lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Fc1ccccc1-c1n[nH]c2cnc(-c3cnccc3OCC3CCCNC3)cc12
InChIKey: FCRIHMREFBZVTE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-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.
Measurement: Ki = 1.52 nM (pChEMBL 8.819999694824219)
Descriptors: MW 403.46, ALogP 4.2, HBA 5, HBD 2, PSA 75.72, QED 0.52
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#N)c1nn2c(C#N)ccc2c(=O)n1-c1ccccc1
InChIKey: IKFBISWCWDKEJI-LBPRGKRZSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Inhibition of PI3Kdelta in human THP1 cells assessed as reduction in MCSF-stimulated akt phosphorylation at Thr308 preincubated for 30 mins followed by MCSF stimulation for 3 mins by ELISA
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 397.4, ALogP 1.77, HBA 10, HBD 2, PSA 150.71, QED 0.53
Compound (SMILES): CS(=O)(=O)Nc1nc(-c2cccc3[nH]ccc23)nc2c1OCC1COCCN21
InChIKey: XZCOOLQYMDVNQA-UHFFFAOYSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: ADP Hunter Plus Assay: The kinase activity was measured by using the commercial ADP Hunter Plus assay available from DiscoveRx (#90-0083), which is a homogeneous assay to measure the accumulation of ADP, a universal product of kinase activity. The enzyme, DNA-PK was purchased from Promega (#V5811). The assay was done following the manufacturer recommendations with slight modifications: Mainly the kinase buffer was replace by 15 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM NaCl, 1 mM EGTA, 0.1 mg/mL BGG, 0.02% Tween 20. The DNA-PK was assayed in a titration experiment to determine the optimal protein concentration for the inhibition assay. To calculate the 1050 of the Compounds, serial 1:3 dilutions of the compounds were added to the enzyme at a fixed concentration (2 U/uL). The enzyme was preincubated with the inhibitor and 200 uM DNA substrate and then ATP was added to a final 75 uM concentration. Reaction was carried out for 1 hour at 37° C. Reagent A and B were sequentially added to the wells and plates were incubated for 30 min at RT. Fluorescence counts were read in a EnVision instrument (Perkin Elmer) with the recommended settings (550 and 590 nm as excitation and emission wavelengths, respectively). Values were normalized against the control activity included for each enzyme (i.e., 100% DNA_PK kinase activity, without compound). These values were plotted against the inhibitor concentration and were fit to a sigmoid dose-response curve by using the model sigmoidal Four-Parameter Logistc implement for Activity base - software.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 401.45, ALogP 1.59, HBA 7, HBD 2, PSA 109.44, QED 0.69
Compound (SMILES): COc1ccccc1-n1nnc2c1CCN(C(=O)c1cccc(C(F)(F)F)c1Cl)C2
InChIKey: AJKNIOFHRYQCIV-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 158.5 nM (pChEMBL 6.800000190734863)
Descriptors: MW 436.82, ALogP 4.15, HBA 5, HBD 0, PSA 60.25, QED 0.62
Compound (SMILES): CC1(C)Cc2c(-c3ccc(C(F)(F)F)c(F)c3)cncc2C1O
InChIKey: NSJPFIVMVWXUKY-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Cellular Enzyme Assay: G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 ug/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 791.5 nM (pChEMBL 6.099999904632568)
Descriptors: MW 325.3, ALogP 4.52, HBA 2, HBD 1, PSA 33.12, QED 0.78
Compound (SMILES): Clc1ccc2c(c1)CCC1CCNCCCN21
InChIKey: QGARNHVKTVUAKJ-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2B (SINGLE PROTEIN), Homo sapiens
UniProt: P41595
Assay: Agonist activity at 5-HT2B receptor (unknown origin) expressed in HEK293 cells assessed as [3H]inositol phosphate accumulation after 2 hrs by scintillation counting
