lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)C5CCN(Cc6ccccc6)C5)CC4)cc3c2C(C)C)ccn1
InChIKey: UINUWNPYGSAVPG-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 520.72, ALogP 6.89, HBA 3, HBD 1, PSA 52.23, QED 0.3
Compound (SMILES): COc1ccc(-c2ccc(C(=O)O[C@H]3C[C@@H](N=C=S)C3)nc2)cc1
InChIKey: SFTUCCXGXHUCLX-FZNQNYSPSA-N
Target: N-acylethanolamine-hydrolyzing acid amidase (SINGLE PROTEIN), Homo sapiens
UniProt: Q02083
Assay: Inhibition of Test Compounds NAAA Assay: In order to have an assay method more conducive to high-throughput screening than those published for measuring the NAE hydrolyzing activity of NAAA, the fluorogenic PEA analog N-(4-methyl coumarin)palmitamide (PAMCA), which is hydrolyzed to fluorescent 7-amino-4-methyl coumarin (AMC) and palmitic acid, was developed. For three point concentration inhibition assays with hNAAA the following procedure was used. Purified activated NAAA (final concentration of 0.25 μg/mL) was incubated in assay buffer (100 mM citrate-phosphate buffer, pH 4.5, 3 mM DTT, 0.1% Triton X-100, 0.05% BSA, and 150 mM NaCl) made up to a total volume of 180 μL, followed by addition of the compound dissolved in 10 μL DMSO (along with DMSO neat for the control sample) with the final concentrations for each compound of 100, 10, and 1 μM, in triplicate on a 96 well plate. These samples were allowed to incubate for 15 min at room temperature and then 10 μL of a PAMCA stock solution in DMSO (final PAMCA concentration [5 μM]) was added. After 5 minutes of agitation on a shaking plate, the reaction was allowed to proceed at 37° C. for 120 minutes, with fluorescence readings taken every 10 minutes at a wavelength of 460 nm (using an excitation wavelength of 360 nm) on a Synergy HT Plate Reader using Gen5 software from Bio-Tek. The enzyme activity was calculated by converting the relative fluorescence units to AMC formed, using a standard curve of AMC.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 340.4, ALogP 3.55, HBA 6, HBD 0, PSA 60.78, QED 0.47
Compound (SMILES): Cc1cc(S(=O)(=O)N2C[C@H](CCC(=O)O)Oc3ccc(NC(=O)OC(C)(C)C(F)(F)F)cc32)c(Cl)cc1F
InChIKey: XDBMTWYCLYOVAT-AWEZNQCLSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET Assay: To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 7.31 nM (pChEMBL 8.140000343322754)
Descriptors: MW 582.96, ALogP 5.5, HBA 6, HBD 2, PSA 122.24, QED 0.41
Compound (SMILES): CC(C)(C)n1nc(-c2ccc3cc(C(=O)NC4CCC4)[nH]c3c2)c2c(N)ncnc21
InChIKey: GTINSPBQCWCEQS-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET and KDR Enzyme Assay: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer was used for the KDR biochemical assay with the addition of 2 mM MnCl2.Inhibitors were pre-incubated in the plate for 15 mins with 5 μL kinase and assay buffer at the following concentrations; 13 pM RET (Carna Biosciences; 08-159) and 150 pM KDR (Millipore; 14-630). The reaction was initiated by the addition of 5 μL ATP and substrate at 2× final reaction concentrations. For RET, this was 18 μM and 2 μM; for KDR, this was 16 μM and 1 μM, respectively. Reactions were performed at ATP Km for each target. The assay was allowed to proceed at room temperature for 20 mins before terminating with the addition of 10 μL HTRF detection buffer containing EDTA supplemented with TK-antibody labelled with Eu3+-Cryptate (1:100 dilution) and streptavidin-XL665 (128 nM). Following incubation at room temperature for 1 hour, FRET signal was measured using the Pherastar FS Microplate Reader.
