lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(Nc1nc2ccccc2s1)c1cccc2c1CN(c1ccc(-c3ccc4c(c3)CCN(CCC3CCCCC3)C4)c(C(=O)O)n1)CC2
InChIKey: KXXFAOMISJDSGF-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 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 671.87, ALogP 8.2, HBA 7, HBD 2, PSA 98.66, QED 0.17
Compound (SMILES): Cc1cnc(Nc2cnn(C3CCN(C)CC3)c2)nc1CCc1ccccc1C1(C(N)=O)CC1
InChIKey: HSBIEJWYEOZFNZ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P35916
Assay: VEGFR3 Biochemical Assay: A biotin labelled peptide is used as substrate (amino acid sequence: Biotin-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2). VEGFR3 cytoplasmic domain (amino acids 798-1298) was purchased as N-terminal GST-fusion protein (the enzyme). The 15 uL assay reactions are run in Greiner brand white 384-well low volume plates. All reactions contained 10 mM HEPES pH 7.4, mM MgCl2, 0.01% (v/v) Tween-20, 50 uM Na3VO4, 0.01% (w/v) albumin from chicken egg white, 1 mM Dithiothreitol, 111 nM peptide substrate, 500 uM ATP, and 3.8 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 90 minutes at 30 degree Celsius.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 459.6, ALogP 3.29, HBA 7, HBD 2, PSA 101.96, QED 0.54
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: ACHQYPZSYFFLFI-SVOOYOIKSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 2445.81
Compound (SMILES): Cn1c(Nc2c(F)cccc2Cl)nc2cc(C(=O)Nc3ccc(C#CC4CC4)c(F)c3)c3c(c21)OCCO3
InChIKey: NDHWTYVEETWKDT-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition of human recombinant mPGES-1 expressed in CHO cells assessed as reduction in conversion of PGH2 to PGE2 incubated for 10 mins followed by substrate addition measured after 1 min in presence of GSH
Measurement: IC50 = 4.19 nM (pChEMBL 8.380000114440918)
Descriptors: MW 534.95, ALogP 6.03, HBA 6, HBD 2, PSA 77.41, QED 0.31
Compound (SMILES): Cc1c(COc2ccccc2)nn2c1CN(C(=O)c1ccc(F)cc1)CC2
InChIKey: HOFPALVKNDSITL-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Cell-Based Functional Assay: HEK cells transfected with the human mGluR5a receptor (H10H cell line) were plated at 15,000 cells/well in clear-bottomed poly-D-lysine-coated assay plates (BD Falcon) in glutamate-glutamine-free growth medium and incubated overnight at 37° C. and 5% CO2. The following day, the growth medium was removed and the cells were washed with assay buffer containing 1× Hank's balanced salt solution (Invitrogen, Carlsbad, Calif.), 20 mM HEPES, 2.5 mM probenecid, pH 7.4 and left with 20 μL of this reagent. Following this step, the cells were loaded with calcium indicator dye, fluo-4 AM, to a final concentration of 2 μM and incubated for 40-45 min at 37° C. The dye solution was removed and replaced with assay buffer.
Measurement: EC50 = 3020 nM (pChEMBL 5.519999980926514)
Descriptors: MW 365.41, ALogP 3.57, HBA 4, HBD 0, PSA 47.36, QED 0.71
Compound (SMILES): O=c1[nH]c(CN2CC(c3ncccn3)C2)nn2c(C3CCOCC3)ncc12
InChIKey: XEFLPTQXULEBHZ-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition Assay: A PDE9 assay may for example, be performed as follows: The assay is performed in 60 uL samples containing a fixed amount of the relevant PDE enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 225 pCi of 3H-labelled cyclic nucleotide substrate, tritium labeled cAMP to a final concentration of 5 nM and varying amounts of inhibitors. Reactions are initiated by addition of the cyclic nucleotide substrate, and reactions are allowed to proceed for one hr at room temperature before being terminated through mixing with 15 uL 8 mg/mL yttrium silicate SPA beads (Amersham). The beads are allowed to settle for one hr in the dark before the plates are counted in a Wallac 1450 Microbeta counter.
