lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3CC4CCC(C3)O4)nc(-c3ccc(NC(=O)NCC4CCCO4)c(F)c3)c2C1=O
InChIKey: UZHJTCHFFQORJK-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The test detects the phosphorylation of the substrate GFP-4EBP1 using a terbium-labelled antibody that is specific for the phospho-Thr46 epitope. Binding of the Tb-antibody to the phosphorylated GFP peptide allows the transfer of energy from Tb, excited at 340 nm, to GFP with an increase in the fluorescence emission of the GFP at 520 nm. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 ul. To 1 ul of inhibitor in 100% DMSO are added (final concentrations) 400 nM of GFP-4EBP1 (Invitrogen PV4759), 8 uM of ATP, 200 ng/ml of enzyme (human recombinant aa mTOR 1360-2549, Invitrogen PV4753) in a buffer of HEPES 50 mM pH 7.5, EGTA 1 mM, MnCl2 10 mM, BSA 0.1 mg/ml, glycerol 0.5%, NV-10 0.1 mg/ml, DTT 2 mM. The mixture is incubated for 30 minutes at room temperature. 10 ul of solution are taken up and the reaction is quenched by adding 10 ul of Tb-antibody solution (Invitrogen PV4757)/EDTA.
Measurement: IC50 = 3475 nM (pChEMBL 5.460000038146973)
Descriptors: MW 568.65, ALogP 3.58, HBA 8, HBD 2, PSA 118.15, QED 0.54
Compound (SMILES): Cc1cn(-c2ccc3n(c2=O)CCN([C@@H]2CC[C@H]2Oc2ccc(F)c4c2CCC4)C3=O)cn1
InChIKey: KXUOSHAFWYNJPL-AUSIDOKSSA-N
Target: Presenilin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P49768
Assay: Cell-Based Assay: The ability of compounds to modulate production of amyloid beta protein Abeta(1-42) was determined using human WT-APP overexpressing CHO cells. Cells were plated at 22,000 cells/100 uL well in 96 well tissue culture treated, clear plates (Falcon) in DMEM/F12 based medium and incubated for 24 hours at 37° C. Compounds for testing were diluted in 100% DMSO to achieve an eleven points, half log, dose response for IC50 determinations. Compounds were added in fresh medium to achieve 1% final DMSO. Appropriate vehicle or inhibitor controls were added into control wells individually to obtain minimum or maximum inhibition values, respectively, for the assay signal window before the plates were incubated for 24 hours at 37° C. This procedure produces conditioned media in each well which is tested for Abeta(1-42) levels in the ELISA detection step described next. The remaining cell cultures in each well are also tested for cell toxicity as described below.
Measurement: IC50 = 162 nM (pChEMBL 6.789999961853027)
Descriptors: MW 448.5, ALogP 3.04, HBA 6, HBD 0, PSA 69.36, QED 0.62
Compound (SMILES): COCCCC(c1ccccc1)n1cc(-c2ccc(N3CCc4cccc(C(=O)Nc5nc6ccccc6s5)c4C3)nc2C(=O)O)cn1
InChIKey: XCAYLVULFKHTMI-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 658.78, ALogP 7.08, HBA 9, HBD 2, PSA 122.47, QED 0.14
Compound (SMILES): CCCNc1ncc(-c2ccc(Oc3ncc(NC(=O)Nc4cc(C(F)(F)F)ccc4S(C)(=O)=O)cn3)cc2)s1
InChIKey: UKKDPXNMPMCVTP-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: TrkA kinase-inhibiting activity in cell systems was measured using CHO-K1 cells expressing human TrkA and NFAT-bla (CellSenser TrkA-NFAT-bla CHO-K1 cells, Invitrogen).On the day before the assay, CellSenser TrkA-NFAT-bla CHO-K1 cells were suspended in an assay medium (Opti-MEM1 Reduced Serum Medium (Invitrogen) containing 0.5% dialysed fetal bovine serum (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen) and antibiotics (100 U/mL penicillin and 100 ug/mL streptomycin (Invitrogen)) and plated at a density of 2.4x104 cells/40 uL/well in a 96-well clear bottom plate (Corning, Catalogue No.: 3882). In some wells were added only the assay medium at 40 uL/well (Cell-free). On the day of the assay, 10 mM of the present compound (DMSO solution) was distributed in a 96-well plate (Costar, Catalogue No.: 3363) and serially diluted with DMSO with the geometrical ratio of 3. The serial dilutions were diluted with the assay medium to 100-fold to prepare a solution of the present compound with a 10-times concentration (DMSO concentration: 1%).