Measurement: IC50 = 302 nM (pChEMBL 6.519999980926514)
Descriptors: MW 403.49, ALogP 3.59, HBA 6, HBD 3, PSA 114.51, QED 0.48
Compound (SMILES): CC(C)Nc1cc(-n2ncc3cc(C#N)cnc32)ncc1-n1cc(CF)nn1
InChIKey: CBELNQDWSSWNEI-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and 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. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 377.39, ALogP 2.56, HBA 9, HBD 1, PSA 110.13, QED 0.57
Compound (SMILES): Cc1cc(OCCCc2c3n(c4c(-c5c(C)nn(C)c5C)c(Cl)ccc24)[C@H](C)CN(c2cc(C(=O)N4CCN(C)CC4)cc4cc(C(=O)O)n(C)c24)C3=O)cc(C)c1Cl
InChIKey: RJAMIHVPZMLQFA-AREMUKBSSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: TR-FRET Binding Assay: Measurements were carried out in OptiPlate-384 White Opaque 384-well plates (Perkin Elmer. Shelton, Conn.). The assay was run using a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) that was purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification, 300 nM FITC-Bak peptide, 1 nM recombinant Maltose Binding Protein (MBP) tagged Mcl-1 (residues 172-327) and 1 nM terbium conjugated-MBP antibody were added to assay buffer (4.5 mM Monobasic potassium phosphate, 15.5 mM Dibasic potassium phosphate, 1 mM Sodium EDTA, 50 mM NaCl, 1 mM DTT, 0.05% Pluronic F-68, pH 7.5). Compounds are diluted in DMSO in a 16-point, semilog serial dilution scheme. For the BAK FITC TR-FRET assay 50 nL compound is added to 20 μL of assay buffer containing Bak-FITC. MBP tagged Mcl-1 protein and antibody, for a final DMSO concentration of 0.25% and a top concentration of 10 μM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 3 hours at room temperature. TR-FRET signal (Delta F) is measured on a Biotek Cytation 3 multimode plate reader equipped with a filter cube containing an Ex 340/30 Em 620/10 nm filter and an Ex 340/30 Em 520 filter.
Measurement: Ki < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 838.84, ALogP 8.39, HBA 9, HBD 1, PSA 118.07, QED 0.15
Compound (SMILES): O=c1[nH]c(CSC2CCOCC2)nc2cc(NC[C@@H]3CCOC3)c(F)c(F)c12
InChIKey: MVZUWHNEJPXDIK-NSHDSACASA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Assay: Enzymatic inhibition of PARP14 was measured using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) monitoring the auto-modification of PARP14 by biotinylated nicotinamide adenine dinucleotide (biotin-NAD). 1 μL of a dose response curve of each test compound was spotted in 384-well nickel-coated white microplates (Thermo) using a Mosquito (TTP Labtech). Reactions were performed in a 50 μL volume by adding 40 μL of PARP14 in assay buffer (20 mM HEPES pH==8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%0 Tween20), incubating with test compound at 25° C. for 30 min, then adding 10 μL of biotin-NAD (Biolog). The final concentrations of PARP14 and biotin-NAD are 50 nM and 3 μM, respectively. Reactions proceeded at 25° C. for 3 h, then were quenched with 5 μL of 10 mM unmodified nicotinamide adenine dinucleotide (Sigma-Aldrich). The quenched reactions were washed 3 times with 100 μL of TBST wash buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween20). Next, to the washed and dried plate was added 25 μL of DELFIA Europium-N1 streptavidin (Perkin Elmer) diluted in DELFIA assay buffer (Perkin Elmer) After a 30 min incubation at 25° C., the plate was washed 5 times with TBST wash buffer. Finally, 25 μL of DELFIA enhancement solution was added. After a 5 min incubation the plate was read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 340 nm and emission of 615 nm to measure the amount of Europium present in each well, informing on the amount of biotin-NAD that was transferred in the automodification reaction.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 411.47, ALogP 3.06, HBA 6, HBD 2, PSA 76.24, QED 0.76
Compound (SMILES): CNc1cc(Nc2cc([C@]34C[C@H]3COC4=O)nn2C2CC2)ccc1C(F)(F)F
InChIKey: SHVCEWSORKSOPL-YPMLDQLKSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay: Materials:LRRK2 G2019S enzymeSubstrate (LRRKtide)ATPTR-FRET dilution bufferpLRRKtide antibody384-well assay plateDMSOEnzyme Reaction Conditions50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35.2 mM DTT5 nM LRRK2134 μM ATP60 minute reaction time23° C. reaction temperature10 μL total reaction volumeDetection Reaction Conditions1× TR-FRET dilution buffer10 mM EDTA2 nM antibody23° C. reaction temperature10 μL total reaction volumeCompounds were prepared by initially diluting to 1 mM with DMSO. 35 μL of reference compound solution, 35 μL of test compound solution, and 35 μL HPE were successively added to the source plate (384-well