lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(C)[C@H]1CC[C@@H](C(=O)N2Cc3cccnc3Nc3ccccc32)CC1
InChIKey: ZXCSLPXFDWVAOE-MAEOIBBWSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Enzymatic Assay: Each test compound (10 mM stock in DMSO) is diluted in DMSO to make a 10-point, 3-fold dilution series. 125 nL of each dilution or DMSO alone is dispensed to a 384-well Greiner Lumitrac 200 assay plate using an Echo® Liquid Handler. To each well of the plate is added 20 uL of enzyme in assay buffer or assay buffer alone. Assay buffer consists of 50 mM sodium phosphate, pH 7.0, 50 mM magnesium chloride, 50 mM sodium chloride, and 0.01% (w/v) bovine serum albumin. When present, the R132H mutant IDH1 enzyme is at a working concentration of 1.875 nM (final concentration in assay of 1.5 nM). The assay plate is allowed to incubate for 30 minutes at room temperature and 5 uL of 5× substrate mixture (2.5 uM nicotinamide adenine dinucleotide phosphate, 100 uM adenosine diphosphate, 7.5 mM glyceraldehyde-3-phosphate, 7.5 ug/mL of spinach glyceraldehyde-3-phosphate dehydrogenase, 25 nM phosphoglycerate kinase, and 5 mM alpha-ketoglutarate in assay buffer) is added to all wells. The reaction plate is incubated for 60 minutes followed by addition of 25 uL of Promega Kinase-GLO reagent to all wells and 10-minute incubation.Luminescence is measured using a PerkinElmer Envision plate reader. The percent activity of each dilution is determined as the ratio of background corrected signal to the background corrected signal of wells receiving only DMSO. IC50 values are determined by fitting percent activity data to a four-parameter logistic dose response equation.
Measurement: IC50 = 595 nM (pChEMBL 6.21999979019165)
Descriptors: MW 363.51, ALogP 5.52, HBA 3, HBD 1, PSA 45.23, QED 0.72
Compound (SMILES): Cc1cc(Cn2cc(-c3ccccn3)cn2)n(C)n1
InChIKey: NMVFTNHCPXMRAP-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of Specific Binding to the hERG-Receptor (HERGBD): After thawing on the day of the assay, the membrane suspension was diluted further with 20 mM HEPES/0.1 mM KCl/pH 7.2.The incubation mixture of 200 μl contained 1.5 nmol/l 3H-Dofetilide, optimized amount of membrane preparation, 20 mM HEPES/0.1 mM KCl/(pH 7.2) and inhibitor in 1% DMSO. Nonspecific binding was estimated in the presence of 10 M Dofetilide. The samples were incubated for 90 min. at RT.Binding was terminated by filtration of the incubated membrane preparations using Filtermat B (Pharmacia, Uppsala Sweden) and a Micro Cell Harvester (Skatron, Lier, Norway). The Filtermat B had been presoaked with 1% polyethylen imine and carefully washed with 0.05 M Tris/HCl-buffer pH=7.7 after the filtration to separate free and bound radioactivity. The filters were counted in a scintillation counter (Betaplate 1205, Berthold, Wildbad, Germany) in order to determine the specific binding of [3H]-Dofetilide.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 253.31, ALogP 2.04, HBA 5, HBD 0, PSA 48.53, QED 0.72
Compound (SMILES): Cc1ccnc(OCc2cn(-c3ccc(F)c(Br)c3)nn2)n1
InChIKey: PEZBPEZARHKLSI-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P19793
Assay: NR2B standard assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ¿EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 364.18, ALogP 2.85, HBA 6, HBD 0, PSA 65.72, QED 0.71
Compound (SMILES): N#CN1CC[C@@H](NC(=O)c2ccc(Oc3ccccc3)cc2)C1
InChIKey: NEOICUABAWDZJA-OAHLLOKOSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 30 (SINGLE PROTEIN), Homo sapiens
UniProt: Q70CQ3
