lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(=O)N1CCc2c(c(-c3cccc(C(=O)Nc4cncnc4)c3)nn2C2CCOC2)C1
InChIKey: CLCUNXXKTNTKAU-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6xHis antibody (6xHis is disclosed as SEQ ID NO: 3) (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 123 nM (pChEMBL 6.909999847412109)
Descriptors: MW 432.48, ALogP 2.46, HBA 7, HBD 1, PSA 102.24, QED 0.68
Compound (SMILES): CC(NC(=O)[C@H]1CN(CC2CC2)CC[C@@H]1NC(=O)c1cc(-c2ccc(F)cc2F)on1)C1CCCO1
InChIKey: XUUNUNAPBCDMLJ-KTGJFSLUSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: Tango CXCR7-bla U2OS cells are detached from culture dishes with 0.05% trypsin-EDTA and collected in growing medium (McCoy's 5A 90% (v/v), dialyzed FCS 10% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), 1 mM sodium pyruvate, P/S 1% (v/v) 50 μg/ml Hygromycin, 100 μg/ml Geneticin, 200 μg/ml Zeocin), spinned down and resuspended in assay medium (McCoy's 5A 90% (v/v), dialyzed FCS 1% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), P/S 1% (v/v)). 10′000 cells per well (in 30 μl) are seeded in a 384 well plate (black-walled, clear bottom). The plate is incubated at 37° C./5% CO2 for 24 hours. Test compounds are dissolved to 10 mM in DMSO and serially diluted in DMSO to 500× of the final concentration for dose response curves. Compounds are then diluted 1:100 in assay medium to 5× of the final concentration. 10 μl/well of diluted compounds are added to the assay plate and incubated for 15 minutes at 37° C. Thereafter CXCL12/SDF1-α is diluted in assay medium to 5× of the final concentration (its EC80 value for receptor activation) and 10 μl/well are added to the assay plate. The agonist leads to activation of the receptor and therefore to b-arrestin recruitment. Compounds acting as antagonists reduce this activation. The plate is incubated for 22 hrs at 37° C. 10 μl/well of detection reagent (LiveBLAzer-FRET BIG (CCF4-AM) substrate) is transferred to the assay plate and the plate is incubated for 2 hours at room temperature protected from light. Fluorescent counts are determined (Scan1: Ex 409/20 nm, Em 460/30 nm, Scan 2: Ex 409/20 nm, Em 530/30 nm).
Measurement: IC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 502.56, ALogP 3.13, HBA 6, HBD 2, PSA 96.7, QED 0.58
Compound (SMILES): CC(C)c1nc2sc([C@H]3CCCN3C(=O)Oc3ccccc3)nc2c(=O)[nH]1
InChIKey: DMCCELVXAOSELG-CYBMUJFWSA-N
Target: Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q8N5Z0
Assay: KAT-II inhibitory assay: The inhibitory action of the test compound on human recombinant KAT-II was determined by the following method.To a reaction mixture (45 μL) containing 3.0 μmol/L kynurenine, 10 μmol/L pyridoxal phosphate, 2.0 ng/μL human recombinant KAT-II, and 150 mmol/L tris(hydroxymethyl)aminomethane-acetate buffer (pH 8.0) was added a 10% dimethyl sulfoxide solution (5 μL) of each test compound prepared, and the mixture was reacted at 37° C. for 1 hr. After the reaction, 50% trichloroacetic acid (5 μL) was added to terminate the reaction.The resultant kynurenic acid was quantified as follows by high performance liquid chromatography. An enzyme reaction mixture was separated by an octadecylsilane reversed-phase column (SC-50DS, Eicom Corporation; mobile phase: 250 mmol/L zinc acetate, 50 mmol/L sodium acetate, and 5.0% acetonitrile (pH 6.2)) incubated at 30° C., and kynurenic acid was quantified using a fluorescence detector (RF-20Axs, Shimadzu Corporation) at excitation wavelength 354 nm, detection wavelength 460 nm. The analytical curve was drawn every time by an external standard method. Each test compound was tested by dual measurement at each concentration. The kynurenic acid level in the presence of a test compound at each concentration was converted into % relative to kynurenic acid resulting from a reaction with an enzyme alone as 100%, and the obtained values were fitted to S-curve to determine IC50.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 384.46, ALogP 3.84, HBA 6, HBD 1, PSA 88.18, QED 0.74
Compound (SMILES): COc1cc2c(-c3cnn4cc(N5CCC(N6CC[N+](C)([O-])CC6)CC5)cnc34)ccnc2cc1F
