lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(c1nn2c(C3CCOCC3)ncc2c(=O)[nH]1)N1CC(c2ccc(N(C)C)cc2)C1
InChIKey: QBUPOLYZNAIZLK-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition Assay: A PDE9 assay may for example, be performed as follows: The assay is performed in 60 uL samples containing a fixed amount of the relevant PDE enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 225 pCi of 3H-labelled cyclic nucleotide substrate, tritium labeled cAMP to a final concentration of 5 nM and varying amounts of inhibitors. Reactions are initiated by addition of the cyclic nucleotide substrate, and reactions are allowed to proceed for one hr at room temperature before being terminated through mixing with 15 uL 8 mg/mL yttrium silicate SPA beads (Amersham). The beads are allowed to settle for one hr in the dark before the plates are counted in a Wallac 1450 Microbeta counter.
Measurement: IC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 422.53, ALogP 2.54, HBA 7, HBD 1, PSA 78.76, QED 0.68
Compound (SMILES): CN1CCN(CCNC(=O)c2ccc(NC(=O)N3Cc4ccc(F)cc4C3)cc2)CC1
InChIKey: DXPGWRWIFUFEST-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 = 33.2 nM (pChEMBL 7.480000019073486)
Descriptors: MW 425.51, ALogP 2.35, HBA 4, HBD 2, PSA 67.92, QED 0.77
Compound (SMILES): COc1ccccc1CNc1ncnc2c1ncn2[C@@H]1O[C@H](COS(N)(=O)=O)[C@@H](O)[C@H]1O
InChIKey: MJRQITOMRHUDAZ-SCFUHWHPSA-N
Target: NEDD8-activating enzyme E1 catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TBC4
Assay: Inhibition of recombinant human untagged NAEalpha/N-terminal His-tagged NAEbeta expressed in Bac-to-Bac expression system using FLAG-tagged NEDD8 as substrate after 90 mins in presence of GST-tagged Ubc12 by HTRF assay
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 466.48, ALogP -0.71, HBA 12, HBD 4, PSA 183.94, QED 0.33
Compound (SMILES): CC1CCN(C(=O)C(CCn2ccnn2)NS(=O)(=O)c2cc(Cl)c(N)c(Cl)c2)CC1
InChIKey: JIGFQRQBGRUWAJ-UHFFFAOYSA-N
Target: C-C chemokine receptor type 10 (SINGLE PROTEIN), Homo sapiens
UniProt: P46092
Assay: Antagonist activity at human CCR10 expressed in CHOK1 cells coexpressing aequorin/Galphaq assessed as inhibition of human CCL27-dependent calcium flux in presence of coelenterazine H by chemiluminescence assay
Measurement: IC50 > 19952.62 nM (pChEMBL 4.699999809265137)
Descriptors: MW 475.4, ALogP 2.16, HBA 7, HBD 2, PSA 123.21, QED 0.59
Compound (SMILES): CCOC(=O)c1cc2cnc(Nc3ccc(N4CCNCC4)cc3)nc2n(C2CCCC2)c1=O.Cl
InChIKey: FJRMZRFQOLFFKE-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: Inhibition of Cdk4/Cyclin D (unknown origin) using retinoblastoma protein substrate and [gamma32P]ATP incubated for 15 mins by beta plate counter based assay
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 462.55, ALogP 3.24, HBA 9, HBD 2, PSA 101.38, QED 0.54
Compound (SMILES): N#Cc1cccc(Cc2ccc(C(=O)NCc3c[nH]c4ccc(Cl)cc34)cc2)c1
InChIKey: FMVJVHQRHHSMMD-UHFFFAOYSA-N
Target: Alpha-synuclein (SINGLE PROTEIN), Homo sapiens
UniProt: P37840
Assay: In Vitro Inhibition Assay: α-Synucleinopathies are characterised by aggregation of α-synuclein in neurons. Aggregation of purified α-synuclein is performed essentially as described by Gerard et al. FASEB. 20(3):524-6 (2006). 20-100 μg purified α-synuclein (Sigma; S7820) at a concentration of about 2.5 μg/mL is incubated in the presence of spermin (250 μM) or paraquat (32 mM) or 6-hydroxydopamine (400 μM) or vehicle in a 384 well plate. Spermin, paraquat and 6-hydroxydopamine promote the α-synuclein aggregation process. Aggregation kinetics is determined by measuring turbidity at 340 nm, every 1-15 minutes for at least one hour.