Measurement: EC50 = 630.96 nM (pChEMBL 6.199999809265137)
Descriptors: MW 250.77, ALogP 2.84, HBA 2, HBD 1, PSA 15.27, QED 0.76
Compound (SMILES): Cn1ccc2ccc(N(C(=O)c3cc(-c4cc5c(cc4C(=O)N4Cc6ccccc6C[C@H]4CN4CCOCC4)OCO5)n4c3CCCCC4)c3ccc(O)cc3)cc21
InChIKey: MOXKNROBKXVESU-BHVANESWSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 777.92, ALogP 7.69, HBA 9, HBD 1, PSA 101.64, QED 0.18
Compound (SMILES): N=C(N)NCCC[C@@H]1NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC(=O)[C@@H](NC(=O)CC2CCCCC2)CCC(=O)NCC[C@@H](C(=O)N[C@@H](Cc2c[nH]c3ccccc23)C(N)=O)NC1=O
InChIKey: SAQQSSWKBKHBIG-NGXVWMITSA-N
Target: Melanocyte-stimulating hormone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q01726
Assay: Competitive Inhibition Binding Assay: A competitive inhibition binding assay was performed using membrane homogenates prepared from HEK-293 cells that express recombinant hMCR-1a or hMCR-4 (in each instance where the h prefix refers to human), or alternatively membrane homogenates from B16-F10 mouse melanoma cells containing endogenous murine MCR-1. In the examples that follow, all MCR-1 and MCR-4 values are for human recombinant receptors, unless otherwise noted. Assays were performed in 96 well polypropylene round-bottom plates (VWR catalog number 12777-030). Membrane homogenates were incubated with 0.1 nM [I125]-NDP-α-MSH (Perkin Elmer) and increasing concentrations of test peptides of the present invention in buffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.3 mM 1,10-phenanthroline, and 0.2% bovine serum albumin. After incubation for 90 minutes at 37° C., the assay mixture was filtered onto GF/B Unifilter plates (Perkin-Elmer catalog number 6005177) and washed with 3 mL of ice-cold buffer per well. Filters were air dried and 35 uL of scintillation cocktail added to each well. Plates were counted in a Microbeta counter for bound radioactivity. Non-specific binding was measured by inhibition of binding of [I125]-NDP-α-MSH in the presence of 1 uM NDP-α-MSH. Maximal specific binding (100%) was defined as the difference in radioactivity (cpm) bound to cell membranes in the absence and presence of 1 uM NDP-α-MSH. Each assay was conducted in duplicate and the actual mean values are described, with results less than 0% reported as 0%. Ki values for peptides of the present invention were determined using Graph-Pad Prism curve-fitting software.
Measurement: Ki = 0.01 nM (pChEMBL 11)
Descriptors: MW 979.16, ALogP -0.15, HBA 10, HBD 13, PSA 353.16, QED 0.04
Compound (SMILES): Nc1noc2ccc(-c3nn(CCN4CCOCC4)c4ncnc(N)c34)cc12
InChIKey: FYKMSJVXJVVFHQ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition of human PI3Kalpha using PIP2 as substrate by HTRF assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 380.41, ALogP 1.13, HBA 10, HBD 2, PSA 134.14, QED 0.53
Compound (SMILES): CC(=O)Nc1ccccc1OCC(=O)NCC(O)CN1CCc2ccccc2C1
InChIKey: HHAUCTVMLLZAHO-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 = 101 nM (pChEMBL 7)
Descriptors: MW 397.48, ALogP 1.56, HBA 5, HBD 3, PSA 90.9, QED 0.63
Compound (SMILES): CCOc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(Cl)c3)cn2CCN2CCCC2)CC1
InChIKey: IDVXLGVNCNGJLE-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is 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 are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l 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 .mu.M 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 is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is 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.