Measurement: IC50 = 3400 nM (pChEMBL 5.46999979019165)
Descriptors: MW 367.41, ALogP 0.7, HBA 8, HBD 1, PSA 101.3, QED 0.72
Compound (SMILES): CC(C)Oc1ccc(/N=c2\[nH]c(=O)n(Cc3cccc(C(=O)O)c3)c(=O)n2Cc2ccc(Cl)cc2)cc1F
InChIKey: KRVHHMRULNJILW-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683)) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37 C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 uM Fluo-3-AM solution (pH 7.5) containing 20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 uL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.).
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 538.96, ALogP 3.95, HBA 7, HBD 2, PSA 118.68, QED 0.35
Compound (SMILES): CCCS(=O)(=O)Nc1cc(Cl)cc(-c2nn(C(C)C)cc2-c2ccnc(NC[C@H](C)NC(=O)OC)n2)c1F
InChIKey: IFUNYGIIWVIYAY-HNNXBMFYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf V600E mutant in human A375 cells harboring B-Raf V600E mutant assessed as reduction in cell proliferation incubated for 2 days by Bright-Glo luminescence assay
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 568.08, ALogP 4.69, HBA 9, HBD 3, PSA 140.13, QED 0.3
Compound (SMILES): COc1ccc(F)c(C(=O)NCC(c2cnc(C(F)(F)F)nc2)N2CCC(F)(F)CC2)c1Cl
InChIKey: HYVZDKYFJYCWRO-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO42H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates. 93-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1X); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes. Two sets of drug plates were prepared: A) Mixtures of compound plus agonist were prepared as follows, in order to determine close response: BzATP: 11 point ½ log, diluted in buffer, starting from 1 mM. Testing compounds: 11 point ½ log, diluted in 2% DMSO buffer starting from 10 μM. B) Agonist only mixture was prepared with BzATP at a single concentration in buffer (concentration determined by dose response). Compound mixtures (A) were added to assay plates containing cells and placed at room temperature for 30 minutes, then BzATP (B) was added. Fluorescence was read using the Tetra FLIPR® (Molecular Devices, Inc., Sunnyvale, Calif., USA) and IC50 values were calculated by standard methods to determine antagonist activity.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 496.84, ALogP 4.5, HBA 5, HBD 1, PSA 67.35, QED 0.6
Compound (SMILES): COc1cc(Nc2ccc(S(=O)(=O)NCC3CCCO3)cc2F)cc(OC)c1
InChIKey: HOOKOEHLECUOBR-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Binding Assay: The binding assays were performed by testing the ability of the new compounds to displace the radiolabeled synthetic non-selective cannabinoid agonist [3H]CP55940 (168 Ci/mmol; PerkinElmer) from the human CB1 (hCB1) or human CB2 (hCB2) receptors on membranes derived from stably transfected HEK-293 cells (PerkinElmer). Membranes were diluted in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, pH=7.4). The amount of membrane was determined for each batch of membranes according to protein binding assay. The minimum amount of membrane that gave 50% specific binding was used for the binding assay. In most assays, binding was tested using 8 μg and 4 μg protein of hCB1 and hCB2 membranes, respectively. The tested compounds were dissolved in DMSO and diluted in assay buffer to a final concentration of 0.1% solvent. Total binding of [3H]CP55940 was evaluated with 1.5 nM to hCB1 and with 0.5 nM to hCB2, according to Kd affinity of [3H]CP55940 for the respective membranes. The ability of the tested compounds to displace [3H]CP55940 was evaluated first at a single concentration point of 100 nM for binding toward hCB2 or hCB1. In certain cases, the displacement was tested at compound concentrations ranging from 0.03 nM to 10 μM. Non-specific binding was measured by the addition of 1 μM of unlabelled CP55940 to the tubes. Binding assays were performed in triplicate in a total volume of 500 μl for 60 minutes at 30° C., in a shaking bath. Free and bound radioligands were separated by rapid filtration through GF/C filter plates (PerkinElmer) that had been presoaked with 0.1% Polyethylenimine (Sigma). Filters were shaken for 1 hour in 7 ml scintillation fluid (PerkinElmer) and radioactivity was determined by liquid scintillation counter (Wallac; PerkinElmer).