Measurement: EC50 = 132 nM (pChEMBL 6.880000114440918)
Descriptors: MW 399.88, ALogP 5.21, HBA 2, HBD 2, PSA 68.68, QED 0.48
Compound (SMILES): CC1(C)CCCN(C(=O)c2ncc(C#Cc3ccccc3)cn2)C1
InChIKey: UCYCJYJSMSDBIV-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Intracellular Ca2+ Mobilization Assay: A monoclonal HEK-293 cell line stably transfected with a cDNA encoding for the human mGlu5a receptor was generated; for the work with mGlu5 Positive Allosteric Modulators (PAMs), a cell line with low receptor expression levels and low constitutive receptor activity was selected to allow the differentiation of agonistic versus PAM activity. Cells were cultured according to standard protocols (Freshney, 2000) in Dulbecco's Modified Eagle Medium with high glucose supplemented with 1 mM glutamine, 10% (vol/vol) heat-inactivated bovine calf serum, Penicillin/Streptomycin, 50 ug/ml hygromycin and 15 ug/ml blasticidin (all cell culture reagents and antibiotics from Invitrogen, Basel, Switzerland).About 24 hrs before an experiment, 5x104 cells/well were seeded in poly-D-lysine coated, black/clear-bottomed 96-well plates. The cells were loaded with 2.5 uM Fluo-4AM in loading buffer (1xHBSS, 20 mM HEPES) for 1 hr at 37° C. and washed five times with loading buffer.
Measurement: EC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 319.41, ALogP 3.14, HBA 3, HBD 0, PSA 46.09, QED 0.76
Compound (SMILES): CC(C)(CO)n1cc(C(=O)c2cncc(NC(=O)Cn3cc(C4CC4)nn3)c2)c2cnccc21
InChIKey: MCRWPTCPZNERRX-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: HTRF Assay: Isolated TRK Enzyme assays use the HTRF KinEASE-TK kit (Cisbio Cat#62TK0PEJ) with recombinant His-tagged cytoplasmic domains of each TRK receptor sourced from Invitrogen (see table below). This activity-assay measures the phosphorylation of tyrosine residues within a substrate from the HTRF kit which has been validated by Cisbio for a variety of tyrosine kinases including the TRK receptors.Assay Details: Invitrogen Amino FAC FAC Assay ReactionTarget Cat# acids enzyme ATP TimeTRKA PV3144 aa 441-4 nM 40 uM 35 min (NTRK1) 796 TRKB PV3616 aa 526-1 nM 1.4 uM 40 min (NTRK2) 838 TRKC PV3617 aa 510- 10 nM 15 uM 30 min (NTRK3) 8250.5 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135 disclosed in WO2005/116035 of 150 uM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 uM.
Measurement: IC50 = 12.4 nM (pChEMBL 7.909999847412109)
Descriptors: MW 459.51, ALogP 2.5, HBA 9, HBD 2, PSA 127.82, QED 0.39
Compound (SMILES): Cc1nc(C(NC(=O)c2ccc(C3CC3)c(OCC3CC3)n2)C2CC2)no1
InChIKey: LIVIAQCOPGNLQD-UHFFFAOYSA-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.times.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.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul 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 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM 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.