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 592.63, ALogP 6.28, HBA 9, HBD 3, PSA 135.2, QED 0.21
Compound (SMILES): NC(=O)c1ccc2[nH]c(-c3ccc(C(O)c4ccc(Cl)c(Cl)c4)cc3)nc2c1
InChIKey: QXEILDOJDHCHNC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Chk2 (SINGLE PROTEIN), Homo sapiens
UniProt: O96017
Assay: Inhibition of recombinant human CDS1 using N-terminal biotin-labeled SGLYRSPSMPENLNRPR as substrate in presence of [gamma-33P]-adenosine triphosphate after 3 hrs by scintillation counting method
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 412.28, ALogP 4.72, HBA 3, HBD 3, PSA 92, QED 0.45
Compound (SMILES): CN(C)c1ccc(C(=O)Nc2ccc(-c3n[nH]c(=O)c4ccccc34)cc2)cc1
InChIKey: PDOWPLWTKJLFMU-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LabChip 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 384.44, ALogP 3.91, HBA 4, HBD 2, PSA 78.09, QED 0.56
Compound (SMILES): CN(C)CCCNc1ccc(NC(=O)c2ccc(N3CCN(CC4=C(c5ccc(Cl)cc5)CC(C)(C)CC4)CC3)cc2Oc2cccc(F)c2)cc1[N+](=O)[O-]
InChIKey: GLRTXOSJRDRRIJ-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 μM (2× starting concentration; 10% DMSO) and 10 μL were transferred into a 384-well plate. Then 10 μL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probed antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the Envision (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki = 0.016 nM (pChEMBL 10.800000190734863)
Descriptors: MW 769.36, ALogP 9.58, HBA 8, HBD 2, PSA 103.22, QED 0.07
Compound (SMILES): CCc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(C)c3)cn2CCN(CC)C(C)C)CC1
InChIKey: VPNSTYRFYMPBQK-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 493.68, ALogP 5.05, HBA 7, HBD 1, PSA 76.1, QED 0.45
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCN(Cc2ccnc3[nH]ccc23)CC1
InChIKey: YXQRCMVFYFMXCH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 473.52, ALogP 3.04, HBA 8, HBD 3, PSA 141.54, QED 0.35
Compound (SMILES): COC(=O)N1C[C@H](C)N(c2nc3cc(-c4nc(=O)o[nH]4)nc(-c4cncc(Cl)c4)c3n2C[C@H]2CC[C@H](C)CC2)[C@@H](C)C1
InChIKey: SJERQOBZODKGGG-VJANTYMQSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: Methods: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (SEQ ID NO:1: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards the p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1x Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1x assay buffer recombinant HDM2 protein, peptide and Lumi-4-Tb Cryptate-conjugate mouse.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 595.1, ALogP 4.98, HBA 10, HBD 1, PSA 135.27, QED 0.34
Compound (SMILES): O=C(Cn1cnc2ccccc21)N1CCN(c2scnc2-c2nc3ccccc3[nH]2)CC1
InChIKey: BGIPMVWYZIKOEO-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.015%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 nM, is also is used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 443.54, ALogP 3.38, HBA 7, HBD 1, PSA 82.94, QED 0.46
Compound (SMILES): NS(=O)(=O)OC[C@H]1O[C@@H](n2cnc3c(N[C@@H](CO)c4ccccc4)ncnc32)[C@H](O)[C@@H]1O
InChIKey: ZTUPSMZTRCBYRP-URQYDQELSA-N
Target: NEDD8-activating enzyme E1 catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TBC4
Assay: Inhibition of recombinant human untagged NAEalpha/N-terminal His-tagged NAEbeta expressed in Bac-to-Bac expression system using FLAG-tagged NEDD8 as substrate after 90 mins in presence of GST-tagged Ubc12 by HTRF assay
