lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cn1cc(Nc2ncc(C(F)(F)F)c(CCc3ccccc3C3(C(N)=O)CC3)n2)cn1
InChIKey: UMEFYEWFNFNLHP-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 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 430.43, ALogP 3.27, HBA 6, HBD 2, PSA 98.72, QED 0.6
Compound (SMILES): CCn1nc(-c2cccc(Br)c2)cc(NC(=O)Nc2c(Cl)cncc2Cl)c1=O
InChIKey: IJQTXEGGUYZYPT-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Inhibition of human recombinant PDE4B1 assessed as reduction in [3H]cAMP hydrolysis to [3H]AMP incubated for 60 mins by PDE-SPA assay
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 483.15, ALogP 5.04, HBA 5, HBD 2, PSA 88.91, QED 0.54
Compound (SMILES): c1ccc2c(N3CCC4(CC3)COC4)cncc2c1
InChIKey: GSPSKHPXAWDCSR-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibition of human CYP11B2-CLE9 expressed in chinese hamster V79 cells using 11-deoxycorticosterone as substrate preincubated for 1 hr followed by substrate addition measured for 3 hrs by HTRF assay
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 254.33, ALogP 2.85, HBA 3, HBD 0, PSA 25.36, QED 0.78
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](C(=O)N[C@@H](C)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](CS)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)C(C)C
InChIKey: BELQJTFNKNUHSK-PNBJJJQLSA-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 2361.8
Compound (SMILES): Cc1nc2c(-c3ccc(OCCC4CCNCC4)c(C(F)(F)F)c3)nc(C#N)nc2n1C
InChIKey: AZXZSUJTRIWYJA-UHFFFAOYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Inhibition Assay: 5 μL of a human cathepsin S enzyme (R&D1183-CY-010) was added to a 96-well plate so as to be 20 ng/well. Next, 10-fold dilution of the test compound (a 10 mM DMSO solution) was serially performed with a buffer solution for assay (a solution of 50 mM sodium methoxide, 250 mM sodium chloride and 5 mM dithiothreitol (DTT), adjusted to pH=4.5 with 1 N hydrochloric acid) six times such that the final concentration becomes 0.1 nM to 10 μM then, 20 μL of each of the solutions was added to the wells (the final DMSO concentration was 0.1%), and 25 μL of synthetic substrate VVR-AMC (3211-V manufactured by PEPTIDE INSTITUTE, INC.) was added thereto such that the final concentration becomes 40 μM, thereby starting a enzyme reaction. A fluorescence intensity (an excitation wavelength: 380 nm and a fluorescent wavelength: 460 nm) was measured at 37° C. for 10 minutes immediately after the start of the reaction using a fluorescence spectrophotometer (SPECTRA MAX GEMINI, manu
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 444.46, ALogP 4, HBA 7, HBD 1, PSA 88.65, QED 0.64
Compound (SMILES): CC(=O)N1CCc2[nH]nc(Nc3cccc(C(F)(F)F)c3)c2C1
InChIKey: BAHQNJLZVVLSJW-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Displacement of biotinylated ligand from recombinant His-tagged BRD4 bromodomain-1 (unknown origin) measured after 10 mins by TR-FRET assay
Measurement: IC50 = 18000 nM (pChEMBL 4.75)
Descriptors: MW 324.31, ALogP 3.08, HBA 3, HBD 2, PSA 61.02, QED 0.89
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1cnn(Cc2ccc3c(c2)nc(C)n3C)c1
InChIKey: ZGMRJKJEDUMBBI-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 403.49, ALogP 2.65, HBA 7, HBD 2, PSA 103.65, QED 0.53
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H]1CCC(=O)NCC[C@@H](C(=O)N[C@@H](Cc2c[nH]c3ccccc23)C(N)=O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC1=O
InChIKey: AXBBCWVVFWOKFH-OKXJKXJKSA-N
Target: Melanocortin receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P32245