InChIKey: QOFILQWATAUHGE-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Luciferase Reporter Gene Assay for ALK2 and TGFbeta Activity: In this reporter gene cell based ALK2 and TGFβ assay, the C2Cl2 cell line was employed for the measurement of ALK2 activity, using a BRE-Luc SMAD1/5/8 reporter and BMP6 as the agonist. The HEK293T cell line was employed to measure TGFβ activity, using a SBE-Luc SMAD2/3 reporter and TGFβ as the agonist. Luciferase reporter assays were read using Promega Steady-Glo Luciferase Assay System.Cells were plated in 96 well white clear bottom assay plates at 10 k cells/well for C2Cl2-BRE, 15 k cells/well for HEK293-SBE in DMEM containing 2% FBS 1% P/S. Cells were given a minimum of 4 hours in incubator at 37° C./5% CO2 to adhere prior to further treatment. Compounds were diluted in DMSO to a 10-point dilution curve and added to the plate to reach the following final concentrations: 10000, 3000, 1000, 300, 100, and 30 nM in the HEK293-SBE assay and 1000, 300, 100, 30, 10, 3, 1, 0.3 nM in the C2Cl2-BRE assay. Negative and positive control wells received 2 μL DMSO as vehicle treatment. Plates were returned to incubator for 45 minutes and then BMP6 and TGFB were added to a final concentration of 50 ng/mL and 5 ng/mL, respectively. Plates were returned to the incubator and left overnight. After a minimum of 18 hours post BMP6/TGFβ addition, the plates were read using Promega Steady-Glo Luciferase Assay System. A 1:1 mixture of prepared steady-glo and phenol free DMEM was prepared, and 50 μL/well was added to the assay plates whose media had been flicked off. Plates were given 10 minutes post steady-glo addition before luminescence was read on a Spectramax M5e microplate reader. Negative control wells were averaged and subtracted from all other wells on the plate. Inhibition was calculated as the percent of signal loss compared to the averaged positive control wells.
Measurement: IC50 = 631 nM (pChEMBL 6.199999809265137)
Descriptors: MW 491.57, ALogP 3.32, HBA 8, HBD 0, PSA 81.85, QED 0.32
Compound (SMILES): O=C(Nc1cc(-c2ccc(NS(=O)(=O)c3ccccc3)cc2)c2cc[nH]c2n1)C1CC1
InChIKey: ICVNZAXMYOMCCF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 432.51, ALogP 4.38, HBA 4, HBD 3, PSA 103.95, QED 0.42
Compound (SMILES): Cc1cc(-c2cn(CCN3CCC3)c(C3CCN(c4ncnc(N)c4C#N)CC3(F)F)n2)ccc1F
InChIKey: HEIGJIKPAPHGIP-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: Enzyme Assay: AKT: A TTP Mosquito liquid handling instrument was used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components were added to a final volume of 12.5 μl:    0.1 ng/μl His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C).    160 uM ATP (Fluka, 02055)    1 mM DTT (Sigma, D0632)    1 mM MgCl2 (Sigma, M1028)    1 μM substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service.    100 mM HEPES pH 7.5 (Calbiochem, 391338)    0.015% Brij-35 (Sigma, B4184)The reaction was incubated for 90 min at 25 C., and then stopped by the addition of 70 μl of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate was read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure −2.3 psi, upstream voltage −500, and downstream voltage −3000. These conditions caused unphosphorylated substrate and phosphorylated product peptide to resolve as separate peaks allowing direct measurement of percentage of conversion of substrate to product.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 496.54, ALogP 3.58, HBA 8, HBD 1, PSA 99.89, QED 0.56
Compound (SMILES): CC(C)C(=O)Nc1ccc(N(C(=O)Cn2nnc3ccccc32)C(C(=O)NCc2ccccc2)c2cccnc2)cc1
InChIKey: WZYXFLGBGJLBDC-UHFFFAOYSA-N
Target: Replicase polyprotein 1ab (SINGLE PROTEIN), Severe acute respiratory syndrome-related coronavirus
UniProt: P0C6X7
Assay: IC50 Determination: The inhibitor library was first screened for inhibition of each 3CLpro at a concentration of 100 μM in duplicate assays containing the following assay buffer (50 mM HEPES, 0.1 mg/mL BSA, 0.01% TritonX-100, 1 mM DTT). The assays were carried out in Costar 3694 EIA/RIA 96-Well Half Area, Flat Bottom, Black Polystyrene plates from Corning Incorporated. 1 μL of 100× inhibitor stock in DMSO was added to 79 μL of enzyme in assay buffer and the enzyme-inhibitor mixture was incubated for 10 minutes. The reaction was initiated by the addition of 20 μL of 10 μM UIVT3 substrate, a custom synthesized Förster resonance energy transfer substrate peptide with the following sequence: HilyteFluor488-ESARLQSGLRKAK-QXL520-NH2, producing final concentrations of 100 nM and 100 μM for the 3CLpro enzyme and UIVT3 substrate, respectively. The fluorescence intensity of the reaction was then measured over time as relative fluorescence units (RFUt) for a period of 10 minutes, using an excitation wavelength of 485 and bandwidth of 20 nm and monitoring emission at 528 and bandwidth of 20 nm using a BioTek Synergy H1 multimode microplate reader.
