or wild-type EGFR kinase) and Tyr04 substrate diluted with the reaction buffer were added to each well. Then 2.5 μl of ATP was added to initiate the reaction. Wherein, ATP was replaced by the reaction buffer in well C1, no drug was added to well C2, and the phosphorylated substrate was added to well C3 according to the instructions. The mixture was allowed to react at 25° C. in a shaker in dark for 60 min. 5 μL of Development Reagent B (Invitrogen, diluted with TR-FRET dilution buffer) was added and allowed to react at room temperature in the shaker for 60 minutes. The plate was read on a Victor X5 plate reader (PerkinElmer) and the absorbance was measured at excitation wavelengths of 405 nm and emission wavelengths of 450 nm and 520 nm (for example, C3520 nrn indicates the absorbance for well C3 at 520 nm).The inhibition rate was calculated according to the following method (refer to the instructions of Invitrogen, PV3193):1. ER=Coumarin Emission (450 nm)/Fluorescein Emission (520 nm)2. Phosphorylation ratio=(1−(ER×C3520 nm−C3450 nm)/((C1450 nm−C3450 nm)+ER×(C3520 nm−C1520 nm))))×100%3. Inhibition ratio (IR)=(1−(phosphorylation ratio of the test compound)/(phosphorylation ratio of C2))×100%The half-inhibitory concentration IC50 was obtained through fitting calculation by using XLFIT 5.0 software (IDBS, UK).
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 489.62, ALogP 4.56, HBA 8, HBD 2, PSA 87.03, QED 0.4
Compound (SMILES): O=C(O)C12CCCC(CCCOc3ccc(-c4ccn[nH]4)nc3)(C1)C2
InChIKey: NYYUNSRDYIJZQE-UHFFFAOYSA-N
Target: Cytochrome P450 4A11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02928
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 341.41, ALogP 3.67, HBA 4, HBD 2, PSA 88.1, QED 0.75
Compound (SMILES): COc1cccc(-c2nc(Nc3ccncc3F)c3cc(CN4CCC(F)(F)CC4)cn3n2)n1
InChIKey: IIACECDWFPHIQY-UHFFFAOYSA-N
Target: TGF-beta receptor type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P37173
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 > 15000 nM (pChEMBL 4.823999881744385)
Descriptors: MW 469.47, ALogP 4.31, HBA 8, HBD 1, PSA 80.47, QED 0.45
Compound (SMILES): C=CC(=O)N1CCCN(C(=O)c2cc(Sc3cnc(NC(=O)c4cccc(CN[C@H](C)C(C)(C)C)c4)s3)c(C)cc2OC)CC1
InChIKey: ONKCBNMZBWUKKB-HSZRJFAPSA-N
Target: Tyrosine-protein kinase TXK (SINGLE PROTEIN), Homo sapiens
UniProt: P42681
Assay: In Vitro Assay: The potency of inhibition is measured by binding affinity.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 649.88, ALogP 6.25, HBA 8, HBD 2, PSA 103.87, QED 0.25
Compound (SMILES): Cn1cnc2nc(N)n(Cc3nc([C@@H]4CO[C@@H](c5ccc(S(F)(F)(F)(F)F)cc5)C4)no3)c(=O)c21
InChIKey: PZZQZTSBVOVKIW-WCQYABFASA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (90 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 90 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC80 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 519.46, ALogP 4.05, HBA 10, HBD 1, PSA 126.88, QED 0.39
Compound (SMILES): CC(C)CN1CCC(F)(C(=O)Nc2cc3cc(-c4nncn4C)ccc3cn2)CC1
InChIKey: ZKOWFDJIWGKOEU-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: The screening for GSK3beta kinase activity: Each compound is dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 11-point dose-response curves from 10 μM to 0.0003 μM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 1536-well black-walled round bottom plates (Corning). The GSK3β kinase assay is run using the Ser/Thr 09 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as ratio of coumarin emission/fluorescein emission. Briefly, recombinant GSK3β kinase, ATP and Ser/Thr peptide 09 are prepared in 1× Kinase buffer to final concentrations of 0.04 μg/mL, 46 μM, and 4 μM respectively. The mixture is allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 serve as control reactions. After incubation, diluted Development Buffer is added to the reaction and allowed to further incubate for one hour at room temperature. The plate is read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer). The Emission ratio (Em) is calculated as a ratio of the coumarin (C) emission signal (at 445 nm)/Fluorescein (F) emission signal (at 520 nm). The percent phosphorylation is then calculated using the following formula: [1−((Em ratio×F100%)−C100%)/((C0%−C100%)+(Em ratio×(F100%−F0%)))]. Dose-response curves are generated and inhibitory concentration (IC50) values are calculated using non-linear regression curve fit in the Dotmatics' Studies Software (Bishops Stortford, UK).