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 = 510 nM (pChEMBL 6.289999961853027)
Descriptors: MW 1044.19
Compound (SMILES): O=C(NC1CCCC1)c1ccc(NC(=O)N2Cc3ccc(F)cc3C2)cc1
InChIKey: VRIAEEFLBWQLLL-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 40.4 nM (pChEMBL 7.389999866485596)
Descriptors: MW 367.42, ALogP 4.05, HBA 2, HBD 2, PSA 61.44, QED 0.86
Compound (SMILES): Cl.NC(=O)c1nn(-c2ccc3c(c2)OCO3)c2c1CCc1ccc(NC(=O)CNC3CCC3)cc1-2
InChIKey: VRTWHIZEQVSBBA-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 459.51, ALogP 2.55, HBA 7, HBD 3, PSA 120.5, QED 0.52
Compound (SMILES): C[C@@](CCc1ccc(-c2ccc(OCCCN3CCCCC3)cc2)cc1)(C(=O)NO)S(C)(=O)=O
InChIKey: FQEWDHPYFHPPKT-AREMUKBSSA-N
Target: UDP-3-O-acyl-N-acetylglucosamine deacetylase (SINGLE PROTEIN), Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG12228)
UniProt: P47205
Assay: Enzyme Assay: IC50 determination in the LpxC enzyme assay was carried out in a similar manner to that described by Malikzay et al in the 2006 Poster, Screening LpxC (UDP-3-O(R-3-hydroxymyristoyl)-GlcNAc deacetylase) using BioTrove Rapid Fire HTS Mass Spectrometry (aNew Lead Discovery and bInflammation and Infectious Disease, cStructural Chemistry, Schering-Plough Research Institute, Kenilworth, N.J. 07033, (BioTrove, Inc. 12 Gill St., Suite 4000, Woburn, Mass. 01801). Briefly, Pseudomonas aeruginosa LpxC enzyme (0.1 nM) purified from E. coli-overexpressing bacteria was incubated at 25° C. in a final volume of 50 ul containing 0.5 uM UDP-3-O(R-3-hydroxydecanoyl)-N-acetylglucosamine, 1 mg/mL BSA, and 50 mM sodium phosphate buffer, pH 8.0 in the presence and absence of inhibitor compound. At the end of 1 hour, 5 ul of 1N HCl was added to stop the enzyme reaction; the plates were centrifuged, and then processed with the BioTrove Rapidfire HTMS Mass Spectrometry System.
Measurement: IC50 = 0.467 nM (pChEMBL 9.329999923706055)
Descriptors: MW 488.65, ALogP 3.85, HBA 6, HBD 2, PSA 95.94, QED 0.28
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4cc(Cl)c(NCC5CCOCC5)c(C#N)c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: HIVQTQIMMQSXQB-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 = 0.114 nM (pChEMBL 9.9399995803833)
Descriptors: MW 882.91, ALogP 9.28, HBA 10, HBD 3, PSA 152.68, QED 0.11
Compound (SMILES): CC(C)Oc1ccc(Nc2ncc(NC(=O)CCO)c(=O)n2Cc2ccc(Cl)cc2)cc1F
InChIKey: MQZJOFBWROXNIT-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 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 in M MgCl2, 5.0 mM CaCl2, 5.6 D-glucose, 2.5 mM probenecid, 1.0% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence inte
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 474.92, ALogP 3.94, HBA 7, HBD 3, PSA 105.48, QED 0.43
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(C(F)(F)F)c(F)c3)CC2)C1
InChIKey: AJXWDVYAVCKCDI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 552.54, ALogP 4.21, HBA 7, HBD 1, PSA 106.73, QED 0.37
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1ccc(C(F)(F)F)cn1)c1ccccc1-n1nccn1
InChIKey: FUOSBVRFUHTLQH-HOTGVXAUSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 416.41, ALogP 3.44, HBA 6, HBD 2, PSA 84.73, QED 0.67
Compound (SMILES): C[C@@H]1CN(Cc2ccccc2)c2ccccc2[C@H](C)N1C(=O)NC1COC1
InChIKey: JJKNTWZITITEHD-SJORKVTESA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1G (SINGLE PROTEIN), Homo sapiens
UniProt: O43497
Assay: Channel blocking activity at recombinant human CaV 3.1 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 365.48, ALogP 3.57, HBA 3, HBD 1, PSA 44.81, QED 0.9
Compound (SMILES): CCOc1ccc(CC(=O)O)cc1-c1ccc(F)c2c1CN(C(=O)CCc1ccccc1F)CC2