Measurement: IC50 = 10900 nM (pChEMBL 4.960000038146973)
Descriptors: MW 561.65, ALogP 4.51, HBA 7, HBD 2, PSA 122.11, QED 0.26
Compound (SMILES): CC(=O)c1nn(CC(=O)N2C[C@H]3C[C@H]3[C@H]2C(=O)Nc2cccc(Br)n2)c2ccc(-c3cnc(C)nc3)cc12
InChIKey: IQUYAWMKSVZUAC-AZZGJVAJSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration was incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) was recorded at 30 second intervals for 30 minutes using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 574.44, ALogP 3.65, HBA 8, HBD 1, PSA 122.97, QED 0.28
Compound (SMILES): CC1(C)CC(n2cc(Nc3nc(C4CCCCC4)cn4ncnc34)cn2)CC(C)(C)O1
InChIKey: UAWOBBSJRUCUBL-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: In Vitro CSF-1R Kinase Activity: The CSF-1R construct corresponds to recombinant human catalytic domain (aa 538-910), which was purchased from Invitrogen (cat #PV4092). CSF-1R was mixed with peptide substrate (biotin-TYR1, Sequence: Biotin-(Ahx)-GAEEEIYAAFFA-COOH, 4 μM final) at varying inhibitor concentrations in reaction buffer: 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTI, 0.01% BSA, 0.1 mM Na3VO4 and 0.1 mM ATP. After about 60 min incubation at rt, the reaction was quenched by addition of EDTA (final concentration: 100 mM) and developed by addition of detection reagents (final approximate concentrations: 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K (europium labeled anti-phosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, Mass.) and 0.6 μg/mL SAXL (Phycolink streptavidin-allophycocyanin acceptor, cat #PJ25S, Prozyme, San Leandro, Calif.). The developed reaction was incubated in the dark for about 60 min at rt, then read via a time-resolved fluorescence detector (Rubystar, BMG) using a 337 nm laser for excitation and monitoring emission wavelength at 665 nm. Within the linear range of the assay, the observed signal at 665 nm was directly related to phosphorylated product and can be used to calculate the IC50 values.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 423.57, ALogP 5.02, HBA 8, HBD 1, PSA 82.16, QED 0.63
Compound (SMILES): COCCn1c(C(=O)O)cc2c(OC)ccc(N3C[C@@H](C)n4c(c(CCCOc5cc(C)c(Cl)c(C)c5)c5ccc(Cl)c(-c6c(C)ncnc6C)c54)C3=O)c21
InChIKey: HPCOZYOPDMCCCG-XMMPIXPASA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: TR-FRET Binding Assay: Measurements were carried out in OptiPlate-384 White Opaque 384-well plates (Perkin Elmer. Shelton, Conn.). The assay was run using a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) that was purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification, 300 nM FITC-Bak peptide, 1 nM recombinant Maltose Binding Protein (MBP) tagged Mcl-1 (residues 172-327) and 1 nM terbium conjugated-MBP antibody were added to assay buffer (4.5 mM Monobasic potassium phosphate, 15.5 mM Dibasic potassium phosphate, 1 mM Sodium EDTA, 50 mM NaCl, 1 mM DTT, 0.05% Pluronic F-68, pH 7.5). Compounds are diluted in DMSO in a 16-point, semilog serial dilution scheme. For the BAK FITC TR-FRET assay 50 nL compound is added to 20 μL of assay buffer containing Bak-FITC. MBP tagged Mcl-1 protein and antibody, for a final DMSO concentration of 0.25% and a top concentration of 10 μM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 3 hours at room temperature. TR-FRET signal (Delta F) is measured on a Biotek Cytation 3 multimode plate reader equipped with a filter cube containing an Ex 340/30 Em 620/10 nm filter and an Ex 340/30 Em 520 filter.