assay plate, Labcyte). The plates were centrifuged at 2500 rpm for 1 minute and sealed in foil. POD was used to perform a 3.162 fold serial dilution and 100 nL of reference compound solution, test compound solution, HPE and ZPE were transferred to assay plates. The assay plate was centrifuged at 2500 rpm for 1 minute, and sealed with foil.To perform the enzyme reaction, 5 μL of LRRKtide substrate and kinase mixture in assay buffer was added to all wells of the assay plate. The plate was centrifuged to concentrate the mixture at the bottom of the wells. The assay plate was incubated at 23° C. for 20 minutes. Following incubation, 5 μL of 2×ATP in assay buffer was added to each well, and plates were centrifuged to concentrate the mixture at the bottom of the wells. The plate was incubated at 23° C. for 60 minutes.To perform the detection of the reaction, EDTA completely mixed in TR-FRET dilution buffer was added to antibody reagent. 10 μL of detection reagent was added to all wells of each well of the assay plate and the plate was centrifuged to concentrate the mixture at the bottom of the wells. The plate was then incubated at 23° C. for 60 minutes. Plates were read on Perkin Elmer Envision 2104 instrument in TR-FRET mode using 340 nm excitation filter, 520 nm fluorescence emission filter, and 490 or 495 nm terbium emission filter.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 392.38, ALogP 3.84, HBA 6, HBD 2, PSA 68.18, QED 0.76
Compound (SMILES): CCOc1cc2ncc(C#N)c(Nc3ccc(OCc4ncccc4C(F)(F)F)cc3Cl)c2cc1NC(=O)CCCN(C)C
InChIKey: LRLJWMHQZMQXNT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13043
Assay: MST1/MST2 Biochemical LanthaScreen Eu Kinase Binding Assay: MST1 and MST2 biochemical LanthaScreen Eu Kinase Binding Assay was based on the binding and displacement of kinase tracer to the kinase of interest. Compounds in 1000×DMSO stock solution was dispensed using automated dispensing system (Labcyte) to 384 well Corning Microplate at 15 nL, 5 uL of Kinase buffer A was added to each well. Plates were shaken and incubated for 1 min to ensure well dissolution of compounds. Kinase/Antibody mixture was added at a final concentration of 5 nM and 2 nM and kinase tracer 222 solution at a final concentration of 100 nM in a total volume of 20 uL. Plates were incubated for 1.5 hrs in dark at room temperature and assay plates were scanned on Envision plate reader with excitation: 340 nM and kinase Tracer Emission at 665 nM.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 627.07, ALogP 7.18, HBA 8, HBD 2, PSA 112.4, QED 0.17
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N4CCNC(=O)C4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: TWVHQQLEYIZXKU-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 403.49, ALogP 2.95, HBA 6, HBD 2, PSA 91.21, QED 0.55
Compound (SMILES): CCc1nnc2n1CCOc1ncc(NS(=O)(=O)c3cc(Cl)ccc3OC)cc1-2
InChIKey: PDSLCSRJGFMOEX-UHFFFAOYSA-N
Target: Alkaline phosphatase, tissue-nonspecific isozyme (SINGLE PROTEIN), Homo sapiens
UniProt: P05186
Assay: Inhibitory Test of TNAP Activity: COS1 cells (DS Pharma Biomedical Co., Ltd.) were transfected with human TNAP (OriGene Technologies, Inc.) using Lipofectamine LTX & Plus reagent (Invitrogen Corp.). On the next day, the medium was replaced with a fresh medium, and the cells were cultured in an incubator for 3 days. After 3 days, the culture supernatant was collected and concentrated by centrifugation at 5000 G for 30 minutes using Amicon 14, 104 cut (Merck Millipore). The concentrated culture supernatant was dialyzed against 5 L of 50 mM Tris/200 mM NaCl/1 mM MgCl2/20 μM ZnCl2 twice and used as an enzyme source (enzyme solution). The substrate pNPP (ProteoChem Inc.) was adjusted to 3.1 mM with Milli-Q water, and a solution of each test compound dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemical Industries, Ltd.) by 6 serial dilutions at a 5-fold common ratio from 100 μM, or DMSO was added thereto at a final concentration of 1% by volume. The enzyme solution adjusted to 2 μg/mL with an assay buffer (200 mM Tris/2 mM MgCl2/0.04 mM ZnCl2/0.01% Tween 20) was added in the same amount of the substrate solution and incubated at room temperature for 60 minutes. Then, the absorbance (ABS: 405 nm) was measured using a microplate reader (model plus 384, Molecular Devices, LLC), and the concentration of produced p-nitrophenol was calculated. The inhibition of human TNAP activity by the test compound was evaluated on the basis of the concentration IC50 at which each test compound suppressed 50% of p-nitrophenol production.