Measurement: Ki = 320 nM (pChEMBL 6.5)
Descriptors: MW 410.47, ALogP 3.04, HBA 6, HBD 2, PSA 85.89, QED 0.7
Compound (SMILES): COc1n[nH]c2cc(NC(=O)N[C@@H](c3ccccc3)C(C)(C)O)nc(-c3cnco3)c12
InChIKey: JAWCXNDDBQMIPG-SFHVURJKSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 4.3 nM (pChEMBL 8.369999885559082)
Descriptors: MW 422.45, ALogP 3.26, HBA 7, HBD 4, PSA 138.19, QED 0.37
Compound (SMILES): CCOC(=O)C1=C(C)Nc2nc3c(c(N)c2C1c1cccc([N+](=O)[O-])c1)CC(C)(C)CC3
InChIKey: CJOKBSAEXRAEIR-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P22303
Assay: Inhibition Assay: Procedure: 48 Ml 1xReaction Buffer was initially added to each compound test well in the 96-well black plate arranged, followed by addition of 2 Ml 100xcompound solution; the test for each concentration was performed in duplicate. To the positive control well, 2 Ml DMSO+48 Ml 1xReaction Buffer were added; while 100 Ml 20 Mm H2O2 working solution was directly added to the positive validation well. To the negative control well, 2 Ml DMSO+98 Ml 1xReaction Buffer were added, and each test was performed in duplicate. 50 Ml 4xAchE application solution was added to each of the compound test wells and the positive control wells. 100 Ml 2xworking solution was added to each of all the wells and mixed until homogenous. The enzymatic reaction was started with the volume of the overall reaction system reaching 200 Ml, which resulted in the final concentrations of the compound solution of 10 umol/L, 2 umol/L, 0.4 umol/L, 0.08 umol/L, 0.016 umol/L, 0.0032 umol/L and 0.064 umol/L.
Measurement: IC50 = 5960 nM (pChEMBL 5.21999979019165)
Descriptors: MW 436.51, ALogP 4.48, HBA 7, HBD 2, PSA 120.38, QED 0.41
Compound (SMILES): CC1CCN(C(=O)C(CCn2ccnc2)NS(=O)(=O)c2cc(Cl)c(N)c(Cl)c2)CC1
InChIKey: TXCJGZQGRIXSLW-UHFFFAOYSA-N
Target: C-C chemokine receptor type 10 (SINGLE PROTEIN), Homo sapiens
UniProt: P46092
Assay: Antagonist activity at human CCR10 expressed in CHOK1 cells coexpressing aequorin/Galphaq assessed as inhibition of CCL27-dependent calcium flux in presence of coelenterazine H by FLIPR assay
Measurement: IC50 > 19952.62 nM (pChEMBL 4.699999809265137)
Descriptors: MW 474.41, ALogP 2.77, HBA 6, HBD 2, PSA 110.32, QED 0.6
Compound (SMILES): NC(=O)Cc1csc(NC(=O)N2CCC(N3CCc4ccc(F)cc43)CC2)n1
InChIKey: NKZYFQTWPBZWNP-UHFFFAOYSA-N
Target: Acyl-CoA desaturase 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P07308
Assay: SCD1 Enzymatic Assay: The SCD1 enzymatic assay was done in a volume of 50 uL using 10 ug of RLM (prepared as described above) in a 96-well polypropylene plate (enzyme reaction buffer contains 0.1 M K-Phosphate Buffer, 10 mM ATP, 6 mM MgCl2, 1 mM CoA, 1 mM β-NADH, 1.6 mM L-glutathione, 20 uM Stearoyl-CoA). Stearoyl-[9,10-3H]-CoA (ARC-0390, 1 mCi/mL, 60 Ci/mmol,) was added at a final concentration of 2 uCi/mL. Test compound was then added to the reaction mixture at the selected concentration. After incubation at room temperature for 2 hours, 5 uL 1 N HCl was added to stop the reaction, followed by addition of 25 uL of 10% charcoal. The reaction mixture was then transferred to 96-well Multiscreen plate (Millipore, Cat# MSFCN6B50). [3H2O] was collected into Opti-plate (PE, Cat #6005290) by centrifuge, at 1,000 rpm for 2 minutes. 150 uL Microscint 40 (PE, cat #6013641) was then added to each well and counted on Topcount for [3H] counts per minute (cpm).