Measurement: Ki < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 784.74, ALogP 9.18, HBA 9, HBD 1, PSA 120.94, QED 0.12
Compound (SMILES): C[C@@H]1CN(c2ccccc2F)CCN1C(=O)NCCC1CCN(Cc2ccccc2)CC1
InChIKey: UPKBYXADRFVHIP-OAQYLSRUSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 438.59, ALogP 4.35, HBA 3, HBD 1, PSA 38.82, QED 0.73
Compound (SMILES): COc1ccc(S(=O)(=O)Nc2ccc(-c3ncnc4cc(OC)c(OC)cc34)cc2F)cc1
InChIKey: JDMJXCTWASLKHU-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell-Based Assays of c-Kit Mutant Kinase Activity: The c-Kit mutant D816V inhibitors were assessed using an engineered BaF3-FL KIT D816V or BaF3-FL KIT V560G/D816V cell line. The BaF3-FL KIT D816V cell lines were created by introduction of KIT mutant (D816V) full length constructs that render the cells dependent on the introduced kinase for growth. Inhibitors of c-Kit mutant D816V kinase reduce or eliminate the mediated c-kit mutant D816V kinase activation, resulting in reduced cell proliferation of the BaF3-FL Kit mutant D816V cells. This inhibition is measured by the effect of compound concentration on cell growth to assess IC50 values. BaF3-FL KIT D816V cells were seeded at 1×104 cells per well of a 96 well cell culture plate in 50 μl of cell culture medium of RPMI Medium 1× (Invitrogen #11875-093) supplemented with 10% FBS (Invitrogen #10438), 1% Non Essential Amino Acids (Invitrogen #11140), 1% Penicillin Streptomycin (Invitrogen #15140), 1% L-Glutamine (Invitrogen #25030-081). Compounds were dissolved in DMSO at a concentration of 5 mM and were serially diluted 1:3 for a total of eight points and added to the cells to a final maximum concentration of 10 μM in 100 μl cell culture medium (final concentration 0.2% DMSO). Cells were also treated with Dasatinib as a positive control. The cells were incubated at 37° C., 5% CO2 for three days. ATPlite Buffer (Perkin Elmer #6016739) and substrate were equilibrated to room temperature, and enzyme/substrate Recombinant Firefly Luciferase/D-Luciferin was reconstituted. The cell plates were equilibrated to room temperature for 30 minutes, then lysed by addition of 25 uL per well of the ATPlite Reagent. The plate was mixed for 5 minutes on a plate shaker to lyse the cells. The plates were read on a Tecan Safire using Luminescence protocol modified to read 0.1s per well. The luminescence reading assesses the ATP content, which correlates directly with cell number such that the reading as a function of compound concentration is used to determine the IC50 value.
Measurement: Ki = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 469.49, ALogP 4.26, HBA 7, HBD 1, PSA 99.64, QED 0.43
Compound (SMILES): NCCSc1ccc(Cl)cc1NS(=O)(=O)c1ccc(Cl)c(C(F)(F)F)c1
InChIKey: UAJFJACVQHFYQT-UHFFFAOYSA-N
Target: C-C chemokine receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P41597
Assay: Biological Assay: HEK-Gqi5 cells stably expressing CCR2 were cultured in (DMEM high glucose, 10% FBS, 1% PSA, 400 μg/ml geneticin and 50 μg/ml hygromycin. Appropriate positive control chemokines (MCP-1, MIP1A or RANTES) was used as the positive control agonist for screening compound-induced calcium activity assayed on the FLIPRTetra. The drug plates were prepared in 384-well microplates using the EP3 and the MuItiPROBE robotic liquid handling systems.
Measurement: IC50 = 1067 nM (pChEMBL 5.96999979019165)
Descriptors: MW 445.32, ALogP 4.86, HBA 4, HBD 2, PSA 72.19, QED 0.62
Compound (SMILES): NC(=O)c1cnc2ccc(-c3c(-c4ccc(F)cc4)nc4n3CCN(CC(F)F)C4)cn12
InChIKey: VHJMTDJWLOCXKK-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 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-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (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.
Measurement: IC50 = 27.2 nM (pChEMBL 7.570000171661377)
Descriptors: MW 440.43, ALogP 3.18, HBA 6, HBD 1, PSA 81.45, QED 0.52
Compound (SMILES): CC(c1ccc2c(c1)OCC2)N1CCC(Oc2cccnn2)CC1
InChIKey: NLSZBEROHFCXNR-UHFFFAOYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Human O-GlcNAcase Enzyme Inhibition Assay: 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λexc 485 nm; (λemm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).