Measurement: EC50 = 20.4 nM (pChEMBL 7.690000057220459)
Descriptors: MW 368.44, ALogP 3.32, HBA 6, HBD 1, PSA 90.14, QED 0.77
Compound (SMILES): Cn1c(SCCCN2CC[C@]3(C[C@@H]3c3ccc(C(F)(F)F)cc3)C2)nnc1-c1ccc(C#N)cc1
InChIKey: ITIATPRLDJMYTO-RDGATRHJSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]-spiperone from human dopamine D3 receptor expressed in CHO-K1 cell membranes after 90 mins by liquid scintillation counting
Measurement: Ki = 0.263 nM (pChEMBL 9.579999923706055)
Descriptors: MW 497.59, ALogP 5.73, HBA 6, HBD 0, PSA 57.74, QED 0.31
Compound (SMILES): FC(F)(F)c1ccnc(Nc2ccc(N3CCOCC3)c(-c3ccccc3)c2)n1
InChIKey: JDHOIODVEBKRPT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 μM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 1694 nM (pChEMBL 5.769999980926514)
Descriptors: MW 400.4, ALogP 4.74, HBA 5, HBD 1, PSA 50.28, QED 0.69
Compound (SMILES): CC(C)Oc1ccc(Nc2ncc(NCCCO)c(=O)n2Cc2ccc(Cl)cc2)cc1F
InChIKey: WCFYOABKWMKPHV-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 = 724 nM (pChEMBL 6.139999866485596)
Descriptors: MW 460.94, ALogP 4.41, HBA 7, HBD 3, PSA 88.41, QED 0.39
Compound (SMILES): O=C(O)Cn1nnc(-c2cnc(N3CCC4(CC3)COc3ccccc34)s2)n1
InChIKey: SRNNTPKXTMAMJQ-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Enzyme Activity Assay: The potency of compounds of formula I against the stearoyl-CoA desaturase was determined by measuring the conversion of radiolabeled stearoyl-CoA to oleoyl-CoA using rat liver microsome or human SCD1 following previously published procedures with some modifications (Joshi, et al., J. Lipid Res., 18: 32-36 (1977); Talamo, et al., Anal. Biochem, 29: 300-304 (1969)). Liver microsome was prepared from male Wistar or Sprague Dawley rats on a high carbohydrate diet for 3 days (LabDiet #5803, Purina). The livers were homogenized (1:10 w/v) in a buffer containing 250 mM sucrose, 1 mM EDTA, 5 mM DTT and 50 mM Tris-HCl (pH 7.5). After a 100,000xg centrifugation for 60 min, the liver microsome pellet was suspended in a buffer containing 100 mM sodium phosphate, 20% glycerol, 2 mM DTT, and stored at -78° C. Human SCD1 desaturase system was reconstituted using human SCD1 from a baculovirus/Sf9 expression system, cytochrome B5 and cytochrome B5 reductase.
Measurement: IC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 398.45, ALogP 1.81, HBA 9, HBD 1, PSA 106.26, QED 0.71
Compound (SMILES): O=C(Nc1sc(N2CCN(CC(F)(F)F)CC2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: VMANNCSXRMMWLK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.38 nM (pChEMBL 8.859999656677246)
Descriptors: MW 587.96, ALogP 5.04, HBA 9, HBD 1, PSA 87.89, QED 0.3
Compound (SMILES): Cc1ccc(-c2noc(C3CC(F)(F)C3)n2)cc1NC(=O)c1cnc2ccc(COCC(F)F)cn12
InChIKey: UVTVIZDRFNGZGM-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell Proliferation Assay: The compounds was tested for inhibition of SCF dependent proliferation using human Mo7e cells which endogenously express c-kit in a 384 well format. Three-fold serially diluted test compounds (Cmax=10 mM) were evaluated for their antiproliferative activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37° C., cell viability was measured by adding 25 uL of CellTiter Glo (Promega) to the cells and the luminescence was measured by a CLIPR CCD camera (Molecular Devices).
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 503.46, ALogP 5.24, HBA 7, HBD 1, PSA 94.55, QED 0.33
Compound (SMILES): COc1cc2ncnc(Nc3ccc(F)c(Cl)c3)c2cc1NC(=O)/C=C/CN(C(C)C)C(C)C
InChIKey: LJRGBEPLXWZRBM-VOTSOKGWSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Irreversible inhibition of GST-tagged ERBB1 (unknown origin) (Met-668 to Ala-1211 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 485.99, ALogP 5.79, HBA 6, HBD 2, PSA 79.38, QED 0.37
Compound (SMILES): COc1ccc(COc2ccc3nc4ccc(=O)cc-4oc3c2)cc1
InChIKey: PSOPUJORTBRJMH-UHFFFAOYSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: In Vitro Competitive Radioligand Binding Assay: To determine the Ki values, fixed brain tissues prepared from 15-month-old Tg2576 mice (N=5) will be incubated for 30 min with 1 uM methoxy-X34 in the presence of a serial dilution of test compounds (10-9-10-6 M, 6 concentrations/compound). Tissue sections (3 sections per reaction) will be then mounted and coverslipped. Photographs of methoxy-X34 binding images will be taken using a Nikon fluorescent microscope. The fluorescent intensity on cerebral amyloid deposits and neuritic plaques will be separately quantified using Image J software.