InChIKey: ZREWHWRUCPVICU-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 479.52, ALogP 5.18, HBA 3, HBD 1, PSA 66.84, QED 0.48
Compound (SMILES): N[C@H]1CCN(C(=O)c2cc(-c3cc(NC4CC4)n4ncc(/C=C5\NC(=O)NC5=O)c4n3)cs2)C1
InChIKey: VOORUZPHJGXVFC-LDFHSBOYSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 478.54, ALogP 1.39, HBA 9, HBD 4, PSA 146.75, QED 0.32
Compound (SMILES): COc1ccc(C(=O)N2CCN(C(=O)c3cc(Cc4n[nH]c(=O)c5c4NCCC5)ccc3F)CC2)cc1
InChIKey: QZJWKQINLBFEHG-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 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 505.55, ALogP 2.46, HBA 6, HBD 2, PSA 107.63, QED 0.55
Compound (SMILES): COc1ccc(-n2nnc(C(=O)Nc3c(C)n(C)n(C4CCCCC4)c3=O)c2C)cc1
InChIKey: GPKSXWTVPBSUBX-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase SMURF1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HCE7
Assay: Inhibition of SMURF1 (unknown origin) in presence of E1/E2/Ub mix measured after 45 mins by europium-labeled streptavidin based TR-FRET assay
Measurement: IC50 = 3100 nM (pChEMBL 5.510000228881836)
Descriptors: MW 424.51, ALogP 3.15, HBA 8, HBD 1, PSA 95.97, QED 0.68
Compound (SMILES): COc1cc(C2CCN(C3CCN(CCF)CC3)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: VMVAXVQCRBDHOB-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human GST-rTEV-tagged IGF1R (957 to 1367 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 760.86, ALogP 8.35, HBA 9, HBD 2, PSA 99.92, QED 0.14
Compound (SMILES): CC(C)Oc1ccc(Nc2nc(=O)n(CCOC3CCCCO3)c(=O)n2Cc2ccc(Cl)cc2)cc1Cl
InChIKey: HYEDLAYHBGAJBN-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 = 10 nM (pChEMBL 8)
Descriptors: MW 549.46, ALogP 4.83, HBA 9, HBD 1, PSA 96.61, QED 0.39
Compound (SMILES): O=C(Nc1cccc(Cc2n[nH]c(=O)c3c2CCCC3)c1)c1ccccc1Nc1ccccc1
InChIKey: VDBZLAQIWDDTPO-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 = 285 nM (pChEMBL 6.539999961853027)
Descriptors: MW 450.54, ALogP 5.24, HBA 4, HBD 3, PSA 86.88, QED 0.38
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(CCC(C)C)cc1C21N=C(N)N(C)C1=O
InChIKey: RYMHRRJVTCOMJD-VYNBAUGJSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Assay: 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).
Measurement: IC50 = 15.8 nM (pChEMBL 7.800000190734863)
Descriptors: MW 383.54, ALogP 3.39, HBA 4, HBD 1, PSA 67.92, QED 0.87
Compound (SMILES): N#CCC(=O)N1CCCC(c2ncn3cnc4[nH]ccc4c23)C1
InChIKey: RVHXGUCJVOYTRJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 308.35, ALogP 1.83, HBA 5, HBD 1, PSA 90.08, QED 0.78
Compound (SMILES): CC(C)Nc1nnc(-c2ccc3[nH]cc(-c4ncc(C(N)=O)s4)c3c2)o1
InChIKey: QMLTXQZCXWHPPX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 111 nM (pChEMBL 6.960000038146973)
Descriptors: MW 368.42, ALogP 3.26, HBA 7, HBD 3, PSA 122.72, QED 0.5
Compound (SMILES): Cc1cnc(C(=O)Nc2cccc(Oc3ccc4c(/C=C/c5ccccc5)n[nH]c4c3)c2)s1
InChIKey: ITLTXJASMJITNS-JLHYYAGUSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of unphosphorylated human 50 residues truncated cytoplasmic domain of VEGFR2 E990V mutant expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using poly(E4Y1) as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 0.03 nM (pChEMBL 10.520000457763672)
Descriptors: MW 452.54, ALogP 6.54, HBA 5, HBD 2, PSA 79.9, QED 0.31
Compound (SMILES): CC(C)(C)c1cc(NC(=O)Nc2ccc(-c3cn4c(n3)sc3nc(OCC5CCNCC5)ccc34)cc2)no1