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 435.89, ALogP 2.76, HBA 8, HBD 1, PSA 108.23, QED 0.66
Compound (SMILES): CCNc1ncc2c(n1)N(C)CCN(c1cccc(COC(CCNCCF)c3cccs3)c1)C2=O
InChIKey: QRAORHCJNXMYOA-UHFFFAOYSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay: α2δ-1: Binding Assay to Human α2δ-1 Subunit of Cav2.2 Calcium Channel.Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 512.66, ALogP 4.27, HBA 8, HBD 2, PSA 82.62, QED 0.35
Compound (SMILES): COC(=O)N1c2ccc3c(nc(Cc4ccccc4)n3C3CC4(CNC4)C3)c2CC[C@@H]1C
InChIKey: UNPFLUDJJUKGKX-KRWDZBQOSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: HTRF Biochemical Assay for CBP and BRD4 Activity: The assay was performed in a final volume of 6 μL in assay buffer containing 50 mM Hepes (pH 7.5, (0.5M Hepes, pH 7.5 solution; Teknova H1575)), 0.5 mM GSH, 0.01% BGG (0.22 μM filtered, Sigma, G7516-25G), 0.005% BSA (0.22 μM filtered, EMD Millipore Corporation, 126575) and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 33 μM to 1.7 nM, top to lowest dose, respectively. 3 of 2× Protein and 3 μL of 2× Peptide Ligand were added to assay plates (pre-stamped with compound). Plates were incubated for varying times at room temperature prior to measuring the signal. TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) was measured on the PHERAstar (BMG, equipped with HTRF optic module [337/520/490]) or on the Envision (PerkinElmer, equipped with the TRF Laser unit, TRF dual mirror D400/D505 and emission filters M520 and M495). Data were reported as percent inhibition compared with control wells based on the following equation: % inh=1−((TR-FRET ratio−AveLow)/(AveHigh−AveLow)) where TR-FRET ratio=(Fluorescence at 520 nm/Fluorescence at 490 nm)*10000), AveLow=average TR-FRET ratio of no enzyme control (n=32), and AveHigh=average TR-FRET ratio of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm. For all assay formats data were reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured Fluorescence, AveLow=average Fluorescence of no enzyme control (n=32), and AveHigh=average Fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 430.55, ALogP 4.46, HBA 5, HBD 1, PSA 59.39, QED 0.67
Compound (SMILES): C[C@@H]1CN(C(=O)[C@@](C)(O)C(F)(F)F)Cc2cnc(-c3ccc(F)cc3F)n21
InChIKey: RDLMYQJAEROHOM-JDNHERCYSA-N
Target: [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q15119
Assay: Isothermal Titration Calorimetry: ITC measurements were performed with a VP-ITC micro calorimeter (Microcal, LLC/GE Healthcare Bio-Sciences AB, Uppsala, Sweden). In general titrations were performed by titrating the protein (50 μM) to the test compound (5 μM) in 12 μl injections. All binding experiments were carried out at 30° C. In general the test compounds were diluted form DMSO stock solutions into the measurement buffer with a maximum final concentration of 1% DMSO. The measurement buffer was 20 mM HEPES, 135 mM KCl, 1 mM TCEP, 2 mM MgCl2, 15 mM NaH2PO4, pH 7.5. The human PDHK2 (12-407) was produced in E. coli as his-tagged protein and purified by affinity chromatography. The tag was removed by side specific proteolysis. Before titration the protein buffer was changed to the measurement buffer containing the same DMSO concentration as the test compound dilution. ITC data analysis was performed using Origin 7 calorimetry software from the same supplier. For most measurements a binding model of one binding site was assumed. According to the applied mathematical model it is possible to calculate the binding constant (KA), the observed binding enthalpy (ΔHobs) as well as the stoichiometry (N) of the formed complex.