Measurement: EC50 = 185 nM (pChEMBL 6.730000019073486)
Descriptors: MW 410.5, ALogP 3.43, HBA 6, HBD 1, PSA 75.94, QED 0.7
Compound (SMILES): Nc1nn(Cc2ccc(Cn3ccccc3=O)cc2)cc1C(=O)NCCNc1cc(Cl)ccn1
InChIKey: CMGOOMKRMVBEPY-UHFFFAOYSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: Inhibitory Activity Assay: KLK1 inhibitory activity in vitro was determined using standard published methods. Human KLK1 (Callbiochem) was incubated at 25° C. with the fluorogenic substrate H-DVal-Leu-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 > 40000 nM (pChEMBL 4.3979997634887695)
Descriptors: MW 477.96, ALogP 2.61, HBA 8, HBD 3, PSA 119.86, QED 0.32
Compound (SMILES): Cc1ncc(CC(=O)N[C@H]2CC[C@H](CCN3CCc4sc(OC5CC(F)(F)C5)nc4C3)CC2)o1
InChIKey: BTGKFNRKGQRPEP-QAQDUYKDSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Binding Inhibition for Dopamine D2 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl.06HO (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl) (067-31, NAKARAI CHEMICALS, LTD.)Radioligand: (final concentration) 1.2 nM [H]-Methylspiperone ([HN-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer) Non-specific ligand: (final concentration) 10 μM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma] SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well)Incubation time and temperature: 120 min at 25° C.Kd: 0.272 nM(Preparation of Solutions of Non-Specific Ligand or the Compounds of the Present Invention)Butaclamol or the compounds of the present invention were weighed and DMSO was added to make a 10 mM solution. This solution was diluted to each concentration.(Preparation of Radioligand Solution),[H]-Methylspiperone was weighed and the buffer solution was added to make a 3.6 nM solution.(Preparation of SPA Beads Solution) SPA beads were weighed and stirred in water to make a 50 mg/mL solution. Using this solution, a mixture with the cell membranes was prepared.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 494.61, ALogP 4.28, HBA 7, HBD 1, PSA 80.49, QED 0.59
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3ccccc3)ncc2C#N)c1
InChIKey: DIOMEEGYXLOHPP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Omnia Assay: Briefly, 10x stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13×ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 27° C. with a 0.5 μL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 μL of the ATP/Tyr-Sox peptide substrate mix and monitored every 30-90 seconds for 60 minutes at λex360/λem485 in a Synergy4 plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 356.39, ALogP 3.96, HBA 6, HBD 3, PSA 102.73, QED 0.58
Compound (SMILES): Cc1ccncc1-c1cc2cc(NC(=O)[C@@H]3C[C@@H]3F)ncc2c(N)n1
InChIKey: KTBVCQUGVQGNPO-RISCZKNCSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Binding Assay: Lanth: For the binding assay, 4 ul 2×HPK1 and Eu-anti-GST antibody were added to each well of the assay plate using a Multidrop reagent dispenser. The solutions were incubated in a 23 C incubator for 1 h. To each well of the assay plate was added 4 ul 2× Tracer-222 using a Multidrop reagent dispenser. The solutions were again incubated in a 23 C incubator for 1 h. The results of the assay were read using an Envision plate reader with the following parameters: TR_FRET, 340ex/615 and 665em; 100 usec Delay; and 200 usec integration.