Measurement: Ki = 218 nM (pChEMBL 6.659999847412109)
Descriptors: MW 333.34, ALogP 3.88, HBA 5, HBD 0, PSA 61.56, QED 0.53
Compound (SMILES): COCCNc1cc(NC(=O)N2CCCc3cc(C4CCN(C5COC5)CC4)c(C=O)nc32)ncc1C#N
InChIKey: CFHKNWFGGBERAE-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Mus musculus
UniProt: Q03142
Assay: Inhibition of FGFR4 in mouse BAF3 cells assessed as decrease in FGFR4 phosphorylation incubated for 40 mins
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 519.61, ALogP 2.78, HBA 9, HBD 2, PSA 132.71, QED 0.4
Compound (SMILES): c1cc(-c2nc3ccc(N4CCCC(c5ncc6n5CCC6)C4)nc3[nH]2)nc(C2CC2)n1
InChIKey: HYSQCOTYMHPUMU-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: In Vitro DGAT2 Assay: For determination of IC50 values, the reactions were carried out in 384-well white Polyplates (Perkin Elmer) in a total volume of 20 uL. To 1 uL of compounds dissolved in 100% DMSO and spotted at the bottom of each well, 5 uL of 0.04% bovine serum albumin (BSA) (fatty acid free, Sigma Aldrich) was added and the mixture was incubated at room temperature for 20 minutes. To this mixture, 10 uL of hDGAT2 membrane fraction (0.01 mg/mL) diluted in 100 mM Hepes-NaOH, pH 7.4, 20 mM MgCl2 containing 200 nM methyl arachidonyl fluorophosphonate (Cayman Chemical; dried from ethyl acetate stock solution under argon gas and dissolved in DMSO as 5 mM stock) was added. After this mixture was preincubated at room temperature for 2 hours, DGAT2 reactions were initiated by the addition of 4 uL of substrates containing 30 uM [1-14C]decanoyl-CoA (custom-synthesized by Perkin Elmer, 50 mCi/mmol) and 125 uM 1,2-didecanoyl-sn-glycerol (Avanti Polar Lipids) dissolved in 12.5% acetone. The reaction mixtures were incubated at room temperature for 40 min and the reactions were stopped by addition of 5 uL of 1% H3PO4. After the addition of 45 uL MicroScint-E (Perkin-Elmer), plates were sealed with Top Seal-A covers (Perkin-Elmer) and phase partitioning of substrates and products was achieved using a HT-91100 microplate orbital shaker (Big Bear Automation, Santa Clara, Calif.). Plates were centrifuged at 2,000×g for 1 min in an Allegra 6R Centrifuge (Beckman Coulter) and then were sealed again with fresh covers before reading in a 1450 Microbeta Wallac Trilux Scintillation Counter (Perkin Elmer). DGAT2 activity was measured by quantifying the generated product [14C]tridecanoylglycerol in the upper organic phase.
Measurement: IC50 = 252 nM (pChEMBL 6.599999904632568)
Descriptors: MW 426.53, ALogP 3.82, HBA 7, HBD 1, PSA 88.41, QED 0.53
Compound (SMILES): COc1nc2ccc(C(O)(c3ccc(N(C)C)cc3)c3cncn3C)cc2c(Cl)c1Cc1ccc(-n2cccn2)cc1
InChIKey: HTRJODMUEHDFAM-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1 °C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20. Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1×=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
Measurement: Kd = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 579.1, ALogP 5.76, HBA 8, HBD 1, PSA 81.23, QED 0.25
Compound (SMILES): CC1CN(Cc2ccccc2)CC1c1nn2c(C3=CCOCC3)ncc2c(=O)[nH]1
InChIKey: KZOJNVNGLJDSCN-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition Assay: A PDE9 assay may for example, be performed as follows: The assay is performed in 60 uL samples containing a fixed amount of the relevant PDE enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 225 pCi of 3H-labelled cyclic nucleotide substrate, tritium labeled cAMP to a final concentration of 5 nM and varying amounts of inhibitors. Reactions are initiated by addition of the cyclic nucleotide substrate, and reactions are allowed to proceed for one hr at room temperature before being terminated through mixing with 15 uL 8 mg/mL yttrium silicate SPA beads (Amersham). The beads are allowed to settle for one hr in the dark before the plates are counted in a Wallac 1450 Microbeta counter.