Measurement: IC50 = 845 nM (pChEMBL 6.070000171661377)
Descriptors: MW 403.48, ALogP 2.37, HBA 5, HBD 2, PSA 91.56, QED 0.82
Compound (SMILES): NC(=O)C1CCCN(C(=O)c2cc(Cc3n[nH]c(=O)c4c3NCCC4)ccc2F)C1
InChIKey: JXOMBJIVTURHDR-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 413.45, ALogP 1.2, HBA 5, HBD 3, PSA 121.18, QED 0.7
Compound (SMILES): Cc1ncccc1NC(=O)N1CCC(N2CC(CC#N)(n3cc(-c4ncnc5[nH]ccc45)cn3)C2)CC1
InChIKey: PEUAIYBHOLIJBN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 496.58, ALogP 3.15, HBA 8, HBD 2, PSA 131.65, QED 0.43
Compound (SMILES): Cc1cc(Nc2nccc(C(F)(F)F)n2)cc(-c2cccc(NS(=O)(=O)c3cc4ccccc4s3)c2)c1
InChIKey: CLXSIZXMYDKYGA-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 μM 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 EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 4394 nM (pChEMBL 5.360000133514404)
Descriptors: MW 540.59, ALogP 7.23, HBA 6, HBD 2, PSA 83.98, QED 0.24
Compound (SMILES): CCC(=O)Nc1cc2c(Nc3ccc(NCc4ccccc4)cc3)ncnc2cc1OC
InChIKey: UQILHDPEYNQZHG-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Irreversible inhibition of GST-tagged ERBB1 (unknown origin) (Met-668 to Ala-1211 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 383 nM (pChEMBL 6.420000076293945)
Descriptors: MW 427.51, ALogP 5.34, HBA 6, HBD 3, PSA 88.17, QED 0.35
Compound (SMILES): COc1cc(C)c(N2CCOc3cc(S(=O)(=O)Nc4nccs4)ccc32)cn1
InChIKey: DJFLJUUTVYEHEZ-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 = 914 nM (pChEMBL 6.039999961853027)
Descriptors: MW 418.5, ALogP 3.19, HBA 8, HBD 1, PSA 93.65, QED 0.68
Compound (SMILES): COc1ccc2c(c1)CC1Cn3c-2c(C2CCCCC2)c2ccc(cc23)C(=O)NS(=O)(=O)N(C)CCOCCN(C)C1=O
InChIKey: JYIVMQPLCGSFGI-UHFFFAOYSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Inhibition Assay: Measurement of HCV NS5B polymerization activity was performed by evaluating the amount of radiolabeled GTP incorporated by the enzyme in a newly synthesized RNA using heteropolymeric RNA template/primer. The RdRp assay was carried out in 384-well plates using 50 nM of purified NS5B enzyme, which was incubated with 300 nM 5'-biotinylated oligo(rG13)/poly(rC) or oligo (rU15)/poly(rA) primer-template, 600 nM of GTP, and 0.1 μCi of [3H]GTP or [3H]UTP in 25 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 25 mM KCl, 17 mM NaCl and 3 mM of DTT. The 30 μL reaction mixture was incubated at room temperature for 2 h before stopping the reaction by adding 30 μL of streptavidin coated SPA-beads (GE Healthcare, Uppsala, Sweden) in 0.5 M EDTA. The 30 μL reaction was terminated after 2 hours at 25° C. upon addition of 30 μl streptavidin-coated SPA beads (GE Healthcare, Uppsala, Sweden 5 mg/ml in 0.5 M EDTA). After incubation at 25° C. for 30 min, the plate was counted using a Packard TopCount microplate reader (30 sec/well, 1 min count delay) and IC50 values were calculated (Table 1: IC50 1bJ4).