Measurement: IC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 325.41, ALogP 3.02, HBA 5, HBD 0, PSA 47.48, QED 0.86
Compound (SMILES): COC(=O)[C@H](C)Cn1c(=O)[nH]/c(=N\c2ccc(Oc3ccccn3)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: BSISVXMSAZLVNB-QGZVFWFLSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 521.96, ALogP 3.27, HBA 9, HBD 1, PSA 120.57, QED 0.36
Compound (SMILES): O=C(NCC1(O)CCCC1)c1cn(C2COC2)c2cccc(Cl)c12
InChIKey: OYGOLYUDIIFGEU-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Pore Permeation Assay: Agonist-induced pore formation was determined by measuring cellular uptake of YO PRO fluorescence dye in HEK293 transfected with human P2X7 receptor. A HEK293 cell over expressing human P2X7 was harvested using HQTase reagent to detach the cells from T75 cm flask. The harvested cells are centrifuged @1200 rpm for 5 min at room temperature. The viability of cells was determined by Trypan blue dye and the cells are plated @10,000 cell/well in 50 ul volume in a 384W BD Poly lysine coated plate and incubated overnight at 37 C. After overnight incubation, the culture medium was replaced with 35 ul/well assay buffer (5 mM KCl, 0.1 mM CaCl2, 5 mM Glucose, 10 mM HEPES buffer pH7.4 containing 125 mM NaCl. The serial dilution of compounds was performed using Bravo liquid handling instrument and the compounds were added using Bravo to the cell assay plate starting at 2.5 uM with three dilutions for 10 points. The positive control inhibitor compound was added to column 23. The plate was shaken slowly on a plate shaker for 10 seconds. The cells were incubated with the compound for 20 minutes at room temperature. After the incubation period, YO PRO dye (1 uM) along with BzATP (10 uM) were added to cells at 10 ul/well. The plate was centrifuged at 1000 rpm for 5 seconds and incubated at room temperature for 30 minutes. The uptake of YO PRO dye into the cells was measured using Envision Fluorescence plate reader instrument (Perkin Elmer).
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 348.83, ALogP 2.9, HBA 4, HBD 2, PSA 63.49, QED 0.89
Compound (SMILES): O=C(NCc1cc(-c2cnc(C(F)(F)F)nc2)cnc1F)[C@@H]1C[C@@H](F)CN1S(=O)(=O)c1ccc(F)cc1
InChIKey: DDBRUPZDWAODIC-AEFFLSMTSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay: IC50s (effective concentration) of compounds on the human and rat TRPA1 channels were determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37C, and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 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 20 minutes at room temperature prior to adding agonist. Following this incubation, EC80 concentration of cinnamaldehyde (75 uM for human TRPA1 and 45 uM for rat TRPA1) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.
Measurement: IC50 = 211 nM (pChEMBL 6.679999828338623)
Descriptors: MW 545.47, ALogP 3.25, HBA 6, HBD 1, PSA 105.15, QED 0.38
Compound (SMILES): Cn1nc(N)c2ccc(-c3cccc(F)c3OCC(F)(F)C(F)(F)F)cc21
InChIKey: KKHUHDWUJQBCCS-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro Electrophysiology: Electrophysiological studies are performed with an IonWorks Quattro (Molecular Devices Corp.) automated patch-clamp electrophysiology platform as described by (Schroeder K et al. Journal of Biomolecular Screening 2003, 8 (1), 50-64). Buffers for the experiments have the following composition (mM): Internal solution; K-gluconate 100, KCl 40, MgCl2 3.2, HEPES 5, EGTA 3, pH 7.3. To this amphotericin B is added to final concentration of 0.1 mg/ml to generate access solution. External solution; Dulbecco's Phosphate buffered saline (D-PBS) NaCl 137.93, KCl 2.67, KH2PO4 1.47, Na2HPO4 8.06, CaCl2 0.90, MgCl2 0.49. Prior to the experiment the cells expressing the voltage-gated ion channel of interest are detached from the tissue culture flasks, centrifuged and resuspended in D-PBS. Compounds are prepared and serially diluted in DMSO and finally diluted 1:100 in D-PBS. Cells are exposed to compounds through the pipetting system integrated into the platform and the voltage-gated ion channel of interest is activated with specific voltage stimulation protocols. The following voltage stimulation protocol is used for testing compounds against the voltage-gated sodium channel Nav1.7; from a holding potential of −100 mV a train of eight 60 ms depolarising steps to −20 mV at a frequency of 14 Hz are employed followed by a further step to −20 mV for 2000 ms. After which the voltage is returned to −100 mV for 10 ms before another voltage step to −20 mV for 60 ms is applied. The following voltage stimulation protocol is used for testing compounds against the voltage-gated sodium channel Nav1.5; from a holding potential of −120 mV a train of twenty-six 120 ms depolarising steps to −20 mV at a frequency of 5 Hz are employed. Recordings are made before and after compound addition with the compound incubation time being 5 minutes. Percent block was calculated for each concentration in duplicate for peak 1 and peak 10 and peak 1 and 25 for Nav 1.7 and Nav 1.5 respectively in order to assess compound activity at close and inactivated states and IC50 curves were fitted to percent block as a function of concentration.