Measurement: IC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 514.05, ALogP 4.59, HBA 8, HBD 1, PSA 85.33, QED 0.47
Compound (SMILES): COc1ccn2c(-c3ccnc(Nc4cccc(OCCN(C)C)c4)n3)c(-c3ccc(OC)c(C(=O)Nc4c(F)cccc4F)c3)nc2c1
InChIKey: JHAJPTOYCXHOLN-UHFFFAOYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of human GST-rTEV-tagged insulin receptor (979 to 1382 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 665.7, ALogP 6.69, HBA 10, HBD 2, PSA 115.14, QED 0.15
Compound (SMILES): O=C(c1cccc(Cl)c1Cl)N1CCc2c(nnn2-c2ccccc2)C1
InChIKey: DISDTFONGVKWEU-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 76.2 nM (pChEMBL 7.119999885559082)
Descriptors: MW 373.24, ALogP 3.77, HBA 4, HBD 0, PSA 51.02, QED 0.69
Compound (SMILES): Cc1cc(Nc2nccc(C)n2)cc(-c2cnc(C(C)(O)C3CCC(C(=O)O)CC3)s2)c1
InChIKey: QEPRCONRQVQCKN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSyk fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-Syk (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45′ at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 452.58, ALogP 5.06, HBA 7, HBD 3, PSA 108.23, QED 0.48
Compound (SMILES): CC(=O)Nc1ccc(Oc2ccccc2)c(-c2cn(C)c(=O)c3[nH]ccc23)c1
InChIKey: LSWNHXHXFDIKEX-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 5.8 nM (pChEMBL 8.239999771118164)
Descriptors: MW 373.41, ALogP 4.28, HBA 4, HBD 2, PSA 76.12, QED 0.56
Compound (SMILES): Cc1c(C(=O)Nc2c(C)n(C)n(C3CCCCC3)c2=O)noc1[C@H]1C[C@H](C(C)C)CO1
InChIKey: FORDFNXCOFGSRG-FUHWJXTLSA-N
Target: E3 ubiquitin-protein ligase SMURF1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HCE7
Assay: Pharmaceutical Assay: To determine the HECT E3 ligase selectivity of the compounds, a panel of biochemical HECT E3 ligase autoubiquitinylation assays was employed (Smurf-1, Smurf-2, WWP1, WWP2, ITCH, Nedd4, Nedd4L and E6AP). The conjugation of ubiquitin to a protein substrate is a multistep process. In an initial ATP-requiring step, a thioester bond is formed between the carboxyl terminus of ubiquitin and an internal cystein residue of the ubiquitin-activating enzyme (E1). Activated ubiquitin is then transferred to a specific cystein residue of an ubiquitin-conjugating enzyme (E2). E2s donate ubiquitin to a HECT E3 ligase (E3) from which it is transferred to the substrate protein. HECT E3 ligases can auto-ubiquitinylate. This event is detected in the TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) assay used in this panel. The reaction mix contains E1, E2, tagged-E3, biotin-conjugated ubiquitin, the compound and ATP in a suitable buffer and is incubated for 45 minutes to allow auto-ubiquitinylation of the E3 ligase. To measure the extent of ubiquitinylated E3 ligase by TR-FRET, the donor fluorophore Europium cryptate (Eu3+ cryptate), conjugated to streptavidin which subsequently binds to biotinylated ubiquitin, and the modified allophycocyanin XL665 (HTRF primary acceptor fluorophore) coupled to a tag-specific antibody (HA, His or GST), which recognizes the respective E3 ligase fusion proteins, are added after the reaction is complete. When these two fluorophores are brought together by a biomolecular interaction (in this case ubiquitinylation of the E3 ligase), a portion of the energy captured by the Cryptate during excitation is released through fluorescence emission at 620 nm, while the remaining energy is transferred to XL665. This energy is then released by XL665 as specific fluorescence at 665 nm. Light at 665 nm is emitted only through FRET with Europium. Because Europium Cryptate is present in the assay, light at 620 nm is detected even when the biomolecular interaction does not bring XL665 within close proximity.
Measurement: IC50 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 430.55, ALogP 4.28, HBA 7, HBD 1, PSA 91.29, QED 0.76
Compound (SMILES): O=C(Nc1ccccc1O)c1ccccc1[Se][Se]c1ccccc1C(=O)Nc1ccccc1O
InChIKey: KWHUYBHCJSYTIT-UHFFFAOYSA-N
Target: Replicase polyprotein 1ab (SINGLE PROTEIN), Severe acute respiratory syndrome coronavirus 2
UniProt: P0DTD1
Assay: Inhibition of recombinant SARS CoV-2 PL protease incubated for 60 mins by fluorescence based analysis
Measurement: IC50 = 34000 nM (pChEMBL 4.46999979019165)
Descriptors: MW 582.38
Compound (SMILES): CN1CCC[C@H]1COc1cc(O)cc(-c2ccccc2)c1
InChIKey: RIJDXMHWSXMKFE-INIZCTEOSA-N
Target: Neuronal acetylcholine receptor; alpha4/beta2 (PROTEIN COMPLEX), Rattus norvegicus
UniProt: P09483
Assay: Displacement of [3H]-epibatidine from alpha4beta2-nACHR in rat cerebral cortex membranes preincubated for 30 mins followed by overnight incubation with [3H]-epibatine
Measurement: Ki = 528 nM (pChEMBL 6.28000020980835)
Descriptors: MW 283.37, ALogP 3.53, HBA 3, HBD 1, PSA 32.7, QED 0.93
Compound (SMILES): COC(=O)c1cccc(CNC(=O)c2ccc(-c3cn[nH]c3)c(OC)c2)c1
InChIKey: OMQBPULZTUQWCQ-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
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.