Measurement: IC50 = 410 nM (pChEMBL 6.389999866485596)
Descriptors: MW 608.76, ALogP 3.93, HBA 7, HBD 1, PSA 110.18, QED 0.47
Compound (SMILES): CC(C)(C)c1cc(NC(=O)Nc2ccc(Oc3ccnc(Nc4ccc5[nH]ncc5c4)n3)c3ccccc23)n(-c2ccc(=O)n(CCO)c2)n1
InChIKey: DHVBTKDVRAXVKY-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: p38 MAPKα Enzyme Inhibition Assay: The inhibitory activities of test compounds against the p38 MAPKα isoform (MAPK14: Invitrogen), are evaluated indirectly by determining the level of activation/phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPK&dlpha; protein (80 ng/mL, 2.5 uL) is mixed with the test compound (2.5 uL of either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL or 0.004 ug/mL) for 2 hr at RT. The mix solution (2.5 uL) of the p38&dlpha; inactive target MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 uM; a phosphorylation target for MAPKAP-K2) is then added and the kinase reaction is initiated by adding ATP (40 uM, 2.5 uL). The mixture is incubated for 1 hr at RT. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 670.73, ALogP 6.32, HBA 11, HBD 5, PSA 176.9, QED 0.12
Compound (SMILES): CCNC(=O)c1ccc(S(=O)(=O)c2ccc(NC(=O)/C=C/c3cccnc3)cc2)cc1
InChIKey: DJHVDGRDQOIHIG-LHHJGKSTSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 uM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine.
Measurement: IC50 = 14.7 nM (pChEMBL 7.829999923706055)
Descriptors: MW 435.51, ALogP 3.32, HBA 5, HBD 2, PSA 105.23, QED 0.55
Compound (SMILES): Cc1c(-c2c(Cl)cc(C(N)=O)c3[nH]c4cc(C(C)(C)O)ccc4c23)cccc1-n1c(=O)cc2c(F)cccn2c1=O
InChIKey: RXGHNLYVCPQUBS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Assay: To V-bottom 384-well plates were added test compounds, human recombinant Btk (1 nM, Invitrogen Corporation), fluoresceinated peptide (1.5 μM), ATP (20 μM), and assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij 35 surfactant and 4 mM DTT in 1.6% DMSO), with a final volume of 30 μL. After incubating at room temperature for 60 min, the reaction was terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product.
Measurement: IC50 = 0.85 nM (pChEMBL 9.069999694824219)
Descriptors: MW 571.01, ALogP 5.18, HBA 6, HBD 3, PSA 122.59, QED 0.27
Compound (SMILES): Cl.NC(N)=N/N=C/c1cccc(O)c1
InChIKey: MYGIITYIQHSNTN-HMXKFKBXSA-N
Target: Heme oxygenase (SINGLE PROTEIN), Pseudomonas aeruginosa
UniProt: O69002
Assay: Binding affinity to Pseudomonas aeruginosa HemO by intrinsic fluorescence quenching method
Measurement: Kd = 21000 nM (pChEMBL 4.679999828338623)
Descriptors: MW 178.2, ALogP 0, HBA 3, HBD 3, PSA 96.99, QED 0.34
Compound (SMILES): COc1cc2nc(NC(=O)CN3CCN(Cc4ccc(F)cc4)CC3)sc2cc1OC
InChIKey: FLRCPYHCKKZOHI-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Inhibition of electric eel AChE preincubated for 15 mins followed by acetylthiocholine iodide substrate addition measured for 5 mins by Ellman's spectrophotometric method
Measurement: IC50 = 175.4 nM (pChEMBL 6.760000228881836)
Descriptors: MW 444.53, ALogP 3.21, HBA 7, HBD 1, PSA 66.93, QED 0.6
Compound (SMILES): COC(=O)[C@@H](Cc1ccc2c(N)nccc2c1F)N1CC[C@H](NS(=O)(=O)c2ccc(N3CCCC3)nc2)C1=O
InChIKey: HNJFPOPALWJYDN-LEWJYISDSA-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 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 556.62, ALogP 1.61, HBA 9, HBD 2, PSA 147.82, QED 0.39
Compound (SMILES): Clc1ccccc1Nc1ncc(Cl)c(Nc2cc(C3CC3)[nH]n2)n1
InChIKey: FUUIYHGRTFEBKO-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 361.24, ALogP 4.87, HBA 5, HBD 3, PSA 78.52, QED 0.6
Compound (SMILES): Cn1ncc(NC(=O)c2csc(-c3cccc(-c4cccc(Cl)c4F)c3F)n2)c1[C@@H]1CC[C@@H](N)[C@@H](F)CO1
InChIKey: HCJDMTOLVIRAIH-UEXGIBASSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Assay: LC3K 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 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL 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.