InChIKey: HUTSEJJHKPVPCT-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Caliper's mobility shift assay (MSA): FLT3 is a receptor tyrosine kinase involved in survival and proliferation of leukemic cells. Constitutively activating FLT3 mutations has been found in about 30% of all patients with acute myeloid leukemia (AML). The tested compounds were screened for their ability to inhibit FLT3 kinase activity using Caliper's mobility shift assay (MSA). The assay uses a microfluidic chip to measure the conversion of a fluorescent peptide substrate to a phosphorylated product following separation by electrophoresis. The signature of the fluorescence signal over time reveals the extent of the reaction. Protein tyrosine kinase assays were carried out in a final volume of 25 ul containing 0.9 nM purified FLT3 (Carna, Cat 08-154) enzyme protein, 50 mM HEPES [pH=7.5], 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT, 2% DMSO, 97 uM ATP, 1.5 uM peptide 2. Each Compound was added into the reaction to final concentrations from 300 nM to 0.015 nM (10 concentration points in 3 fold serial dilution).
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 545.67, ALogP 5.92, HBA 9, HBD 3, PSA 118.61, QED 0.24
Compound (SMILES): CC(=O)N1CCC(C)(c2ccccc2)c2cc(NC(=O)OCCC(C)C)ccc21
InChIKey: JOEBARIVRGDWFY-UHFFFAOYSA-N
Target: Thyrotropin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P16473
Assay: Antagonist Activity Assay: Antagonist activity of three test compounds against human TSI at the human TSH receptor was tested in Chinese Hamster Ovary (CHO) cells (cultured 16 h in the absence of serum prior to the assay) stably expressing the human TSH receptor and a CRE-driven firefly luciferase reporter gene. TSI was partially purified from the serum of a GD patient by filtration over a 0.45 mm filter, protein G-Sepharose column chromatography, dialysis against phosphate-buffered saline and subsequent concentrating on a 10K Amicon filter. It was confirmed that the TSI preparation does not activate luciferase activity in control CHO cells lacking the human TSHR. The cAMP phosphodiesterase inhibitor rolipram was included in the assay medium (10 μM) to augment TSHR-induced CRE-luciferase synthesis, which was quantified using a luminescence counter. The cells were incubated with compound A, B or C (0.316 nM-10 μM) together with 3.16 mg/ml TSI (or bovine TSH at a equieffective concentration of 18 nM).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 394.52, ALogP 5.34, HBA 3, HBD 1, PSA 58.64, QED 0.74
Compound (SMILES): CCc1cc(CNc2cc(Cl)nn3c(-c4ccc(OC)c(S(=O)(=O)NC5CC5)c4)c(C)nc23)ccn1
InChIKey: LYAFGTQDNCDDFD-UHFFFAOYSA-N
Target: Phosphatidylinositol 4-kinase beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBF8
Assay: Inhibition of PI4KB (unknown origin) by ADP-Glo kinase assay
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 527.05, ALogP 4.38, HBA 8, HBD 2, PSA 110.51, QED 0.33
Compound (SMILES): CC[C@H](C)[C@@H]1NCCOc2ccccc2CCCNC(=O)[C@@H](Cc2ccccc2Cl)NC(=O)[C@@H](C)N(C)C1=O
InChIKey: ABOHBKNCAMQYLH-QIDJRYDTSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki = 0.1 nM (pChEMBL 10)
Descriptors: MW 557.14, ALogP 3.36, HBA 5, HBD 3, PSA 99.77, QED 0.54
Compound (SMILES): CC(CO)Nc1ncc2ccc(C(=O)NC(c3ccc(Cl)c(F)c3)c3cnn(C)c3)cc2n1
InChIKey: KJYWWRQXFYFUCA-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an N-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 1 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 468.92, ALogP 3.47, HBA 7, HBD 3, PSA 104.96, QED 0.38
Compound (SMILES): O=Cc1c(N(Cc2ccc(F)c(C(F)(F)F)c2)S(=O)(=O)c2ccccc2)sc2ccccc12
InChIKey: OPRIFUGIGNUNQH-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 493.5, ALogP 6.27, HBA 4, HBD 0, PSA 54.45, QED 0.24