Measurement: IC50 = 0.85 nM (pChEMBL 9.069999694824219)
Descriptors: MW 365.39, ALogP 2.8, HBA 5, HBD 2, PSA 93.31, QED 0.66
Compound (SMILES): O=C1COc2ccc(C3(O)c4ccccc4C(=O)N3c3cccc(F)c3)cc2N1
InChIKey: FHABBMMGGOBOLS-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 390.37, ALogP 3.01, HBA 4, HBD 2, PSA 78.87, QED 0.71
Compound (SMILES): Cc1cc2cnc(Nc3ccc(N4CCNCC4)cn3)nc2n(C2CCCC2)c1=O.Cl
InChIKey: UQKVGYXAXJDYHU-UHFFFAOYSA-N
Target: CDK2/Cyclin A2 (PROTEIN COMPLEX), Homo sapiens
UniProt: P20248
Assay: Inhibition of Cdk2/Cyclin A (unknown origin) using retinoblastoma protein substrate and [gamma32P]ATP incubated for 15 mins by beta plate counter based assay
Measurement: IC50 = 4050 nM (pChEMBL 5.389999866485596)
Descriptors: MW 405.51, ALogP 2.76, HBA 8, HBD 2, PSA 87.97, QED 0.69
Compound (SMILES): CN1C(=N)N[C@](C)(c2cc(-c3cncc(-c4cnco4)c3)cs2)CS1(=O)=O
InChIKey: YNYYSRGLRMOFMG-KRWDZBQOSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: HTRF FRET Assay: Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1×BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1×BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 μs delay on a RUBYstar HTRF plate reader (BMG Labtechnologies).
Measurement: Ki = 98 nM (pChEMBL 7.010000228881836)
Descriptors: MW 403.49, ALogP 2.48, HBA 7, HBD 2, PSA 112.18, QED 0.69
Compound (SMILES): Cn1cc(-c2cc(NS(=O)(=O)CCF)ccc2Oc2ccc(F)cc2F)c2cc[nH]c2c1=O
InChIKey: SHTZSDMHMQUVAA-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 63.3 nM (pChEMBL 7.199999809265137)
Descriptors: MW 477.46, ALogP 4.32, HBA 5, HBD 2, PSA 93.19, QED 0.41
Compound (SMILES): CCOC(=O)c1cc(-c2ccccc2)sc1NS(C)(=O)=O
InChIKey: RRWTYWVQADTJNO-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Displacement of [3H]-CP-55940 from human CB2 receptor expressed in HEK293 cell membranes after 90 mins by radioligand binding assay
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 325.41, ALogP 2.96, HBA 5, HBD 1, PSA 72.47, QED 0.86
Compound (SMILES): Cn1cc(-c2ccc3nc(NC(=O)c4cccc(C(F)(F)F)n4)cn3n2)c(-c2ccc(F)cc2)n1
InChIKey: JYSMHVFTOIBTHO-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 24.7 nM (pChEMBL 7.610000133514404)
Descriptors: MW 481.41, ALogP 4.6, HBA 7, HBD 1, PSA 90, QED 0.38
Compound (SMILES): C[C@H]1[C@H]2Cc3ccc(C(=O)NCCc4ccc(-c5cn[nH]c5)cc4)cc3[C@@]1(C)CCN2CC1CC1
InChIKey: WXKYXZOJGYCMRZ-RFASMWRLSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: null: The Ki (binding affinity) for u opioid receptors was determined using a competitive displacement assay as previously described in Neumeyer (Journal of Med. Chem. 2012, p 3878), which is incorporated herein in its entirety. Briefly, membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed the cloned human u opioid receptor were incubated with 12 different concentrations of the compound set forth herein in the presence of 0.25 nM [3H]DAMGO (see Tiberi et al., Can. J. Physiol. Pharmacol. 1988, Vol. 66, p 1368, which is incorporated by reference herein in its entirety) in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO (see Gulati et al., Life Sci. 1990, Vol. 47, p 159, which is incorporated by reference herein in its entirety). Nonspecific binding was measured by inclusion of 10 uM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 468.65, ALogP 4.98, HBA 3, HBD 2, PSA 61.02, QED 0.51