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 466.48, ALogP -1.19, HBA 12, HBD 5, PSA 194.94, QED 0.27
Compound (SMILES): CC(C)Oc1ccc(-c2nc(-c3cccc4c3CC[C@@H]4NC(=O)N3CCC(N(C)C)CC3)no2)cc1C#N
InChIKey: GERMNTVMEBHJCQ-VWLOTQADSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 514.63, ALogP 4.79, HBA 7, HBD 1, PSA 107.52, QED 0.5
Compound (SMILES): O=C(Nc1cn(CC(=O)N2CCC(CO)CC2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: JTPKLMJGUFVPRN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.34 nM (pChEMBL 9.470000267028809)
Descriptors: MW 559.96, ALogP 3.33, HBA 9, HBD 2, PSA 126.88, QED 0.34
Compound (SMILES): C=CC(=O)N1CC[C@H](Oc2cc3c(Nc4ccc(Cl)c(Cl)c4F)ncnc3cc2OC)C1
InChIKey: RAHSHQQWQIMPOG-LBPRGKRZSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of N-terminal GST-tagged human EGFR (696 to end residues) expressed in baculovirus infected Sf21 cells using poly (Glu,Tyr)4:1 as substrate preincubated for 10 mins followed by substrate addition measured after 1 hr by fluorescence polarization assay
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 477.32, ALogP 4.99, HBA 6, HBD 1, PSA 76.58, QED 0.4
Compound (SMILES): O=c1[nH]cc(SCc2cccnc2)cc1O
InChIKey: AWUWWINBQXLGBM-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P14920
Assay: Enzyme Assay: The functional activity of compounds inhibiting the DAAO enzyme was determined by utilizing the co-product of the catalysis of D-Serine, H2O2 which can be quantitatively measured using the Amplex Red (Invitrogen) detection. Amplex Red reagent is a colorless substrate that reacts with hydrogen peroxide (H2O2) with a 1:1 stoichiometry in the presence of hydrogen peroxide to produce highly fluorescent resorufin (excitation/emission maxima=570/585 nm). The changes in fluorescence were monitored by a fluorescence plate reader, Envision (Perkin Elmer) and increases in DAAO activity were readily detected upon addition of D-Serine and suppression of this response observed with the application of test compounds.Human DAAO enzyme was supplied by the Takeda Pharmaceutical Company (Osaka) and each batch was tested and used at concentrations giving comparable levels of activity. The Km of D-Serine was measured for each enzyme batch to maintain consistency; this Km was used in subsequent assays.
Measurement: IC50 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 234.28, ALogP 1.77, HBA 4, HBD 2, PSA 65.98, QED 0.79
Compound (SMILES): O=C(Nc1ccnc2ccccc12)c1cc(NC2CCCCC2)ncn1
InChIKey: TZFMZIWHSKGSQT-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: 35S-GTPgammaS Binding: Measurements of 35S-GTPgammaS Binding: Membranes (1 to 10 ug protein) prepared as described above, were incubated in 96-well Scintiplates (PerkinElmer) with test compounds diluted in DMSO, in 180 ul of 20 mM HEPES, pH 7.4, 10 mM MgCl2, 2 ug/well Saponin, 0.2% fatty acid free BSA (Assay buffer), 140 mM NaCl and 1.7 uM GDP. The assay was initiated with the addition of 20 ul of 1.5 nM [35S]-GTPgammaS (1100 Ci/mmol; GE Healthcare) in assay buffer. After 60 min incubation at 30° C. on a shaker, plates were centrifuged for 10 min at 2000 RPM. Supernatant was discarded and membrane bound radioactivity was measured on a PerkinElmer 1450 MicroBeta counter. Triplicate samples were averaged and expressed as % response relative to S1P activation in absence of compound (n=2).