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 389.3, ALogP 4.54, HBA 4, HBD 1, PSA 53.07, QED 0.67
Compound (SMILES): COC(=O)c1ccc(-c2n[nH]c3cc(F)ccc23)cc1
InChIKey: IEDHWGFYBSOPRB-UHFFFAOYSA-N
Target: Dual specificity mitogen-activated protein kinase kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P45985
Assay: ADP-Glo Kinase Activity Assay: Kinase activity of recombinant active full-length human MEK4 (Carna Biosciences) was measured using the ADP-Glo assay (Promega) with 3 μM recombinant full-length human p38α (MAPK14). The concentration of ATP used for high throughput screening was 3 μM (equal to the ATP KM value for MEK4). For ATP competition studies, the ATP concentration was varied (1, 4, 12, or 45 μM). Standard ADP-Glo assay buffer contained 25 mM Tris-HCl, 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, and 10 mM MgCl2 at pH 7.5. Triton X-100 was added at a final concentration of 0.01% (w/v) for the aggregation studies. For MEK family profiling, recombinant active full-length human MEK1, 2, 3, 4, 5, 6, and 7 (Carna Biosciences) were used together with 3 μM recombinant full-length substrates. p38α was used for MEK3, 4, and 6. Jnk1B (MAPK8) was used for MEK4 and 7. Erk2 (MAPK1) was used for MEK1 and 2. Erk5 (MAPK7) was used for MEK5. All reactions included 0.6 μM ATP, a concentration that was at least 2-fold below the empirically determined ATP KM values for all MEKs.
Measurement: IC50 = 420 nM (pChEMBL 6.380000114440918)
Descriptors: MW 270.26, ALogP 3.16, HBA 3, HBD 1, PSA 54.98, QED 0.73
Compound (SMILES): N#C/C(=C\C1C2COCC12)C(=O)N1CCC[C@@H](n2c(=O)n(-c3ccc(Oc4ccccc4)cc3)c3c(N)nccc32)C1
InChIKey: CZYGKRBKDILPIY-IGKCTRRQSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzymatic Activity Assay: A Caliper-based kinase assay (Caliper Life Sciences, Hopkinton, Mass.) was used to measure inhibition of BTK kinase activity of a compound of the present disclosure. Serial dilutions of test compounds were incubated with human recombinant BTK (0.5 nM), ATP (16 μM) and a phosphoacceptor peptide substrate FAM-GEEPLYWSFPAKKK-NH2(1 μM) at room temperature for 3 h. The reaction was then terminated with EDTA, final concentration 20 mM and the phosphorylated reaction product was quantified on a Caliper Desktop Profiler (Caliper LabChip 3000).
Measurement: IC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 562.63, ALogP 4.07, HBA 9, HBD 1, PSA 128.4, QED 0.28
Compound (SMILES): C=CC(=O)N1CCCN(C(=O)c2cc(Sc3cnc(NC(=O)C4CC4)s3)c(C)cc2C)C[C@H]1CC
InChIKey: OLKAUBUUBHWFHE-LJQANCHMSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: In Vitro Assay: The potency of inhibition is measured by binding affinity.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 512.7, ALogP 4.9, HBA 6, HBD 1, PSA 82.61, QED 0.53
Compound (SMILES): O=C(CCC(=O)c1ccccc1F)c1ccc2c(c1)OCCO2
InChIKey: NWPUADFLMHBJPW-UHFFFAOYSA-N
Target: Catenin beta-1/Protein Wnt-3a (PROTEIN COMPLEX), Homo sapiens
UniProt: P56704
Assay: Evaluation of Biological Activity: Compounds that enhance the Wnt activity, or Activators, were assayed as follows. Reporter cell lines were generated by stably transducing cells of colon cancer cell lines (with a lentiviral construct that included 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 also 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.Cultured SW480 cells bearing a reporter construct were distributed at approximately 10,000 cells per well into 384- or 96-well multiwell plates. Compounds from a small molecule compound library were then added to the wells in half-log dilutions using a maximum concentration of three or ten micromolar. A series of control wells for each cell type received only buffer and compound solvent DMSO. Twenty-four hours after the addition of compound, reporter activity for luciferase 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 any activities above DMSO were considered activation. Compounds were considered activators if reporter activities were 2× fold or greater than DMSO. EC50 is the concentration at half maximal activation.