Measurement: Kd < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 389.32, ALogP 3.04, HBA 4, HBD 1, PSA 58.36, QED 0.8
Compound (SMILES): Cc1c(F)cccc1[C@]1(C(=O)NO)Cc2nc(-c3ccc(F)cc3)sc2C1
InChIKey: XESWLHUWEZFSSE-FQEVSTJZSA-N
Target: Histone deacetylase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UKV0
Assay: HDAC9 Biochemicle Assay: The potency of the compounds is quantified by measuring the Histone Deacetylase 9 (HDAC9) enzymatic activity using the fluorogenic substrate, Boc-Lys(Tfa)-AMC. The substrate is deacetylated to Boc-Lys-AMC by HDAC9. Cleavage by trypsin results in the release of the fluorophore AMC from the deacetylated substrate. The fluorescence of the sample is directly related to the histone deacetylase activity in the sample.
Measurement: IC50 = 193 nM (pChEMBL 6.710000038146973)
Descriptors: MW 386.42, ALogP 3.94, HBA 4, HBD 2, PSA 62.22, QED 0.53
Compound (SMILES): O=C(c1cc(-c2ccccc2)on1)N1CC2(C1)CN(c1ccccc1)C2
InChIKey: ORCOAQUKULWFKN-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Compound Profiling Methods: Strains expressing SCD1 or SCD5 as the sole desaturase, the human SCD1 and SCD5 genes were used to evaluate inhibition of SCD1/SCD5 using reduced growth as a surrogate for SCD inhibition. These yeast strains express human SCD1 or SCD5 from a plasmid harbored in a strain in which the yeast OLE1 gene is deleted.All compound profiling experiments were performed using the same basic protocol. Yeast were cultured using standard techniques in complete synthetic media lacking uracil and containing yeast nitrogen base supplemented with 2% (w/v) glucose (SD-Ura) Starter cultures were inoculated in 3 mL SD-Ura media containing 0.01% tween and 0.2 mM palmitoleic and oleic acid. Cultures were incubated overnight in a 30° C. shaker incubator (225 rpm). Saturated morning cultures were centrifuged, washed in SD-Ura media lacking TWEEN-20 and fatty acids, and then diluted 1:20 in fresh SD-Ura media also lacking TWEEN-20 and fatty acids. Cells were grown for 6 h to an  (optical density) of 0.4-0.8 at 30° C. with shaking.Compound stocks (10 mM in 100% DMSO) were arrayed into 384-round well, v-bottom polypropylene plates and diluted according to indicated dilution factors. Compound administration was performed in two separate steps. First, 15 μL of SD-Ura was dispensed into clear 384-well assay plates using a MULTIDROP Combi reagent dispenser. The diluted compound stock plates were then applied to the assay plates using an automated workstation (Perkin Elmer JANUS) outfitted with a 384-pin tool containing slotted pins that deliver 100 nL of compound. The cultures described above were centrifuged and washed with media lacking TWEEN-20 or oleic and palmitoleic acids. Cultures were then resuspended at a 2-fold concentrated  of 0.02 (final  of 0.0.01) in SD-Ura. 15 μL of diluted culture was then dispensed into the pinned assay plate to achieve 30 μL of the 1× culture (0.01) and a top drug concentration of 33.3 μM.After yeast delivery, assay plates were incubated under humidified conditions at 30° C. for 40 h. Yeast growth was monitored by reading the  of each well using a microplate reader (Perkin Elmer EnVision). Data were analyzed as follows. Raw data were processed by background subtracting and converting values to a percent of the nontreated condition for that strain [(EXP-0.035)/(DMSO-0.035)×100%].
Measurement: IC50 > 45000 nM (pChEMBL 4.3470001220703125)
Descriptors: MW 345.4, ALogP 3.3, HBA 4, HBD 0, PSA 49.58, QED 0.73
Compound (SMILES): O=C(O)c1csc(-c2ccc(/C=C/C34CCC(OCc5c(-c6c(Cl)cncc6Cl)noc5C5CC5)(CC3)CC4)cc2)n1
InChIKey: JMHHCBCASLRABU-BQYQJAHWSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/ml Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 622.57, ALogP 9.07, HBA 7, HBD 1, PSA 98.34, QED 0.2
Compound (SMILES): COc1nc(Nc2cnn([C@@H]3CCn4ccnc43)c2C)ncc1C(F)(F)F
InChIKey: CXWKIVRKCQHGDS-GFCCVEGCSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay : 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT, 5 nM LRRK2, 134 μM ATP, 60 minute reaction time, 23° C. reaction temperature, and 10 μL total reaction volume.Detection Reaction Conditions:1×TR-FRET dilution buffer, 10 mM EDTA, 2 nM antibody, 23° C. reaction temperature, and 10 μL total reaction volume.Compound solutions were prepared by initially diluting to 1 mM with DMSO. 35 μL of reference compound solution, 35 μL of test compound solution, and 35 μL HPE were successively added to the source plate (384-well assay plate, Labcyte). The plates were centrifuged at 2500 rpm for 1 minute and sealed in foil. POD was used to perform a 3.162 fold serial dilution and 100 nL of reference compound solution, test compound solution, HPE and ZPE were transferred to assay plates. The assay plate was centrifuged at 2500 rpm for 1 minute, and sealed with foil.