Measurement: Ki = 0.533 nM (pChEMBL 9.270000457763672)
Descriptors: MW 562.02, ALogP 5.91, HBA 7, HBD 2, PSA 95.06, QED 0.32
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(CCC3CC3)cc1C21N=C(N)N(Cc2ccccn2)C1=O
InChIKey: GMRAAWWRBRNSQO-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 15.8 nM (pChEMBL 7.800000190734863)
Descriptors: MW 458.61, ALogP 4.11, HBA 5, HBD 1, PSA 80.81, QED 0.71
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NC5CCN(CC(F)F)CC5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: JKNVNSREOKPFNI-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 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 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 probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (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 = 0.017 nM (pChEMBL 10.770000457763672)
Descriptors: MW 917.48, ALogP 8.96, HBA 11, HBD 3, PSA 166.04, QED 0.07
Compound (SMILES): NC(=O)c1nn(-c2ccc(S(N)(=O)=O)cc2)c2c1CCc1ccc(NS(=O)(=O)CC(F)(F)F)cc1-2
InChIKey: CHAJYXZNMFDDQP-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: Inhibition of human recombinant C-terminal FLAG-epitope-tagged IKK2 expressed in baculovirus using biotinylated IkappaBalpha peptide as substrate assessed as decrease in [gamma-33P]-ATP incorporation in substrate after 30 mins by scintillation counting method
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 529.52, ALogP 1.69, HBA 7, HBD 3, PSA 167.24, QED 0.44
Compound (SMILES): COc1ccc(NC(=O)Nc2ccc(-c3nc(N4CC5CCC(C4)O5)nc4c3C(=O)N(C3CCOCC3)CC(C)(C)O4)cc2)cn1
InChIKey: FHFAEZOOTGEWKW-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 = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 629.72, ALogP 4.35, HBA 10, HBD 2, PSA 140.27, QED 0.4
Compound (SMILES): COc1ccc(/C(=C(\CCCO)c2ccc3cnn(C)c3c2)c2ccc(OCCN(C)C)cc2)cc1.Cl
InChIKey: BNJUABSPVHGUPZ-QIRPVNSRSA-N
Target: Estrogen-related receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P62508
Assay: Inhibition of fluorescien-conjugated coactivator PGC1a binding to GST-tagged human ERRgamma LBD after 1 hr by TR-FRET assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 485.63, ALogP 5.25, HBA 6, HBD 1, PSA 59.75, QED 0.29
Compound (SMILES): COc1ccc(-c2cc3c(N[C@H](C)C4CCCCC4)ncnc3o2)cc1
InChIKey: GUNJQUPJXFVBLR-CQSZACIVSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human GST-tagged EGFR cytoplasmic domain (668 to 1210 residues) expressed in baculovirus by Z'-LYTE assay
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 351.45, ALogP 5.28, HBA 5, HBD 1, PSA 60.18, QED 0.68
Compound (SMILES): COC(=O)N1CCC(c2nc(C(F)(F)F)c(-c3cc(C(=O)N4CC(c5ccc(C#N)cc5)C4)ccc3C)[nH]2)CC1
InChIKey: LFUMOOOOCGSGFQ-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 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 551.57, ALogP 5.46, HBA 5, HBD 1, PSA 102.32, QED 0.46
Compound (SMILES): CCC(O)(c1nnnn1Cc1ccc2c(-c3ccc(F)cc3)cc(C(N)=O)nc2c1)C(F)(F)F
InChIKey: WUDOPQXWPIFCFE-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr-cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity. Then 2,500 nM glutamate is added and the antagonist response is monitored. The maximum calcium response at each concentration of compound for agonist or antagonist were plotted as dose responses and the curves were fitted with a four parameter logistic equation giving IC50 and Hill coefficient using the iterative non linear curve fitting software ADA (Merck & Co).