Measurement: EC50 = 1070 nM (pChEMBL 5.96999979019165)
Descriptors: MW 347.42, ALogP 4.02, HBA 5, HBD 2, PSA 79.8, QED 0.74
Compound (SMILES): Cc1ccc(Cl)cc1COc1ccc(S(=O)(=O)N2C[C@H](O)CC(C)(C)[C@@H]2C(=O)NO)cc1
InChIKey: WYKNWJFOHUYOQU-XLIONFOSSA-N
Target: Interstitial collagenase (SINGLE PROTEIN), Homo sapiens
UniProt: P03956
Assay: Inhibition of trypsin-activated recombinant human collagenase 1 preincubated with protein followed by addition of DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(NMA)-NH2 as substrate measured after 20 mins by fluorescence analysis
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 482.99, ALogP 2.88, HBA 6, HBD 3, PSA 116.17, QED 0.43
Compound (SMILES): NS(=O)(=O)OC[C@H]1O[C@@H](n2cnc3c(C#Cc4ccc(Cl)cc4)ncnc32)[C@H](O)[C@@H]1O
InChIKey: FAUBHMYGSSEKOC-GFOCRRMGSA-N
Target: NEDD8-activating enzyme E1 catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TBC4
Assay: Inhibition of recombinant human untagged NAEalpha/N-terminal His-tagged NAEbeta expressed in Bac-to-Bac expression system using FLAG-tagged NEDD8 as substrate after 90 mins in presence of GST-tagged Ubc12 by HTRF assay
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 465.88, ALogP -0.28, HBA 10, HBD 3, PSA 162.68, QED 0.44
Compound (SMILES): c1cc(N2CCCNCC2)nc(-n2ncc3cnc(-c4cn[nH]c4)cc32)c1
InChIKey: CYOQMLSODZDBGH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.122 nM (pChEMBL 9.90999984741211)
Descriptors: MW 360.43, ALogP 2.01, HBA 7, HBD 2, PSA 87.55, QED 0.58
Compound (SMILES): COc1cc(OC)cc(N(CCCNCC(F)(F)F)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: IQAIXSLQDLQWKO-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 3235.94 nM (pChEMBL 5.489999771118164)
Descriptors: MW 500.53, ALogP 4.73, HBA 8, HBD 1, PSA 77.33, QED 0.32
Compound (SMILES): Cc1c([C@H]2CN3CCN(C(=O)Cc4ccc(-n5cnnn5)nc4)C[C@H]3CS2)ccc2c1COC2=O
InChIKey: RWZHZCMTEWARFB-LAUBAEHRSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: The ROMK channel functional thallium flux assay is performed in 384 wells, using the FLIPR-Tetra instrument. HEK-hKir1.1 cells are seeded in Poly-D-Lysine microplates and kept in a 37° C.-10% CO2 incubator overnight. On the day of the experiment, the growth media is replaced with the FluxOR™ reagent loading buffer and incubated, protected from light, at ambient temperature (23-25° C.) for 90 min. The loading buffer is replaced with assay buffer±test compound followed by 30 min incubation at ambient temperature, where the Thallium/Potassium stimulant is added to the microplate. The fluorescence intensity of wells containing 3 μM of a standard control ROMK inhibitor of the present invention is used to define the ROMK-sensitive component of thallium flux. Fluorescence in the presence of test compounds is normalized to control values to provide % fluorescence change. IC50 values represent the concentration of compound that inhibits 50% of the ROMK thallium flux signal.