Assay: USP30 Biochemical Fluorescence Intensity IC50 Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl.Either 1 μl of 50% DMSO or diluted compound was added to the plate. USP30 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.001 μl/well and 10 μl of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of Ubiquitin-Rhodamine 110 (U-555; Boston Biochem) at a final concentration of 100 nM. Reactions were read immediately after addition of substrate and following a 2 h incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). 2 Excitation 487 nm; 2 Emission 535 nm.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 307.35, ALogP 2.76, HBA 4, HBD 1, PSA 65.36, QED 0.88
Compound (SMILES): C[C@@H]1CN(c2nccc(-c3cnc4ccc(Cl)cn34)n2)C[C@H](c2cn[nH]c2)O1
InChIKey: SOQNOKWAORAMTK-SJKOYZFVSA-N
Target: eIF-2-alpha kinase GCN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P2K8
Assay: Enzyme Inhibition Assay: An assay buffer containing GCN2 kinase and (RS)7 was prepared. 4.7 μL of this stock solution was placed per well of a black, low volume, 384-well microtitre plate (e.g. catalogue number 3676, Corning Inc., NY). To this was added 0.65 μM of DMSO containing serial dilutions of the test compound (typical final concentrations of test compound were 0 to 8 μM). The plate was incubated for 10 minutes at 25° C. prior to the addition of 4.7 μL of ATP stock buffer to initiate the enzyme reaction. The reaction was allowed to proceed for 1 hour at 25° C., prior to the addition of 10 μL detection buffer (consisting of appropriate concentrations of ADP2 antibody and ADP Alexa633 tracer in 1× stop and detect buffer as supplied by BellBrook Labs). The reaction was left to incubate for 1 hour at 25° C., prior to measuring the fluorescence polarisation signal (mP) in each well using a PHERAstar FS reader (BMG Labtech, Germany).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 395.85, ALogP 3.13, HBA 7, HBD 1, PSA 84.23, QED 0.57
Compound (SMILES): CCn1nc(C(=O)NC[C@H]2CC[C@H](S(C)(=O)=O)CC2)c(Cl)c1-c1cnc(N2CC(CC(F)(F)F)C2)cc1OC
InChIKey: JVHTUFYUHQIANO-JCNLHEQBSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: For the RORγt construct used in the ThermoFluor® assay, numbering for the nucleotide sequences was based on the reference sequence for human RORγt, transcript variant 2, NCBI Accession: NM 001001523.1 (SEQ ID NO:1). Nucleotides 850-1635 (SEQ ID NO:2) coding for the wild type human RORγt ligand binding domain (RORγt LBD) were cloned into the pHIS1 vector, a modified pET E. coli expression vector (Accelagen, San Diego), containing an in-frame N-terminal His-tag and a TurboTEV protease cleavage site (ENLYFQG, SEQ ID NO:3) upstream of the cloned insert sequence. The amino acid sequence for the RORγt construct used in the Thermofluor® assay is shown as SEQ ID NO:4.ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Development, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.
Measurement: Kd = 3300 nM (pChEMBL 5.480000019073486)
Descriptors: MW 592.08, ALogP 4.35, HBA 8, HBD 1, PSA 106.42, QED 0.46
Compound (SMILES): Cc1cc(Oc2cnc(C(=O)N[C@H](C)C(=O)N3[C@H](c4cccc(Cl)c4)CC[C@@H]3C(C)(C)C#N)cc2C)c(=O)[nH]n1
InChIKey: ONOIDEBGFPNFFK-WSUBETFTSA-N
Target: Calcitonin gene-related peptide type 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q16602
Assay: Determination of Binding Affinity for CGRP Receptor by Radioligand Binding Assay in SK-N-MC Cell Membranes: The binding affinity assay of compounds for human CGRP receptor was carried out by inhibition of radiolabeled ligand [125I]-CGRP binding in human neuroblastoma cell line SK-N-MC cell membranes.Cell membranes prepared from SK-N-MC cells expressing CGRP receptors endogenously (Muff et al., Ann N Y Acad Sci. 1992; 657: 106-116) were used for radioligand binding assay. Radioligand binding assay was performed using 96-well microplate in a total volume of 200 μL in each well. A 2.5 μL of serial dilution of compound dissolved in dimethylsulfoxide (DMSO) was mixed with SK-N-MC cell membranes (40 μg membrane protein per well) and [125I]-CGRP (PerkinElmer NEX354, final concentration of 150 pM) in an