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 314.31, ALogP 3.44, HBA 4, HBD 0, PSA 52.6, QED 0.79
Compound (SMILES): COC(=O)c1c(O)cc(O)c(Cl)c1CCc1ccc(O)cc1Cc1ccccc1
InChIKey: XGKANVNEYRKCOT-UHFFFAOYSA-N
Target: Endoplasmin (SINGLE PROTEIN), Canis lupus familiaris
UniProt: P41148
Assay: Fluorescence Polarization Assay: The assay was performed in 96-well black, flat-bottom plates with a final volume of 100 μL. 25 μL of assay buffer (20 mM HEPES, pH 7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, 2 mM DTT, 0.1 mg/mL BGG, and 0.01% NP-40) were added, followed by 25 μL of assay buffer containing 6 nM FITC-GDA (fluorescent tracer, stock in DMSO, diluted in assay buffer) and 50 μL of assay buffer containing 10 nM of either Grp94 or Hsp90α were added to each well. For each plate, wells containing buffer only (background), tracer in buffer only (low polarization control) and protein, tracer, and 1% DMSO (final concentration, high polarization control) were included. Compounds were then added with a final concentration of DMSO=1%. Plates were incubated at 4° C. with rocking for 24 h. Polarization values (in mP units) were measured at 37° C. with an excitation filter at 485 nm and an emission filter at 528 nm. Polarization values were correlated to % tracer bound and compound concentrations. The concentration at which the tracer was 50% displaced by compound of interested were calculated and reported as apparent Kd's.
Measurement: Kd = 330 nM (pChEMBL 6.480000019073486)
Descriptors: MW 412.87, ALogP 4.62, HBA 5, HBD 3, PSA 86.99, QED 0.51
Compound (SMILES): COc1cc(-c2cnc(N)c(-c3ccc(NC(=O)c4cn(CC5CCOCC5)cc(-c5ccc(C)cn5)c4=O)cc3F)c2)ccc1OC[C@@H]1COCCO1
InChIKey: ZOEILZXFYPPMTR-PMERELPUSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Cell-Free Axl Kinase Inhibitory Activity: A kinase dilution solution containing 170 ng/ml AXL (the 464th to 885th amino acids of the intracellular domain of human AXL expressed as a fusion protein with glutathione transferase in a baculovirus expression system and purified by glutathione Sepharose chromatography; Carna Biosciences, Inc., catalog No. 08-107) was prepared using a kinase reaction buffer solution (100 mM HEPES (pH 7.4), 0.003% Brij-35, 0.004% Tween-20, 1 mM DTT, and 10 mM MgCl2) and added at 19 μl/well to a 384-well plate.Next, each test compound was diluted with DMSO, and this dilution solution was added at 1 μl/well to the plate.After preincubation at room temperature for 30 minutes, a solution containing a substrate peptide (FL-Peptide 30 (5FAM-KKKKEEIYFFF-CONH2), Caliper Life Sciences, catalog No. 760430) and ATP at 1.5 μM and 10 μM, respectively, was prepared and added at 5 μl/well to the plate to start kinase reaction. The plate was incubated at 28° C. for 1.5 hours, and the reaction was terminated by the addition of a termination buffer solution (100 mM HEPES (pH 7.4), 0.015% Brij-35, 40 mM EDTA, and 0.1% Coating Reagent 3) at 40 μl/well.The substrate peptide and the phosphorylated peptide in the reaction mixture were separated and quantified using EZ Reader II (Caliper Life Sciences).The kinase reaction was evaluated on the basis of a product ratio (P/(P+S)) calculated from the peak height (S) of the substrate peptide and the peak height (P) of the phosphorylated peptide.The rate of inhibition (Inhibition) was determined according to the following expression (automatically calculated using the software of the EZ Reader II system).