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 474.42, ALogP 3.33, HBA 7, HBD 2, PSA 119.81, QED 0.41
Compound (SMILES): O=C1c2c(F)cc(C3CC3)cc2OCCN1c1cccc(-c2ccnc3[nH]c(-c4ccc(OCCN5CCC5)cn4)cc23)c1CO
InChIKey: GXWLXBZIKKRBCP-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 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 619.7, ALogP 5.92, HBA 7, HBD 2, PSA 103.81, QED 0.21
Compound (SMILES): O=C(NC1CCCCC1)c1cc(-c2ccco2)nc2ccc(-c3ccc4scnc4c3)cc12
InChIKey: BGFNRQGSBMGCQC-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Biotin Assay: A streptavidin-coated plate (Thermo Scientific, NUNC #436014) was washed three times with 300 μL of PBS-0.05% Tween solution. A biotinylated twenty-six amino acid peptide, corresponding to the BH3 domain of Bim, with the sequence biotin-(β)A-D-M-R-P-E-I-W-I-A-Q-E-L-R-R-I-G-D-E-F-N-A-Y-Y-A-R-R-amide (SEQ ID No. 14), hereafter referred to as biotin-Bim, was obtained (Tufts). The biotin-Bim peptide was diluted to 0.018 μg/mL (5 nM) in SuperBlock blocking buffer in PBS (Thermo Scientific, #37515), and 100 μL of this solution was incubated in the streptavidin-coated plate for 2.5 hours while shaking. Separately, a compound of Formula I or Formula II, which had been prepared in a 10 mM stock DMSO solution, was then incubated with 20 nM GST-Mcl-1 fusion protein in PBS. Compounds were tested in six three-fold dilutions ranging from 20 μM to 0.6 μM, 10 μM to 0.3 or 5 μM to 0.15 μM. In addition, Bim peptide (New England Peptide) was utilized as a control and tested in six-fold dilutions ranging from 100 nM to 3.3 nM. The streptavidin-coated plate which had previously been treated with biotin-Bim peptide was then washed three times with 300 μL of PBS-0.05% Tween solution. A solution of GST-MCL-1 and a compound of Formula I or Formula II (100 μL) was then added to the streptavidin-coated plate, with two wells utilized as controls (PBS buffer only, no GST-MCL-1 or biotin-Bim) and two wells as an alternative control (containing GST-MCL-1 but no biotin-Bim), and four three-fold dilutions of DMSO (0.2%, 0.06%, 0.02%, and 0.008%). All wells containing compounds of Formula I or Formula II were loaded in duplicate. The plate was incubated for 2 hours at RT, then washed three times with 300 μL of PBS-0.05% Tween solution. Anti-GST HRP (GE Healthcare, #RPN1236) is diluted 1:20,000 in freshly prepared PBS, 0.1% Tween20, and 0.5% BSA was then added to the plate and the plate was incubated for 30 minutes. The plate was then washed five times with 300 μL of PBS-0.05% Tween solution. A solution of color reagents A (stabilized peroxide solution) and B (stabilized chrommogen solution) (R&D Systems, #DY999) is mixed in a 1:1 ratio and added to each well in a quantity of 80 μL. The wells were allowed to develop until control wells containing DMSO are blue (approximately 5-10 minutes). A solution of ELISA stop solution (1M sulfuric acid, 20 μL per well) was then added. Absorbance at 450 nM was read on a Tecan GeniosPro plate reader.