Compound (SMILES): Cc1c([C@H]2CN3CCN(C(=O)Cc4ccc(-n5cnnn5)nc4)C[C@@H]3CO2)ccc2c1COC2=O
InChIKey: YPUSXXSDSCDEJP-DYESRHJHSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Displacement of 35S-MK499 from human ERG expressed in HEK293 cell membranes
Measurement: IC50 = 53400 nM (pChEMBL 4.269999980926514)
Descriptors: MW 475.51, ALogP 0.86, HBA 10, HBD 0, PSA 115.57, QED 0.51
Compound (SMILES): O=C(c1c(F)ccc(NS(=O)(=O)c2c(F)cccc2F)c1F)c1c[nH]c2nccc(Cl)c12
InChIKey: LZXVWIKLBIDGKX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf V600E mutant (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 483.83, ALogP 4.8, HBA 4, HBD 2, PSA 91.92, QED 0.31
Compound (SMILES): N#Cc1nc(C(=O)NCCCCC(=O)O)c(O)c2cc(Oc3ccccc3)ccc12
InChIKey: PLDDEHBMYMKSPF-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 65150 nM (pChEMBL 4.190000057220459)
Descriptors: MW 405.41, ALogP 3.59, HBA 6, HBD 3, PSA 132.54, QED 0.49
Compound (SMILES): N#Cc1ccc2ccc(Oc3ccc(F)cc3OCCn3ccc(=O)[nH]c3=O)cc2c1
InChIKey: ZPHDCAUTJFTFSV-UHFFFAOYSA-N
Target: Human immunodeficiency virus type 1 reverse transcriptase (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: Q72547
Assay: Inhibition of HIV1 reverse transcriptase K103N/Y181C double mutant infected in human MT2 cells assessed as protection against viral infection by MTT assay
Measurement: EC50 = 9400 nM (pChEMBL 5.03000020980835)
Descriptors: MW 417.4, ALogP 3.57, HBA 6, HBD 1, PSA 97.11, QED 0.52
Compound (SMILES): CC(C)(c1nc(-c2ccnc3[nH]ccc23)nc2c1OCC1COCCN21)S(C)(=O)=O
InChIKey: NBTONHDHNXEIPE-UHFFFAOYSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: ADP Hunter Plus Assay: The kinase activity was measured by using the commercial ADP Hunter Plus assay available from DiscoveRx (#90-0083), which is a homogeneous assay to measure the accumulation of ADP, a universal product of kinase activity. The enzyme, DNA-PK was purchased from Promega (#V5811). The assay was done following the manufacturer recommendations with slight modifications: Mainly the kinase buffer was replace by 15 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM NaCl, 1 mM EGTA, 0.1 mg/mL BGG, 0.02% Tween 20. The DNA-PK was assayed in a titration experiment to determine the optimal protein concentration for the inhibition assay. To calculate the 1050 of the Compounds, serial 1:3 dilutions of the compounds were added to the enzyme at a fixed concentration (2 U/uL). The enzyme was preincubated with the inhibitor and 200 uM DNA substrate and then ATP was added to a final 75 uM concentration. Reaction was carried out for 1 hour at 37° C. Reagent A and B were sequentially added to the wells and plates were incubated for 30 min at RT. Fluorescence counts were read in a EnVision instrument (Perkin Elmer) with the recommended settings (550 and 590 nm as excitation and emission wavelengths, respectively). Values were normalized against the control activity included for each enzyme (i.e., 100% DNA_PK kinase activity, without compound). These values were plotted against the inhibitor concentration and were fit to a sigmoid dose-response curve by using the model sigmoidal Four-Parameter Logistc implement for Activity base - software.