Measurement: IC50 > 60 nM (pChEMBL 7.2220001220703125)
Descriptors: MW 379.35, ALogP 2.94, HBA 8, HBD 1, PSA 82.68, QED 0.75
Compound (SMILES): CCn1ccc(-c2nnc(NS(=O)(=O)[C@@H](C)[C@@](O)(c3ncc(C)cn3)C(F)(F)F)n2-c2c(OC)cccc2OC)n1
InChIKey: SZAYJDTVNFMIBI-NPMXOYFQSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager (PerkinElmer, Inc.). All the results presented are means of several independent experiments and analyzed by non-linear regression methods using the commercially available program Prism (GraphPad, San Diego, Calif.) providing the EC50 values detailed in Table 40.
Measurement: EC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 596.59, ALogP 2.85, HBA 12, HBD 2, PSA 159.17, QED 0.28
Compound (SMILES): c1ccc(-c2ccnc3[nH]c(-c4n[nH]c5ccc(-c6cncc(CNCC7CCCC7)c6)cc45)nc23)nc1
InChIKey: YKNORYLVRMYGTC-UHFFFAOYSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: The screening assay for Wnt activity: Reporter cell lines can be generated by stably transducing cancer cell lines (e.g., colon cancer) or primary cells (e.g., IEC-6 intestinal cells) with a lentiviral construct that includes a Wnt-responsive promoter driving expression of the firefly luciferase gene.SW480 colon carcinoma cells were transduced with a lentiviral vector expressing luciferase with a human Sp5 promoter consisting of a sequence of eight TCF/LEF binding sites. SW480 cells stably expressing the Sp5-Luc reporter gene and a hygromycin resistance gene were selected by treatment with 150 μg/ml of hygromycin for 7 days. These stably transduced SW480 cells were expanded in cell culture and used for all further screening activities. For Sp5-Luc reporter gene assays, the cells were plated at 10,000 cells/well in 96-well plates with growth medium containing 10% fetal calf serum and incubated overnight at 37° C. and 5% CO2. Each compound was dissolved in DMSO as a 10 mM stock in standard j-vials and used to prepare compound source plates in dose-response format with 3-fold serial dilutions and a 10 mM top concentration. Compound transfer from serially diluted source plates to assay plates containing the cells was accomplished using a pintool (Multimek 96, Beckman equipped with V&P Scientific FP1S50H pins) based liquid handling protocol. This protocol used a slotted pin to transfer 50 nl of compound from a source plate well to an assay plate well containing 50 μl of cells in growth medium. The 1000-fold dilution resulted in a final DMSO concentration of 0.1% on the cells in each well. Control wells received 50 nl of DMSO treatment for normalization and calculating IC50 values. The treated cells were incubated at 37° C. and 5% CO2 for an additional forty-two hours. Following incubation, the growth medium was removed and 50 μl of BrightGlo luminescence reagent (Promega) was added to each well of the 96-well assay plates. The plates were placed on an orbital shaker for 5 min and then luminescence was quantified on the Victor3 (Perkin Elmer) plate reader. Readings were normalized to DMSO only treated cells, and normalized activities were utilized for IC50 calculations using the dose-response log (inhibitor) vs. responsevariable slope (four parameters) nonlinear regression feature available in GraphPad Prism 5.0 or 6.0.