Measurement: IC50 = 420 nM (pChEMBL 6.380000114440918)
Descriptors: MW 491.58, ALogP 1.58, HBA 10, HBD 0, PSA 106.34, QED 0.5
Compound (SMILES): [C-]#[N+]c1c(F)ccc([C@H]2CN3CCN(C(=O)Cc4cnc(-n5cnnn5)c(F)c4)C[C@H]3CO2)c1C
InChIKey: QRDHTEJMUGASER-OXJNMPFZSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: The ROMK channel functional thallium flux assay is performed in 384 wells, using the FLIPR-Tetra instrument. HEK-hKir1.1 cells are seeded in Poly-D-Lysine microplates and kept in a 37° C.-10% CO2 incubator overnight. On the day of the experiment, the growth media is replaced with the FluxOR™ reagent loading buffer and incubated, protected from light, at ambient temperature (23-25° C.) for 90 min. The loading buffer is replaced with assay buffer±test compound followed by 30 min incubation at ambient temperature, where the Thallium/Potassium stimulant is added to the microplate. The fluorescence intensity of wells containing 3 μM of a standard control ROMK inhibitor of the present invention is used to define the ROMK-sensitive component of thallium flux. Fluorescence in the presence of test compounds is normalized to control values to provide % fluorescence change. IC50 values represent the concentration of compound that inhibits 50% of the ROMK thallium flux signal.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 480.48, ALogP 2.02, HBA 8, HBD 0, PSA 93.63, QED 0.53
Compound (SMILES): Cc1ccc(C(=O)N2CCC(c3ccc(C#N)cc3)CC2)cc1-c1[nH]c(C2CC2)nc1Cl
InChIKey: HRPYBVAQACGPDX-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 444.97, ALogP 5.81, HBA 3, HBD 1, PSA 72.78, QED 0.55
Compound (SMILES): CC(C)(C)CN1CCC2(CC1)CN(c1ncccc1NC(=O)Nc1ccc(OC(F)(F)F)cc1)c1c(O)ccc(C(F)(F)F)c12
InChIKey: KQAKTHLPOBSXSE-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 uL containing 15 fmol of P2Y1 receptor (1.7 ug of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 uM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium Chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 ug of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 uL of the aqueous soluttion was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKERSM (Amersham) Image Reader. Dose-response curves (IC50) were fit by non-linear regression (Toolset an in house data processing program) and binding constants (Ki) calculated using the Cheng-Prusoff relationship (Ki=IC50/(1+L/Kd) in which a Kd for 2MeS-ADP to the P2Y1 receptor was determined to be 14 nM.
Measurement: Ki = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 637.63, ALogP 7.88, HBA 6, HBD 3, PSA 89.96, QED 0.25
Compound (SMILES): CS(=O)(=O)c1ccccc1N1CCOc2cc(S(=O)(=O)Nc3nccs3)ccc21
InChIKey: OABDPQHPULPVDW-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 33600 nM (pChEMBL 4.46999979019165)
Descriptors: MW 451.55, ALogP 2.88, HBA 8, HBD 1, PSA 105.67, QED 0.64
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)nc(NCCc4ccccn4)nc23)N1
InChIKey: JYZOWQBBPNLCQG-ZROIWOOFSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 405.42, ALogP 0.93, HBA 9, HBD 4, PSA 138.23, QED 0.34
Compound (SMILES): CCn1c(=O)oc2cc(Cc3cncc4cc(OC)c(OC)cc34)ccc21
InChIKey: KCEBELPCJQSUSM-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Diaxonhit Phosphodiesterase Assay: The phosphodiesterase assay was developed using the LANCE cAMP kit (PerkinElmer). The assay buffer contained HBSS with 5 mM HEPES, 0.1% BSA, and 1.5 mM MgCl2, pH 7.4. PDE10A (BPS Bioscience) was used at 200 pg/well (with a specific activity of 3200 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min) and PDE4D3 (BPS Bioscience) was used at 100 pg/well (with a specific activity of 32713 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min). The Biotin-cAMP tracer, supplied in 10 mmol/L Tris-HCl buffered (pH 8.0) salt solution with 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1% bovine serum albumin (BSA), and 0.05% sodium azide, is used at a dilution of 1/375. The assay detection mixture contained the LANCE Eu-W8044 labeled streptavidin 1/2250 (supplied in 50 mmol/L Tris-HCl buffer).