Measurement: IC50 = 2820 nM (pChEMBL 5.550000190734863)
Descriptors: MW 429.5, ALogP 1.9, HBA 8, HBD 1, PSA 110.3, QED 0.67
Compound (SMILES): CN(CCO)c1cc(Nc2nccc(-c3cnc(N4CC[C@H](F)C4)c(C#N)c3)n2)ccn1
InChIKey: DTVMGWZBDSGYJR-KRWDZBQOSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 434.48, ALogP 2.53, HBA 9, HBD 2, PSA 114.09, QED 0.58
Compound (SMILES): NCCC1CNC(=O)c2cc(-c3cccc(C(F)(F)F)c3)c(-c3ccc(O)cc3)n21
InChIKey: PMKXIEFCLZIQLZ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Trans-Phosphorylation Assay: The inhibitory activity of putative kinase inhibitors and the potency of selected compounds were determined using a trans-phosphorylation assay.Specific peptide or protein substrates were trans-phosphorylated by their specific ser-thr or tyr kinase in the presence of ATP traced with 33P-gamma-ATP, and in the presence of their own optimal buffer and cofactors. At the end of the phosphorylation reaction, more than 98% unlabeled ATP and radioactive ATP were captured by an excess of the ion exchange dowex resin; the resin then settled down to the bottom of the reaction plate by gravity. Supernatant was subsequently withdrawn and transferred into a counting plate, then evaluated by beta-counting. Kinase Buffer (KB):The buffer for PIM2 assay was composed of HEPES 50 mM, at pH 7.5, with 1 mM MgCl2, 1 mM DTT, 3 uM Na3VO4, and 0.2 mg/mL BSA.Â  Full-length human PIM2 was expressed and purified as described in Fedorov O, et al., PNAS 2007 104, 51, 20523-28.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 415.42, ALogP 4.18, HBA 4, HBD 3, PSA 80.28, QED 0.6
Compound (SMILES): O=c1ccc2c(OCCCOc3cccc(C(F)(F)F)c3)c3ccoc3cc2o1
InChIKey: BRUCNEJBTJLSAA-UHFFFAOYSA-N
Target: Potassium voltage-gated channel subfamily A member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P22460
Assay: Inhibition of Kv1.5 (unknown origin)
Measurement: EC50 = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 404.34, ALogP 5.41, HBA 5, HBD 0, PSA 61.81, QED 0.32
Compound (SMILES): CC(C)CC(CC(=O)O)NC(=O)c1nc(C#N)c2cc(Oc3ccccc3)ccc2c1O
InChIKey: ZCLCWANYQTWHIW-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 53520 nM (pChEMBL 4.269999980926514)
Descriptors: MW 433.46, ALogP 4.22, HBA 6, HBD 3, PSA 132.54, QED 0.48
Compound (SMILES): Cc1c(-c2ncnc3[nH]c(-c4ccc(C(=O)N5CCCCC5)cc4)cc23)cccc1N1CCOc2cc(C3CC3)ccc2C1=O
InChIKey: VQLMDGYDOIQWNF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Recombinant Human BTK Inhibition Assay: The inhibition assays were performed using the generic tyrosine kinase assay kit from C is Bio (HTRF KinEASE-TK #62TK0PEB). The assay is based on the principle of fluorescence resonance energy transfer (FRET) between the Europium Cryptate labeled anti-phosphotyrosine antibody and streptavidin labeled fluorophore, XL-665. In an uninhibited kinase reaction the biotinylated peptide substrate (Tk-Biotin) gets phosphorylated at an intermediate tyrosine site which is captured by anti-phosphotyrosine antibody which eventually acts as a FRET donor. The streptavidin labeled XL-665 binds strongly to the Tk-Biotin substrate and act as a FRET acceptor. The resultant FRET signal is measured at 665 nm. A ratio of this specific fluorescence (665 nm) to the background fluorescence (620 nm), conferred by the free antibody, is directly proportional to the kinase activity. Inhibition of the kinase activity by a pharmacological intervention such as a Btk inhibitor of the present disclosure will result into decreased FRET signal.