Measurement: Ki = 0.793 nM (pChEMBL 9.100000381469727)
Descriptors: MW 337.36, ALogP 2.88, HBA 5, HBD 2, PSA 93.79, QED 0.77
Compound (SMILES): CC#CC(=O)N[C@@H](C)c1nc(-c2ccc(C(=O)Nc3ccccn3)c(OC)c2)c2c(N)nccn12
InChIKey: LHFAKDSSXLAPAE-HNNXBMFYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: IMAP (Immobilized Metal Ion Affinity-Based Fluorescence Polarization) Assay: EGFR enzyme activity is measured using the IMAP (immobilized metal ion affinity-based fluorescence polarization) assay as outlined below.EGFR enzyme (Invitrogen catalog #PR7295B), is diluted to 2.5 μg/mL in KR buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.1% NaN3, 1 mM DTT, 2 mM MnCl2, pH 7.5).Serial dilution log 10 from 1 mM to 31.6 nM of test compounds are made in 100% DMSO. The dilutions in DMSO are then diluted 25-fold in KR-buffer of which 5 μL is used in the assay, leading to a final compound concentration range in the assay from 10 μM to 0.316 nM.The assay is performed as follows: 5 μL/well of test compound in KR buffer (final DMSO concentration in the assay is 1%) is mixed with 5 l/well of 2.5 μg/mL EGFR enzyme (final concentration in the assay is 625 ng/mL). Test compounds and EGFR enzyme are pre-incubated 60 min at room temperature, before adding 5 μL/well of 200 nM Fluorescein labeled substrate peptide (PDGFR-tide substrate peptide RP7084, Molecular Devices) in KR-buffer. Final peptide substrate concentration in assay is 50 nM. The kinase assay is started by adding 5 L/well of 8 μM ATP in KR-buffer (final ATP concentration is 2 μM, Km ATP in EGFR IMAP assay). Following incubation for 60 min at room temperature in the dark the enzyme reaction is stopped by adding 40 μL/well IMAP Progressive Binding Solution (according to suppliers (Molecular Devices) protocol using 20% 1× buffer A and 80% 1× buffer B with 600× diluted beads). After 60 min incubation at room temperature in the dark the FP signal is read. Fluorescence at 535 nm is measured using parallel and perpendicular filters to determine differences in rotation due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmPi) of the controls with and without ATP.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 469.51, ALogP 2.83, HBA 8, HBD 3, PSA 136.53, QED 0.37
Compound (SMILES): CC[C@]1(O)CC[C@@]2(C)[C@@H](CC[C@@H]3[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)[C@H](O)CC4CCCC4)CC[C@@H]32)C1
InChIKey: FOTKEMKUACPHAO-NYHHFTEHSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: Whole-Cell Patch Clamp: Whole-Cell Patch Clamp of Mammalian Cells (Ionworks Barracuda (IWB)):The whole-cell patch-clamp technique was used to investigate the effects of positive allosteric modulating activity of test compounds on_GlunN1/GluN2A and GluN2B glutamate receptors expressed in mammalian cells.
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 444.74, ALogP 7.36, HBA 2, HBD 2, PSA 40.46, QED 0.46
Compound (SMILES): C=C1CC(c2nc3cc(Cl)c(C(F)(F)F)cc3[nH]2)N(C(=O)c2cc(C)sc2-c2ccc(C)c(C)c2)C1
InChIKey: ZTAVKFHMSOWRCW-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 208 nM (pChEMBL 6.679999828338623)
Descriptors: MW 530.02, ALogP 8.03, HBA 3, HBD 1, PSA 48.99, QED 0.27