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 364.4, ALogP 3.77, HBA 6, HBD 0, PSA 66.49, QED 0.54
Compound (SMILES): Cc1c(-c2ccc(N3CCc4cccc(C(=O)Nc5nc6ccccc6s5)c4C3)nc2C(=O)O)cnn1CC1(OCCO)CCCCC1
InChIKey: QTDHFNWYZXYUOP-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 666.8, ALogP 6.09, HBA 10, HBD 3, PSA 142.7, QED 0.16
Compound (SMILES): Nc1ncc(Cl)cc1-c1ccc(Oc2ncc(NC(=O)Nc3cc(C(F)(F)F)ccc3Cl)cn2)cc1
InChIKey: GAJBJKWFCCHZHO-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Activity Assay: On the day before the assay, CellSenser TrkA-NFAT-bla CHO-K1 cells were suspended in an assay medium (Opti-MEM1 Reduced Serum Medium (Invitrogen) containing 0.5% dialysed fetal bovine serum (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen) and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin (Invitrogen))) and plated at a density of 2.4x104 cells/40 uL/well in a 96-well clear bottom plate (Corning, Catalogue No.: 3882). In some wells were added only the assay medium at 40 uL/well (Cell-free). On the day of the assay, 10 mM of the present compound (DMSO solution) was distributed in a 96-well plate (Costar, Catalogue No.: 3363) and serially diluted with DMSO with the geometrical ratio of 3. The serial dilutions were diluted with the assay medium to 100-fold to prepare a solution of the present compound with a 10-fold concentration (DMSO concentration: 1%). To the plate where cells were plated was added the present compound at 5 L/well and the plate was incubated in a CO2 incubator with 5% CO2, 95% Air at 37° C. for 30 minutes. For a control and a blank, the assay medium containing 1% DMSO was added at 5 L/well in place of the solution of the present compound. Subsequently the assay medium containing NGF (Mouse 2.5s, Natural, Invitrogen) was added to the plate at 5 L/well (final concentration of NGF: 50 ng/ml) and the plate was incubated in a CO2 incubator with 5% CO2, 95% Air at 37° C. for 5 hours. For the blank group, the assay medium was added in place of NGF at 5 L/well. A reporter assay detection reagent (10 L/well) was added to the plate which was then incubated in the dark at room temperature for 120 minutes. The reporter assay detection reagent was prepared from LiveBLAzer -FRET B/G Loading Kit (Invitrogen). On the Analyst GT (Molecular Devices Japan, K.K.) the wells were irradiated with excitation light at 405 nm and the fluorescence intensities at 460 nm and 530 nm were measured.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 535.31, ALogP 6.88, HBA 6, HBD 3, PSA 115.05, QED 0.26
Compound (SMILES): O=[N+]([O-])c1ccccc1CS(=O)(=O)Nc1ccc(F)c(Nc2ncccc2-c2ncnc3[nH]cnc23)c1F
InChIKey: MAXUDKMCUTVIEB-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Enzyme Assay: To check the B-Raf kinase inhibitory activity of the compounds of the present invention, the following experiments were conducted.(1) Serial Signaling: 20 ul of diluting solvent (20 mM MOPS, pH 7.2, 25 mM Î²-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol) and 10 ul of Mg/ATP mixed solution (500 uM ATP, 75 mM magnesium chloride) were added into a centrifuge tube, the derivative compound of Formula 1 was added or the compounds of the Examples were not added as a control group, and then 1 ng of activated B-Raf, 0.4 ng of inactivated MEK1, and 1 ug of inactivated MAPK2 were added. The solutions in the tube were collected on the bottom via centrifugation and were reacted at 30° C. for 30 minutes. After taking 4 ul of the mixed solution, the test proceeded to the next step.(2) Phosphorylation of Matrix Protein MBP by MAPK2: 10 ul of diluting solvent, 20 ug of MBP used as a matrix.