Measurement: IC50 = 281 nM (pChEMBL 6.550000190734863)
Descriptors: MW 391.48, ALogP 2.46, HBA 6, HBD 1, PSA 75.52, QED 0.74
Compound (SMILES): CN(Cc1cccc(-c2cccnc2)c1)C(=O)OC(C(F)(F)F)C(F)(F)F
InChIKey: ODXJGFKCMDXJSM-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08914
Assay: In Vitro Competitive Activity-Based Protein Assay: Proteomes (mouse brain membrane fraction or cell lysates) (50 uL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37 C. After 30 min, FP-Rh (1.0 uL, 50 uM in DMSO) was added and the mixture was incubated for another 30 min at 37 C. Reactions were quenched with SDS loading buffer (50 uL-4x) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL, ABHD6 and FAAH using ImageJ 1.43u software.Preparation of Mouse Brain Proteomes from Inhibitor Treated Mice. Inhibitors were administered to wild-type C57Bl/6J by oral gavage in a vehicle of polyethylene glycol. Each animal was sacrificed 4 h following administration and brain proteomes were prepared and analyzed according to previously established methods (See Niphakis, M. J., et al. (2011) ACS Chem. Neurosci. and Long, J. Z., et al. Nat. Chem. Biol. 5:37-44).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 392.3, ALogP 4.81, HBA 3, HBD 0, PSA 42.43, QED 0.7
Compound (SMILES): CC(C)(CO)NC(=O)c1ccc(N2CC(F)(F)C2)c(OCC2CC2)n1
InChIKey: JLMSBICRLNNQIJ-UHFFFAOYSA-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.times.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.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul 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 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM 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.
Measurement: EC50 = 84.1 nM (pChEMBL 7.079999923706055)
Descriptors: MW 355.39, ALogP 1.83, HBA 5, HBD 2, PSA 74.69, QED 0.78
Compound (SMILES): O=C(c1nnc(NCCc2ccncc2)o1)c1nc2ccc(-c3ccccc3)cc2s1
InChIKey: XUKWIIFDGKRAFB-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Enzymatic Assay: The enzymatic assay was measured using white, opaque 96-well half area plates. Each well contained 60 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 2 ul of a DMSO solution containing compound of interest. Conditioned media obtained from HT-1080 cells, which were transformed by RAGE technology (Athersys) to overexpress endogenous EL, was added and the reaction was allowed to incubate for 20 min at 37° C. with gentle agitation. The reaction was started by the addition of 20 μL of a 1:4 dilution of vesicles. The final total reaction volume was 100 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 488 nm and an emission of 530 nm. Readings were taken every 20 seconds for 10 min with agitation between each reading.