Measurement: IC50 = 1462 nM (pChEMBL 5.829999923706055)
Descriptors: MW 500.61, ALogP 5.91, HBA 6, HBD 3, PSA 108.06, QED 0.25
Compound (SMILES): C=CC(=O)N1CC(Nc2cncc(-c3cnc(Oc4cccc(F)c4)nc3)n2)C1
InChIKey: PBZDGAXQEUUOPY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTK and EGFR in Vitro Assay: The BTK and EGFR biochemical assays utilize LANTHASCREEN® Eu Kinase Binding Assays from Thermo Fisher Scientific, measuring the binding of the kinase with a fluorinated tracer. The assay buffer consists of 50 mM HEPES pH 7.5, 0.01% BRU-35, 10 mM MgCl2, 1 mM EGTA, and 1 mM DTT. The test compounds are diluted and added to the assay plate with a Labcyte ECHO® 555 liquid handler. The compounds are tested in 10 point dose response curves (100 μM-0.005 μM) with a maximum DMSO concentration of 1%. The assays are performed in 20 μL in low-volume 384-well white proxiplates. For the BTK assay, the concentration of full-length His-labeled BTK in the assay is 5 nM, the Eu-anti-His antibody is 2 nM, and the kinase tracer 236 is 60 nM. For the EGFR assay, the concentration of truncated (amino acids 668-1210) GST-labeled EGFR is 5 nM, the Eu-anti-GST antibody is 2 nM, and the kinase tracer 199 is 10 nM. The components of the assay (compound, enzyme/antibody, tracer) are assembled and incubated for 30 min prior to being read on a Perkin-Elmer EnVision with excitation at 340 nM, tracer emission at 665 nM, and antibody emission at 615 nM. The signal ratio is converted to percent inhibition using the following equation: % Inhibition=100−[(Test Compound−Median Minimum)/(Median Maximum−Median Minimum)×100].
Measurement: IC50 = 19.3 nM (pChEMBL 7.710000038146973)
Descriptors: MW 392.39, ALogP 2.67, HBA 7, HBD 1, PSA 93.13, QED 0.64
Compound (SMILES): COc1cnc2c(Nc3ccc(F)c([C@]4(C)CS(=O)(=O)C(C)(C)C(N)=N4)c3)cccc2n1
InChIKey: PXGUCDPWUSFXIE-QFIPXVFZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vivo Inhibition of Beta-Secretase: Several animal models, including mouse, rat, dog, and monkey, may be used to screen for inhibition of beta-secretase activity in vivo following administration of a test compound sample. Animals used in this invention can be wild type, transgenic, or gene knockout animals. For example, the Tg2576 mouse model, prepared and conducted as described in Hsiao et al., 1996, Science 274, 99-102, and other non-transgenic or gene knockout animals are useful to analyze in vivo inhibition of Amyloid beta peptide (Abeta) production in the presence of inhibitory test compounds. Generally, 2 to 18 month old Tg2576 mice, gene knockout mice or non-transgenic animals are administered test compounds formulated in vehicles, such as cyclodextran, phosphate buffers, hydroxypropyl methylcellulose or other suitable vehicles. One to twenty-four hours following the administration of compound, animals are sacrificed, and brains as well as cerebrospinal fluid (CSF) and plasma are removed for analysis of A-beta levels and drug or test compound concentrations (Dovey et al., 2001, Journal of Neurochemistry, 76, 173-181) Beginning at time 0, animals are administered by oral gavage, or other means of delivery such as intravenous injection, an inhibitory test compound of up to 100 mg/kg in a standard, conventional formulation, such as 2% hydroxypropyl methylcellulose, 1% Tween80. A separate group of animals receive 2% hydroxypropyl methylcellulose, 1% Tween80 alone, containing no test compound, and serve as a vehicle-control group. At the end of the test period, animals are sacrificed and brain tissues, plasma or cerebrospinal fluid are collected. Brains are either homogenized in 10 volumes (w/v) of 0.2% diethylamine (DEA) in 50 mM NaCl (Best et al., 2005, Journal of Pharmacology and Experimental Therapeutics, 313, 902-908), or in 10 volumes of 0.5% TritonX-100 in Tris-buffered saline (pH at about 7.6). Homogenates are centrifuged at 355,000 g, 4° C. for 30 minutes. CSF or brain supernatants are then analyzed for the presence of A-beta peptide by specific sandwich ELISA assays based on ECL (Electrochemiluminescence) technology.
Measurement: IC50 = 11.4 nM (pChEMBL 7.940000057220459)
Descriptors: MW 457.53, ALogP 3.3, HBA 8, HBD 2, PSA 119.56, QED 0.62
Compound (SMILES): CC(c1ccc2c(c1)OCO2)N1CCN(c2cc(Cl)ncn2)CC1
InChIKey: YETOTNSLBAWPQH-UHFFFAOYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Human O-GlcNAcase enzyme inhibition assay: 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λexc 485 nm; (λemm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).