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 538.5, ALogP 4.29, HBA 9, HBD 3, PSA 168.69, QED 0.19
Compound (SMILES): Cn1cc2c(nc(NC(=O)Nc3ccc(CC(=O)NCCCC(=O)OC(C)(C)C)cc3)n3nc(-c4ccco4)nc23)n1
InChIKey: OIQIWYIQBQXEQV-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Radioligand Binding Assays: Radioligand binding assays at hA1, hA2A, and hA3ARs were performed according to the procedures described in Gao, Z. G., et al., Biochem Pharmacol. 2003; 65: 1675-84, using the radiolabeled agonists [3H]R-PIA from Moravek Biochemicals (Brea, Calif.) or [3H]CGS21680 and [125I]I-AB-MECA from PerkinElmer (Waltham, Mass.) for the hA1, hA2AAR, and hA3AR assays, respectively.
Measurement: Ki = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 573.61, ALogP 3.7, HBA 11, HBD 3, PSA 170.57, QED 0.18
Compound (SMILES): CN(C)c1ccc(S(=O)(=O)N[C@H]2CCN([C@H](Cc3ccc4c(N)nccc4c3F)C(=O)O)C2=O)cn1
InChIKey: VYTBURKHXBGAQX-ZWKOTPCHSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: Inhibition Assay: The compounds according to the invention are tested in a range of concentrations (4.9 nM to 5 µM final) and deposited in a proportion of 25 µl per well. After the deposition of 50 µl of Spectrozyme 229 substrate (American Diagnostica), at the final concentration of 625 µM, the reaction is triggered by the addition of 25 µl of FIXa (human factor IXa supplied by Enzyme Research Laboratories (ERL)), at the final concentration of 2.5 U/ml. Reading is carried out at 405 nm for 15 min at 37°C. The percentage of inhibition of enzymatic activity (expressed as maximum rate of cleavage of the substrate) is calculated with respect to the enzymatic activity in the absence of inhibitor.
Measurement: IC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 516.56, ALogP 0.99, HBA 8, HBD 3, PSA 158.82, QED 0.4
Compound (SMILES): CC(C)(O)CNc1nc(C#CCO)cn2c(-c3ccc(C(=O)NC4CC4)cc3)cnc12
InChIKey: KRTBEZVYGAAVCC-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Mps-1 Kinase Assay: For the assay 50 nL of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 ul of a solution of Mps-1 in assay buffer [0.1 mM sodium- ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 ul of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 uM=> final conc. in the 5 ul assay volume is 10 uM) and peptide substrate (1.67 uM=> final conc. in the 5 ul assay volume is 1 uM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 ul assay volume). The reaction was stopped by the addition of 3 ul of a solution of HTRF detection reagents (100 mM Hepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nM anti-phospho(Ser/Thr)-Europium-antibody [#AD0180, PerkinElmer LAS, Rodgau-Jugesheim, Germany]. The resulting mixture was incubated 1 h at 22° C. to allow the binding of the phosphorylated peptide to the anti-phospho(Ser/Thr)-Europium-antibody. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Europium-labelled anti-phospho(Ser/Thr) antibody to the Streptavidin-XLent. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a Viewlux TR-FRET reader (PerkinElmer LAS, Rodgau-Jugesheim, Germany).