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 427.49, ALogP 4.63, HBA 8, HBD 1, PSA 93.8, QED 0.38
Compound (SMILES): NCc1ccc(CNC(=O)[C@H](Cc2ccccc2)NC(=O)[C@@H](CC2CCCCC2)NCC(=O)O)cc1
InChIKey: CFVWQRMYGPSOPB-RPBOFIJWSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Inhibition Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stiirzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 494.64, ALogP 2.5, HBA 5, HBD 5, PSA 133.55, QED 0.29
Compound (SMILES): CC(=O)Oc1cc2cccc(OC(C)=O)c(=O)c2c(O)c1OC(C)=O
InChIKey: HJIVTYBHKLKJSH-UHFFFAOYSA-N
Target: Toll-like receptor 1/2 (PROTEIN COMPLEX), Mus musculus
UniProt: Q9EPQ1
Assay: Antagonist activity at TLR1/2 in mouse RAW264.7 cells assessed as inhibition of Pam3CSK4-induced receptor activation by measuring NO production after 24 hrs
Measurement: IC50 = 1420 nM (pChEMBL 5.849999904632568)
Descriptors: MW 346.29, ALogP 1.68, HBA 8, HBD 1, PSA 116.2, QED 0.66
Compound (SMILES): CC(C)n1cc(-c2nc(-c3ccc4c(c3)CCN4C)cnc2N)nn1
InChIKey: OMIVQNOGOUDDNN-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Kinase Activity Assay: The kinase reaction buffer consists of 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, and 2 mM DTT. In the assay, 1 nM LRRK2 WT or 250 pM LRRK2 G2019S kinase is incubated with the test compound (typically at 0 to 30 μM) for 30 minutes before the kinase reaction is initiated by addition of 1.3 mM ATP and 0.4 μM fluorescein-LRRKtide. The reaction mixture (20 μl total volume) is incubated for 2 hours at 30 °C. before the reaction is terminated by addition of 10 mM EDTA and 1 nM terbium-labelled anti-phospho-LRRKtide antibody (final volume 20 μl). The mixture is further incubated for 30 minutes at RT. TR-FRET is measured by excitation of the terbium-donor with 340 nm light and subsequent (delay time 100 μs) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a time window of 1000 μs. The measurement is repeated 10 times for fluorescein and 10 times for terbium emission with a 2000 μs time window between repeats. TR-FRET measurements are performed on a Biomek Synergy plate. The TR-FRET signal is calculated as the emission-ratio at 520 nm over 495 nm.
Measurement: IC50 = 340 nM (pChEMBL 6.46999979019165)
Descriptors: MW 335.42, ALogP 2.56, HBA 7, HBD 1, PSA 85.75, QED 0.79
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)nc(NCc4ccc(Cl)nc4)nc23)N1
InChIKey: MJPBZUYTZIPIGM-XGICHPGQSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 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; 75 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 the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 425.84, ALogP 1.54, HBA 9, HBD 4, PSA 138.23, QED 0.26
Compound (SMILES): CC(=O)N[C@H](CC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C)Cc1ccccc1
InChIKey: NVVMMXTZCXILDY-SDWRPPRWSA-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 = 366 nM (pChEMBL 6.440000057220459)
Descriptors: MW 2461.9
Compound (SMILES): Cc1n[nH]c(C)c1S(=O)(=O)N1CCC(=C(C#N)c2c(F)cccc2F)CC1
InChIKey: SIKDDFXGAULCSX-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TCW9
Assay: Antagonist activity at human PKR1 expressed in RBL2H3 cells assessed as inhibition of prokineticin1-mediated intracellular calcium release preincubated for 10 mins followed by PK1 addition by FLIPR assay
Measurement: IC50 = 3680 nM (pChEMBL 5.429999828338623)
Descriptors: MW 392.43, ALogP 3.07, HBA 4, HBD 1, PSA 89.85, QED 0.81
Compound (SMILES): NC(=O)c1cc(-c2ccc(F)cc2)c2ccc(CCc3cncnc3)cc2n1
InChIKey: YSZAXBGUMAMQQB-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Binding affinity to human mGluR2 expressed in cell membrane incubated for 1 hr in presence of tritium labeled [3H]-7-((2,5-dioxopyrrolidin-1-yl)methyl)-4-(4-fluorophenyl)quinoline-2-carboxamide by competitive radioligand binding assay
Measurement: Ki = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 372.4, ALogP 3.72, HBA 4, HBD 1, PSA 81.76, QED 0.58
Compound (SMILES): COc1cc(CN(C)C(C)c2ccc3c(c2)CCO3)ccc1OCCN1C(=O)CSC1=O
InChIKey: MHCRYSMPKCIWGG-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 456.56, ALogP 3.9, HBA 7, HBD 0, PSA 68.31, QED 0.57
Compound (SMILES): O=C(c1cc(Cc2n[nH]c(=O)c3c2NCCC3)ccc1F)N1CCN(S(=O)(=O)c2ccccc2)CC1
InChIKey: DRKOYYKZTFWVOO-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 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 511.58, ALogP 2, HBA 6, HBD 2, PSA 115.47, QED 0.54
Compound (SMILES): FC(F)(F)c1cc(-c2noc(CCC3(c4ccccc4)CCCCC3)n2)cc(C(F)(F)F)c1
InChIKey: ROGFJMYMAMLLTI-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: GTPgammaS Binding Assay: Compounds were loaded in a 384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Ga15-bla HEK293T cells were added to the compound plate (40 ul/well, final protein 3 ug/well) with MULTIDROP. [35S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 10 uM GDP, 0.1% fatty acid free BSA, and 10 ug/ml Saponin to 0.4 nM. 40 ul of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11 Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 ul) was added to each well.