Measurement: IC50 = 6400 nM (pChEMBL 5.190000057220459)
Descriptors: MW 453.57, ALogP 6.83, HBA 5, HBD 1, PSA 68.02, QED 0.32
Compound (SMILES): COC(=O)CC1CCC(N2CC=C(c3[nH]c4nccc5c4c3CN(C)c3cc(F)c(F)cc3-5)CC2)CC1
InChIKey: HWLKAHSXHHYJBZ-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay : CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were diluted in 100% DMSO then diluted with 1:10 in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. Equal volume of the compound dilutions was added to a final reaction mixture containing LANCE detection buffer (PerkinElmer CR97-100), 100 nM ULight MBP (PerkinElmer TRF0109M), 1000 μM ATP, and CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 1 hour before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer. The reaction was incubated for 1 hour and the signal was read in Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 506.6, ALogP 5.67, HBA 5, HBD 1, PSA 61.46, QED 0.46
Compound (SMILES): C[C@H]1CCCN1C1CC[C@H](c2ccc(N3CCC4(CCCCC4)C3=O)cc2)C1
InChIKey: VKYUGIUREVMUGA-XTIMDYPVSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: Radioligand Binding Assay: Rhesus H3 radioligand binding assay was performed using rhesus H3 receptor membranes (prepared as described above), [3H]-Methylhistamine (Perkin Elmer) and WGA SPA beads (wheat germ agglutinin scintillation proximity assay) beads (Amersham). The assay was performed in 96-well Opti-Plates (Packard). Each reaction contained 50 μl rhesus H3 membranes (20-30 μg total protein), 50 μl WGA SPA beads (0.1 μg) and 50 μl of 83 Ci/mmol [3H]-Methylhistamine (final concentration 2 nM) and 50 μl of tested compound. The compounds of this invention and/or vehicle were diluted with binding buffer from 10 mM DMSO stocks. Assay plates were sealed with TopSeal (Perkin Elmer) and mixed on shaker (25° C., 1 hour). Assay plates were read on TopCount scintillation counter (Packard).
Measurement: Ki = 12.8 nM (pChEMBL 7.889999866485596)
Descriptors: MW 380.58, ALogP 5.49, HBA 2, HBD 0, PSA 23.55, QED 0.69
Compound (SMILES): NC[C@@H]1CCN(c2ncc(C(=O)Nc3ccc(OC(F)(F)Cl)cc3)cc2-c2cncnc2)C1
InChIKey: NVNUBAJYACXNCY-AWEZNQCLSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Radio ABL1 (64-515) Assay: For determination of ABL kinase activity, the radiometric filter-binding assay was used. The assay was performed by mixing 10 uL of the compound pre-diluted with 10 uL of ATP (20 uM ATP with 0.1 uCi [γ-33P]-ATP) with the phospho-acceptor peptide poly[Ala6Glu2LysHBr5Tyr1]=polyAEKY) in 20 mM Tris/HCl pH 7.5, 1 mM DTT, 10 mM MgCl2, 0.01 mM Na3VO4, 50 mM NaCl. 10 uL of enzyme (ranging between 5 nM to 20 nM) was added to initiate the reaction. Pre-incubation of enzyme with compounds (when stated) was performed by exposing the enzyme to compounds prior to addition of the substrate mixture (ATP and/or peptide substrate). After 15 mM at room temperature, the reaction was stopped by the addition of 50 uL 125 mM EDTA, and the peptide-bound 33P separated on filter-plates (PVDF or MAIP; Millipore, Volketswil, Switzerland) prepared according to the manufacturer's instructions. Filter-plates were washed 3x with 0.5% H3PO4, followed by addition of 30 uL scintillation cocktail (Microscint, Perkin Elmer) per well and then analysed in a TopCount NXT scintillation counter (Perkin Elmer).
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 474.9, ALogP 3.74, HBA 7, HBD 2, PSA 106.26, QED 0.5