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 419.49, ALogP 1.82, HBA 7, HBD 4, PSA 111.78, QED 0.45
Compound (SMILES): CNS(=O)(=O)c1ccc(OC/C(=C/F)CN)cc1
InChIKey: RVCAAPOJOTYELI-RMKNXTFCSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: MAO-B Inhibition Assay: Recombinant human MAO-B (0.06 mg/mL; Sigma Aldrich) was used as source of MAO-B enzyme activities. The assay was performed in a similar way as for human SSAO/VAP-1 (Example 5) except, the substrate benzylamine was used at 100 μM. Briefly, in a standard 96 well plate assay 50 μL of purified human SSAO/VAP-1 (0.25 μg/mL) in 0.1 M NaPO4 buffer (pH 7.4) was added into each well. Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 4-9 data points, typically in the micromolar or nanomolar range after incubation with human SSAO/VAP-1 for 30 min at 37 °C. After 30 min incubation, 50 μL of the reaction mixture containing 600 μM benzylamine (Sigma Aldrich), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 0.1 M NaPO4 buffer (pH 7.4) were added to the corresponding well. The fluorescence unit (RFU) was read every 2.5 min for 30 min at 37 °C. excitation 565 nm and emission 590 (Optima; BMG labtech). The slope of the kinetics for each well was calculated using MARS data analysis software (BMG labtech) and this value was used to deduce the IC50 value (Dotmatics).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 274.32, ALogP 0.79, HBA 4, HBD 2, PSA 81.42, QED 0.8
Compound (SMILES): NC1=N[C@](CF)(c2cc(NC(=O)c3ccc(Cl)cn3)cc(F)c2F)C[C@@H](C(F)(F)F)O1
InChIKey: LKJAOKBSWHVMOZ-SUMWQHHRSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 466.77, ALogP 4.1, HBA 5, HBD 2, PSA 89.6, QED 0.66
Compound (SMILES): COc1cc(OC)cc(N(CCCN2CCCC2(C)C)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: MXVLJTMBTSLDPM-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 11.48 nM (pChEMBL 7.940000057220459)
Descriptors: MW 500.65, ALogP 5.45, HBA 8, HBD 0, PSA 68.54, QED 0.3
Compound (SMILES): Cl.N=C(N)c1ccc(COc2ccc3cc(CC(NC(=O)c4ccc([N+](=O)[O-])cc4)C(=O)O)ccc3c2)cc1
InChIKey: NHOZVENFFYKUHD-UHFFFAOYSA-N
Target: Integrin alpha-IIb/beta-3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P08514
Assay: Antagonist activity at GP2b/GP3a receptor in human platelet rich plasma assessed as inhibition of ADP-induced platelet aggregation preincubated for 1 min followed by ADP addition by aggregometric method
Measurement: IC50 = 2100 nM (pChEMBL 5.679999828338623)
Descriptors: MW 512.52, ALogP 4.04, HBA 6, HBD 4, PSA 168.64, QED 0.11
Compound (SMILES): C[C@H](NC(=O)C1CC1)c1ccc(CN2Cc3ccc(OCC4CC4(F)F)cc3C(O)C2)cc1
InChIKey: LEVAWBGDUZEKER-FERLNZQHSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.Each assay microtiter plate contains wells with vehicle instead of compound as controls for the non-inhibited enzyme (100% CTL; HIGH) and wells without acetyl-CoA as controls for non-specific NADH degradation (0% CTL; LOW).
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 456.53, ALogP 4.36, HBA 4, HBD 2, PSA 61.8, QED 0.62
Compound (SMILES): NS(=O)(=O)c1ccc(SCc2ccccc2-c2ccccc2)s1
InChIKey: HUPWCJRQUJXEQL-UHFFFAOYSA-N
Target: Carbonic anhydrase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P00918
Assay: Inhibition of human recombinant cytosolic carbonic anhydrase 2 preincubated for 10 mins prior to testing by stopped-flow CO2 hydration assay
Measurement: Ki = 280 nM (pChEMBL 6.550000190734863)
Descriptors: MW 361.51, ALogP 4.35, HBA 4, HBD 1, PSA 60.16, QED 0.69