Measurement: EC50 = 280 nM (pChEMBL 6.550000190734863)
Descriptors: MW 468.44, ALogP 7.61, HBA 3, HBD 0, PSA 38.92, QED 0.36
Compound (SMILES): COc1cc(N2CCC(N3CCC(O)CC3)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: JVTDZPUCHJEKQC-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 730.82, ALogP 7.17, HBA 10, HBD 3, PSA 120.15, QED 0.14
Compound (SMILES): COc1c(C)cccc1-c1cccc(C(CC(=O)O)c2nnc(C)o2)c1
InChIKey: PYZVIINPOKEBHH-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37° C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.
Measurement: IC50 = 571 nM (pChEMBL 6.239999771118164)
Descriptors: MW 352.39, ALogP 3.97, HBA 5, HBD 1, PSA 85.45, QED 0.72
Compound (SMILES): O=C(O)[C@@H]1[C@@H]2C[C@@H](C(=O)N3CC[C@](c4ccc(C(F)(C(F)(F)F)C(F)(F)F)cc4)(S(=O)(=O)c4ccc(F)cc4)C3)C[C@@H]21
InChIKey: HNOZQRKROBDUDI-VYNBFOJNSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 1283 nM (pChEMBL 5.889999866485596)
Descriptors: MW 625.53, ALogP 5.37, HBA 4, HBD 1, PSA 91.75, QED 0.34
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](CC(=O)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: NMYVGSYWXNPPHW-KQUOGIPMSA-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 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 2473.82
Compound (SMILES): CC(C)Oc1ccc(-c2nc(-c3cccc4c3CC[C@H]4N[C@H](C)C(=O)O)no2)cc1C#N
InChIKey: IYWOYGVPVALNEZ-JLTOFOAXSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 < 0.05 nM (pChEMBL 10.300999641418457)
Descriptors: MW 432.48, ALogP 4.11, HBA 7, HBD 2, PSA 121.27, QED 0.57
Compound (SMILES): CC(c1nc2c(cnn2C2CCOCC2)c(=O)[nH]1)N1CC(Oc2ccc(C#N)cc2F)C1.O=C(O)C(F)(F)F
InChIKey: LFDNXZPAFBNKCN-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition of human recombinant PDE9 expressed in insect Sf9 cells using [3H]-cGMP as substrate after 30 mins by scintillation proximity assay
Measurement: IC50 = 33.2 nM (pChEMBL 7.480000019073486)
Descriptors: MW 438.46, ALogP 2.31, HBA 8, HBD 1, PSA 109.06, QED 0.65
Compound (SMILES): CN1CCN(C2CCN(C(=O)c3cc(Cc4n[nH]c(=O)c5c4NCCC5)ccc3F)CC2)CC1
InChIKey: MWJFQDMAYIJAOX-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 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 468.58, ALogP 1.71, HBA 6, HBD 2, PSA 84.57, QED 0.71
Compound (SMILES): CC(C)(C)OC(=O)CNc1nnc(C(=O)c2nc3ccc(-c4ccccc4)cc3s2)o1
InChIKey: RZYJFECIKZWMKO-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Enzymatic Assay: The enzymatic assay was measured using white, opaque 96-well half area plates. Each well contained 60 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 2 ul of a DMSO solution containing compound of interest. Conditioned media obtained from HT-1080 cells, which were transformed by RAGE technology (Athersys) to overexpress endogenous EL, was added and the reaction was allowed to incubate for 20 min at 37° C. with gentle agitation. The reaction was started by the addition of 20 μL of a 1:4 dilution of vesicles. The final total reaction volume was 100 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 488 nm and an emission of 530 nm. Readings were taken every 20 seconds for 10 min with agitation between each reading.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 436.49, ALogP 4.33, HBA 9, HBD 1, PSA 107.21, QED 0.35
Compound (SMILES): CCc1ccc(Cc2c(O[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)n[nH]c2C)cc1
InChIKey: GBVDTKXHUHCPPZ-ILYVXUQDSA-N
Target: Sodium/glucose cotransporter 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31639