Measurement: IC50 = 133 nM (pChEMBL 6.880000114440918)
Descriptors: MW 597.72, ALogP 7.14, HBA 5, HBD 1, PSA 91.42, QED 0.23
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OC[C@@H]1CN2CCC[C@H]2CO1
InChIKey: FSJDACURTSWGHC-HOTGVXAUSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: Inhibition of recombinant human N-terminal His6-tagged EphA2 (N598 to R890 residues) expressed in baculovirus using poly-EY as substrate after 180 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 475.38, ALogP 4.93, HBA 7, HBD 1, PSA 68.74, QED 0.54
Compound (SMILES): CC1Cc2c(nnn2-c2ncc(F)cn2)CN1C(=O)c1cccc(Cl)c1Cl
InChIKey: CYNWLFOLQKOWDD-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 10.5 nM (pChEMBL 7.980000019073486)
Descriptors: MW 407.24, ALogP 3.09, HBA 6, HBD 0, PSA 76.8, QED 0.65
Compound (SMILES): O=C1/C(=C/c2ccc(Cl)c(Cl)c2)Cc2cc(O)ccc21
InChIKey: KAKGHBSBMFOMRF-VZUCSPMQSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Inhibition of recombinant human MAO-B using kynuramine as substrate after 20 mins by fluorescence spectrophotometric analysis
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 305.16, ALogP 4.52, HBA 2, HBD 1, PSA 37.3, QED 0.79
Compound (SMILES): CO[C@@H](C)C(=O)Nc1ccc(F)c([C@]23COC[C@H]2S(=O)(=O)C(C)(C)C(=N)N3)c1
InChIKey: HDZXDROMEIJJHI-DGVXXNCNSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1×BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1×BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% fin
Measurement: Ki = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 413.47, ALogP 1.17, HBA 6, HBD 3, PSA 117.58, QED 0.68
Compound (SMILES): C[C@H](c1nc2c(cnn2C2CCOCC2)c(=O)[nH]1)N1CC(Oc2ccc(F)cc2)C1
InChIKey: XYRJHICAYZEKEV-CYBMUJFWSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition of human recombinant PDE9 expressed in insect Sf9 cells using [3H]-cGMP as substrate after 30 mins by scintillation proximity assay
Measurement: IC50 = 10.7 nM (pChEMBL 7.96999979019165)
Descriptors: MW 413.45, ALogP 2.43, HBA 7, HBD 1, PSA 85.27, QED 0.69
Compound (SMILES): CC(=O)C(CCC(=O)NCCOCCOCCNS(=O)(=O)c1cccc([C@@H]2CN(C)Cc3c(Cl)cc(Cl)cc32)c1)(CCC(=O)NCCOCCOCCNS(=O)(=O)c1cccc([C@@H]2CN(C)Cc3c(Cl)cc(Cl)cc32)c1)CCC(=O)NCCOCCOCCNS(=O)(=O)c1cccc([C@@H]2CN(C)Cc3c(Cl)cc(Cl)cc32)c1
InChIKey: PCLFRHOSFRIFIN-NLXGDKCYSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 630.96 nM (pChEMBL 6.199999809265137)
Descriptors: MW 1727.62
Compound (SMILES): Cc1c(C(=O)Nc2cccc(C(F)(F)F)c2)cc(-c2ccccc2)n1CCCN1CCCCC1
InChIKey: FDXOSOUYTCWDST-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 3: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) Frozen cells were thawed and subcultured with the medium (MEM-α, 10% FBS, 200 mmol/L Glutamine, 50 unit/mL penicillin, 50 μg/mL streptomycin, 1 mg/mL G418). (3) The 96-well plates which hTRPV4/CHO cells were seeded at densities of 2×10^4 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for 24 h was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 1 mol/L HEPES, 250 mmol/L probenecid, pH7.4). (5) Fluo-3, fluorescent dye for Ca influx assay was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour (final conc.; 5 μmmol/L Fluo-3). (6) The assay plate was washed with the assay buffer, and the buffer was remained at 30 μL/well. Then the assay plate was incubated for 10 min at 37° C. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the fluorescence analysis system FDSS 3000 (Hamamatsu Photonics). (8) 50 μL of 4α-PDD solution (concluding 0.1% Pluronic F-127) was applied to each well of assay plate and mixed. (9) The fluorescent intensity was measured by FDSS 3000 for 8 min from the point of time addition the compound solution, at Ex 480 nm, Em 540 nm wavelength.
Measurement: Ki = 327 nM (pChEMBL 6.489999771118164)
Descriptors: MW 469.55, ALogP 6.61, HBA 3, HBD 1, PSA 37.27, QED 0.42
Compound (SMILES): Cn1cc2c(nc(NC(=O)Nc3ccc(CC(=O)NCCCCCN)cc3)n3nc(-c4ccco4)nc23)n1
InChIKey: BKRMHMBQECVLAH-UHFFFAOYSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
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 = 2.66 nM (pChEMBL 8.569999694824219)
Descriptors: MW 516.57, ALogP 2.7, HBA 10, HBD 4, PSA 170.29, QED 0.21