Measurement: IC50 = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 346.82, ALogP 2.74, HBA 6, HBD 0, PSA 50.72, QED 0.8
Compound (SMILES): C=CC(=O)N1CC[C@@H](c2nc(-c3ccc(C(=O)Nc4ccccn4)cc3)c3cnccn23)C1
InChIKey: JWWQEEHPCNMZMX-LJQANCHMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assay: Btk and Other Kinases: Btk kinase activity was determined using a homogenous time resolved fluorescence (HTRF) methodology. Measurements were performed in a reaction volume of 15 μL using 384-well assay plates. Kinase enzyme, inhibitor, ATP and 1 μM peptide substrate were incubated in a reaction buffer compose of Hepes50 mM (pH7.0), NaN3 0.02%, BSA 0.01%, Orthocanadate 0.1 mM. After one hour, the kinase reaction was quenched by the addition of Eμ-labeled antibody and XL-665 in 1×Detection buffer containing 60 mM EDTA (Cisbio), and the mixture was allowed to incubate for one hour. The HTRF signal was measured on a multimode plate reader (EnVision® Multilabel Reader, Perkin Elmer) with an excitation wavelength (2Ex) of 330 nm and detection wavelengths (λEm) of 615 and 665 nm. Activity was determined by the ratio of the fluorescence at 665 nm to that at 615 nm. For each compound, enzyme activity as measured at various concentrations of compound, Negative control reactions were performed in the absence of inhibitor in two replicates and eight no enzyme controls were used to determine baseline fluorescence levels.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 438.49, ALogP 3.55, HBA 6, HBD 1, PSA 92.49, QED 0.48
Compound (SMILES): COC1=CC=C(C)N2N=C(/C=C\c3nc(-c4cccs4)cn3C)NC12
InChIKey: FNFVWGFTSINNHN-HJWRWDBZSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: PDE10A Inhibition Assay: 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 HEPES7.6; 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. The measured signal can be converted to activity relative to an uninhibited control (100%) and IC50 values can be calculated using the Xlfit extension to EXCEL.
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 353.45, ALogP 3.15, HBA 7, HBD 1, PSA 54.68, QED 0.92
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(-c3ccc(=O)n(C)n3)nn2-c2ccccc2)[C@H](c2ccc(F)cc2)C1
InChIKey: SVFQUMDXMLLEOP-UKILVPOCSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Kinase Binding Assay: TrkA binding activity was determined in a TrkA LanthaScreen Eu Kinase Binding Assay. 5 nM His-tagged recombinant human TrkA (6HIS tagged cytoplasmic domain from Invitrogen, Catalog No. PV3144) was incubated with 4 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat. No. PV5592), 2 nM biotinylated anti-His (Invitrogen Cat. No. PV6090), and 2 nM europium-labeled Streptavidin (Invitrogen Cat. No. PV5899), in buffer (25 mM MOPS, pH 7.5, 5 mM MgCl2, 0.005% Triton X-100). Three fold serial dilutions of compounds of the invention in DMSO were added to a final percentage of 2% DMSO. After 60-minute incubation at 22° C., the reaction was measured using the EnVision mutlimode plate reader (PerkinElmer) via TR-FRET dual wavelength detection at 615 nM and 665 nM. The percent of control was calculated using a ratiometric emission factor.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 545.62, ALogP 3.32, HBA 8, HBD 2, PSA 106.31, QED 0.35
Compound (SMILES): Cc1cc(-c2n[nH]c3cc4c(cc23)CN(Cc2cc(F)cc(F)c2)C(=O)N4)cc(C)n1
InChIKey: WXGIRMKNHMIAJO-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Z′-LYTE Kinase Assay: The inhibition of ERK activity by the compounds disclosed herein was determined using the Z′-LYTE kinase assay kit (Life Technologies) with a Ser/Thr 3 peptide substrate (Life Technologies) according to manufacturers' instructions. The assay was run with an ERK2 enzyme (Life Technologies) concentration of 0.47 ng/μL at 100 μM ATP (approximately the ATP Km for ERK2). The IC50 values for the compounds were determined with 3-fold serial dilutions in duplicate. The compounds were first diluted in 1:3 dilutions in 100% DMSO at 100× the desired concentration, and then further diluted (1:25) in 20 mM HEPES buffer (Invitrogen) to make 4× solutions prior to adding to the enzyme solution. The final DMSO concentration in the assay was 1%. Final reaction volume was 20 μL/well in 384-well plates. Kinase reactions were conducted or 1 hour followed by the assay development reaction (1 hour) in a 20 ul/well in a 384 well plate format.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 419.44, ALogP 5.07, HBA 3, HBD 2, PSA 73.91, QED 0.49