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc(-c3ccnc(-c4ccccc4)c3)cc2F)CS1(=O)=O
InChIKey: OUOVRDXFJWCQLN-FQEVSTJZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 μl are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 132 nM (pChEMBL 6.880000114440918)
Descriptors: MW 430.53, ALogP 3.63, HBA 5, HBD 2, PSA 86.15, QED 0.66
Compound (SMILES): COCCCNC(=O)CCc1ccc(Nc2nccc(Nc3cc(C4CC4)n[nH]3)n2)cc1
InChIKey: RQBJSVSFCXBHIB-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 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 435.53, ALogP 3.65, HBA 7, HBD 4, PSA 116.85, QED 0.32
Compound (SMILES): CC(C)(O)C(=O)NCc1cncc(-c2cnc3c(c2)CCCN3C(N)=O)c1
InChIKey: NCVXNIXOLFYZFJ-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Aldosterone Synthase Inhibition Assay: The aldosterone synthase inhibition assay employs cynomolgus adrenal gland mitochondria as the source of aldosterone synthase (CYP11B2). Mitochondria are prepared from frozen cynomolgus monkey adrenal glands according to Method A described in by J. D. McGarry et al. (Biochem. J., 1983, 214, 21-28), with a final resuspension in the AT buffer described in R. Yamaguchi et al. (Cell Death and Differentiation, 2007, 14, 616-624), frozen as aliquots in liquid nitrogen and stored at -80° C. until use. Assays are performed in 96-well format in a final volume of 60 uL/well, containing 100 mM potassium phosphate, pH 7.4, 1% (v/v) DMSO, and additionally, 2 uM of corticosterone and 6 mg of mitochondrial protein. Reactions are started by the addition of NADPH to 1 mM and allowed to proceed for 60-90 minutes at 37° C. Reactions are terminated by the addition of 60 uL of acetonitrile. One hundred microliters are then transferred to a glass filter plate and centrifuged at 570xg.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 369.43, ALogP 1.36, HBA 5, HBD 3, PSA 121.44, QED 0.75
Compound (SMILES): Cc1noc(C)c1Cn1cc(N2C(=O)N(Cc3cc(O)cc(O)c3)C(C)(C)C2=O)cn1
InChIKey: BGSMGFCADYXOKT-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 425.45, ALogP 2.69, HBA 8, HBD 2, PSA 124.93, QED 0.6
Compound (SMILES): O=C(O)CC1CCC(c2ccc(-c3ccc4nc(C(=O)NCc5ccc(OC(F)(F)F)cc5)cn4c3)cc2)CC1
InChIKey: KFGSQOIWOCDWAS-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Inhibition Assay: Â Assay buffer [20 mM HEPES (pH 7.4), 100 mM MgCl2 and 0.04% bovine serum albumin (BSA)] containing 500 uM of enzyme substrate (didecanoyl glycerol) and 7.5 uM radiolabeled acyl-CoA substrate ([14C]decanoyl-CoA) was added to each well of a phospholipid-coated radiometric assay plate. A small aliquot of cell lysate having human DGAT1 activity (5 ug/well) obtained in above, of human liver microsomes having human DGAT1 activity (5 ug/well), or of mouse liver microsomes having mouse DGAT1 activity (5 ug/well) was added to the assay plate to start the reaction, and the reaction was allowed to proceed for 60 minutes at 25° C. The reaction was terminated upon the addition of an equal volume (100 uL) of isopropanol. The plates were sealed, incubated overnight and counted the next morning on a liquid scintillation counter and luminometer.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 551.57, ALogP 6.58, HBA 5, HBD 2, PSA 92.93, QED 0.26
Compound (SMILES): C[C@H](NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](C)NC(=O)CC1CCCC1)C(N)=O