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 735.81, ALogP 6.15, HBA 11, HBD 2, PSA 149.05, QED 0.16
Compound (SMILES): CC(C)(C(=O)NC1CC1)c1ccc(-c2ccsc2)c(NC(=O)Nc2cccc(C(F)(F)F)c2)c1
InChIKey: LQHAQXWDPBRSSA-UHFFFAOYSA-N
Target: Isocitrate dehydrogenase [NADP], mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P48735
Assay: IDH2 Inhibitory Activity: The inhibitory activity of a compound against IDH2 (R172K, 40-end) was determined by the decrease of a helper factor NADPH. The test compound was pre-incubated with an enzyme and NADPH, and then a reaction was initiated by the addition of a-KG, and performed for 120 minutes under a linear condition. Then, the reaction was terminated by the addition of diaphorase (lipoamide dehydrogenase) and the corresponding substrate resazurin. Diaphorase terminated the IDH2m reaction by decreasing the available helper factor NADPH, which oxidized NADPH to NADP, and reduced resazurin to highly fluorescent resorufin. The amount of remaining helper factor NADPH after a specific reaction time was quantified via an easily detectable fluorophore.Specifically, 2.5 μl of a 3-fold gradient diluted test compound was added to a 384-well plate, and then 5 μl of a reaction buffer (20 mM Tris-HCl, PH7.5; 150 mM NaCl; 10 mM MgC12; 10 mM MnCl2; 0.4 mg/ml BSA and 2 mM DTT) containing 80 nM IDH2 (R172K, 40-end) and 40 μM NADPH was added. Then, the resulting test mixture was incubated for 120 minutes at a temperature of 23° C., and then 2.5 μl of the reaction buffer containing 4 mM α-KG was added to initiate the reaction. After incubating for 120 minutes at room temperature, 5 μl of a termination mixture (0.4 U/ml diaphorase and 40 μM resazurin) prepared with the reaction buffer was added to convert resazurin to resorufin to determine the remaining NADPH. After incubating for 10 minutes at a temperature of 23° C., a fluorescence value was determined through Flexstation 3 at Ex535/Em595.
Measurement: IC50 = 65.7 nM (pChEMBL 7.179999828338623)
Descriptors: MW 487.55, ALogP 6.63, HBA 3, HBD 3, PSA 70.23, QED 0.37
Compound (SMILES): COC1CCN(C(=O)c2ccc(-c3cccc4nc(NC(=O)C5CC5)nn34)cc2)CC1
InChIKey: XMGVUFLDOGTFOB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1 Inhibition Assay: Recombinant human JAK1 catalytic domain (amino acids 850-1154; catalog number 08-144) was purchased from Carna Biosciences. 10 ng of JAK1 was incubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (15 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% Tween-20, 10 mM MgCl2, 2 μM non-radioactive ATP, 0.25 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5 μL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom). After 45 min at 30° C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μL/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:Percentage inhibition=((cpm determined for sample with test compound present−cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle−cpm determined for sample with positive control inhibitor))*100%.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 419.49, ALogP 3, HBA 6, HBD 1, PSA 88.83, QED 0.69
Compound (SMILES): COC(=O)CCn1c(=O)[nH]/c(=N\c2ccc(Oc3ccccn3)cc2)n(Cc2ccc(C)cc2)c1=O
InChIKey: NRPWMDSRMHLJBB-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 487.52, ALogP 2.68, HBA 9, HBD 1, PSA 120.57, QED 0.38
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1ccnc(Cc2cc(S(C)(=O)=O)c3ncc(Cl)cc3c2)c1
InChIKey: LOAAVKXHIOVCPQ-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Enzyme Inhibition Assay: The ability of the compounds disclosed herein to inhibit human plasma kallikrein activity was quantified according to the procedures below.A 10 mM solution of the test compound was made in DMSO. This solution was serially diluted 1:5 in DMSO to yield 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256 and 0.00512 μM compound test solutions. A control tube containing only DMSO is included. 16 μL of each compound test solution was combined with 384 μL of assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Triton X-100) to yield a 4× test compound buffer stock.Separately, a 40 nM solution of human Plasma Kallikrein (Abcam) and a 93.6 μM solution Pro-Phe-Arg-AMC (Bachem) were made using assay buffer. These solutions are hereby referred to as 4×hPK and 2×PFR-AMC, respectively.60 μL of each 4× test compound buffer stock was combined with 60 μL of 4×hPK to yield 120 μL of 2× test compound buffer stock/2×hPK. 50 μL was removed from this mixture and placed into duplicate wells on a Microfluor 1Black U-bottom microtiter plate (Thermo Scientific). This plate was incubated for 5 minutes at 37° C. To each well, 50 μL of pre-warmed 2×PFR-AMC was added to start the enzymatic reaction. Cleavage of PFR-AMC was monitored in a Biotek Synergy H4 reader set at 37° C. Readings are taken every 43 seconds for 1 hour. The highest mean velocity over 20 reads (15 minutes) is used to calculate the IC50.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 510.02, ALogP 3.8, HBA 7, HBD 2, PSA 127.93, QED 0.4