Assay: Inhibition of human SGLT2 expressed in COS7 cells assessed as reduction in uptake of methyl-alpha-D-(U-14C)glucopyranoside incubated for 2 hrs by microplate scintillation counting method
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 378.43, ALogP 0.05, HBA 7, HBD 5, PSA 128.06, QED 0.48
Compound (SMILES): CN1CCN(Cc2ccc(-c3cnc(N4CCO[C@H](Cn5nnc6ncc(-c7cnn(C)c7)nc65)C4)nc3)cc2)CC1
InChIKey: PQJGYYZYYMVBDF-VWLOTQADSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Lanthanide Fluoro-Immunoassay: The c-Met kinase inhibitory activity was analyzed using dissociation enhanced lanthanide fluoro-immunoassay (DELFIA, Perkin Elmer), which is a kind of time-resolved fluorescence (TRF). 10 mL of the compound prepared in the present invention, as a test compound, was put in a Greiner 96-well V-shaped bottom plate, and a tyrosin kinase buffer (20 mL) mixed with a c-Met enzyme was added thereto, and then the enzyme and the test compound were stirred for 15 minutes, followed by culturing. An ATP solution (10 mL) was added thereto to proceed with a kinase reaction at room temperature for 30 minutes, and then a 50 mM ethylene diamine acetic acid solution (EDTA, 40 mL) was added to stop the reaction. After that, the reaction mixture was transferred to a plate coated with Streptavidin, followed by culturing under shaking. After 2 hours, the cultured material was washed three times with a PBS-T buffer (PBS 0.05% Tween 20). The europium-labeled anti-phosphotyrosine antibody was diluted to 1:2,500, and 100 mL of the diluent was added per well, followed by culturing under shaking. After 1 hour, the cultured material was washed three times with a PBS-T buffer (PBS 0.05% Tween 20). 100 mL of an enhancement solution was added, followed by shaking culturing for 5 minutes, and then the reaction material was read out within a wavelength range of 615/665 nm using a Wallac Envision 2103 device.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 566.67, ALogP 1.73, HBA 13, HBD 0, PSA 119.04, QED 0.29
Compound (SMILES): CC(O)CN1CCN(CCSC(c2cccc(Cl)c2)c2cccc(Cl)c2)CC1
InChIKey: KQOYJPPZVJXGGF-UHFFFAOYSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Rattus norvegicus
UniProt: P23977
Assay: Displacement of [3H]WIN35,428 from DAT in Sprague-Dawley rat brain membranes incubated for 120 mins by radioligand binding assay
Measurement: Ki = 143 nM (pChEMBL 6.840000152587891)
Descriptors: MW 439.45, ALogP 4.81, HBA 4, HBD 1, PSA 26.71, QED 0.64
Compound (SMILES): O=c1cc(COc2ccc(F)cc2)[nH]nc1O
InChIKey: DFARBTKATGLYKL-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P14920
Assay: Inhibition Assay: The DAAO inhibitory activity was measured by assaying the amount of hydrogen peroxide (H2O2) produced by reacting DAAO protein with flavin adenine dinucleotide (FAD) and D-alanine. The amount of H2O2 was determined by measuring the fluorescence generated on conversion of Amplex red (manufactured by Invitrogen Co.) into resorufin by the reaction of H2O2 with horseradish peroxidase (HRP). 4 uL of 4% dimethyl sulfoxide (DMSO) buffer (50 mM sodium phosphate (pH 7.5), 0.02% CHAPS) solution of the test compound was added to 384-well black, low volume plate, a mixed solution (4 uL) of recombinant human DAAO protein (15 nM), which had been expressed in Escherichia coli and purified, and 18 uM FAD was added, and the mixture was incubated at room temperature for 15 min. After incubation, a mixed buffer (4 uL) of 2.25 mM D-alanine, 1.5 U/mL HRP and 150 uM Amplex red was added, the mixture was incubated at room temperature for 30 min.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 236.2, ALogP 1.19, HBA 4, HBD 2, PSA 75.21, QED 0.84