InChIKey: BZRBITFQGJDKTP-OBJOEFQTSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Binding Assay: All the FP based experiments were performed in MICROFLUOR 2 Black, "U" Bottom, 96-well Microtiter Plates (ThermoSci.) and FP was measured as mP units in a microplate reader (Tecan Ultra) with excitation at 485 nm and emission at 530 nm. To dilutions of WDR5Δ23 (2.2-0 uM) in 100 ul assay buffer (0.1M Phosphate, 25 mM KCl, 0.01% Triton, pH 6.5) 20 ul of a fixed concentration of the tracer in the assay buffer was added, followed by an addition of 5 ul DMSO to give 125 ul of total volume. Each assay had two controls: blank (without protein and tracer) and tracer only (without protein). The plates were incubated on a shaker at room temperature to reach equilibrium and mP values were measured at the 3 hour time point.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 425.53, ALogP -1.19, HBA 5, HBD 7, PSA 192.29, QED 0.12
Compound (SMILES): CCNc1n[nH]c2cc(NC(=O)NC(C(=O)N(C)C)c3ccc(F)cc3)ncc12
InChIKey: LXKMXZVZUDSMCQ-UHFFFAOYSA-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. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 413 nM (pChEMBL 6.380000114440918)
Descriptors: MW 399.43, ALogP 2.48, HBA 5, HBD 4, PSA 115.04, QED 0.51
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN(CCCC#N)CCO1
InChIKey: CFLVHNKVJHQBJP-UHFFFAOYSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 502.4, ALogP 5.07, HBA 8, HBD 1, PSA 92.53, QED 0.41
Compound (SMILES): CN(Cc1ccccc1)c1cc(N[C@@H]2C[C@H](COS(N)(=O)=O)[C@@H](O)[C@H]2O)ncn1
InChIKey: WHCPURSXXHCTPI-DTDBQYNISA-N
Target: NEDD8 activating enzyme (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13564
Assay: Inhibition of recombinant human NAEalpha/N-terminal His-tagged NAEbeta expressed in baculovirus infected insect Sf9 cells using N-terminal FLAG tagged Nedd8 as substrate incubated for 105 mins in presence of N-terminal GST-tagged Ubc12 by FRET assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 423.5, ALogP -0.14, HBA 9, HBD 4, PSA 150.9, QED 0.45
Compound (SMILES): NC(=O)[C@@H]1C[C@H](F)CN1C(=O)c1ccc(C2CC2)c(OCC2CC2)n1
InChIKey: AGFOWDLWXFQXDY-WFASDCNBSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37 °C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30 °C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30 °C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 1041.6 nM (pChEMBL 5.980000019073486)
Descriptors: MW 347.39, ALogP 1.79, HBA 4, HBD 1, PSA 85.52, QED 0.85
Compound (SMILES): N#Cc1cnc2c(Nc3ccc(F)c([C@]4(C(F)F)COCC(N)=N4)c3)nccc2c1
InChIKey: AYBCGPRPLJRSKE-FQEVSTJZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE-1 Inhibition Assay: Recombinant BACE-1 (extracellular domain, expressed in baculovirus and purified using standard methods) at 0.1 to 1 nM concentrations is incubated with the test compound at various concentrations for 1 hour at room temperature in 100 mM acetate buffer, pH 4.5, containing 0.1% CHAPS. Activity was measured using a final concentration of 3 uM of the fluorescence-quenched substrate Q-C(HSO3)-Ile-Asp-Leu-Ala-Val-Leu-Asp-HN-CH2-CH2-Mca, where Q=2-nitro-5-amino benzoic acid and Mca=7-methoxy-4-coumarinyl acetic acid. Catalytic turnover was monitored in a Spectramax Gemini fluorescence plate reader (Molecular Devices) in black 96-well microplates using excitation/emission wavelength of 325 nm and 400 nm, respectively. Fluorescence increase was followed for 15 min, in 1 minute's intervals.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 412.38, ALogP 3.23, HBA 7, HBD 2, PSA 109.21, QED 0.68
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(N3CCN(c4ccc(Cl)cc4)CC3)n2)c1
InChIKey: LHJRHRIQFVIMTR-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 3360 nM (pChEMBL 5.46999979019165)
Descriptors: MW 423.91, ALogP 2.95, HBA 7, HBD 2, PSA 86.28, QED 0.65