assay buffer consisting of 50 mM Tris-HCl, 5 mM MgCl2 and 0.1% bovine serum albumin (pH 7.4). The assay plate was incubated with shaking on a plate shaker at room temperature for 90 minutes. The incubation was terminated by filtration through a GF/C glass fiber filter plate (Merck Millipore) pre-soaked with 0.3% polyethyleneimine (PEI). Filters were washed 4 times with 300 μL of ice-cold assay buffer. After drying the filter plate, 100 μL of scintillation fluid (PerkinElmer, MicroScint-20) was added to each well and the radioactivity was counted using a TopCount NXT (PerkinElmer). Non-specific binding was determined in the presence of 1.25 μM unlabelled human α-CGRP (Bachem). The radioactivity was converted to the percent of specific binding using the equation below.% ⁢ ⁢ of ⁢ ⁢ specific ⁢ ⁢ binding = ( Y - Y ⁢ ⁢ min ) ( Y ⁢ ⁢ max - Y ⁢ ⁢ min ) × 100 [ Number ⁢ ⁢ 1 ]In the formulae, Y is observed radioactivity, Ymax is total bound activity, and Ymin is non-specific bound activity.From these data, the concentration of compound required for 50% inhibition of radioligand binding (IC50) was determined using Prism (GraphPad Inc.). The IC50 value is then converted to the equilibrium dissociation constant (Ki) using the Cheng-Prusoff equation below (Cheng & Prusoff (1973) Biochem. Pharmacol. 22, 3099-3108).Ki = IC 50 1 + [ L ] Kd [ Number ⁢ ⁢ 2 ]In the formulae, [L] is the concentration of radioligand, Kd is the apparent dissociation constant of the radioligand for the receptor as determined by saturation binding assay with [125I]-CGRP.
Measurement: Ki = 0.07 nM (pChEMBL 10.149999618530273)
Descriptors: MW 563.06, ALogP 4.63, HBA 7, HBD 2, PSA 141.07, QED 0.43
Compound (SMILES): COC(=O)[C@H](C)[C@H](C)n1c(=O)[nH]/c(=N\c2ccc(Oc3ccccn3)c(C)c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: DQQPIVKGUHDFQJ-MOPGFXCFSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor 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 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 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 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-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 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution 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, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 550.02, ALogP 4.14, HBA 9, HBD 1, PSA 120.57, QED 0.33
Compound (SMILES): C[C@@H]1CCOc2nc3cc(C(=O)Nc4ccc(OC(F)(F)Cl)cc4)cc(-c4cscn4)c3n21
InChIKey: MHURZQOZVLWMLN-GFCCVEGCSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Biochemical Kinase Assay: ABL1 WT protein (64-515aa) containing an N-terminal His tag was produced by co-expression with YopH in Sf9 insect cells. Cells were harvested by centrifugation and resuspended in 50 mM Tris, 500 mM NaCl, 5 mM β-ME, pH 8.2. Cells were lysed by sonication and clarified by centrifugation. ABL1 was purified by affinity chromatography using a HisTrap column with a wash step in 4% wash buffer (50 mM Tris, 500 mM NaCl, 500 mM imidazole, 5 mM β-ME, pH 8.2) and eluted in a linear gradient of the same buffer. Fractions containing ABL1 were pooled, concentrated and further purified using an ion exchange column washed with 50 mM Tris, pH 8.3 and eluted with a linear gradient of elution buffer (50 mM Tris, 1M NaCl, pH 8.3). Purified protein was stored at -80° C. in 50 mM Tris (pH 8.2), 300 mM NaCl, 1 mM DTT and 20% glycerol.
Measurement: IC50 = 0.35 nM (pChEMBL 9.460000038146973)
Descriptors: MW 490.92, ALogP 5.92, HBA 7, HBD 1, PSA 78.27, QED 0.35
Compound (SMILES): COC(=O)Nc1ccc(-c2cnc([C@@H]3CSc4cc(-c5cc(Cl)ccc5-n5cnnn5)cc(=O)n43)[nH]2)cc1
InChIKey: XAFJHYBQQFKSRO-NRFANRHFSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Enzyme Activity Assay: Human Factor XIa (Haematologic Technologies Inc.) activity was measured at an enzyme concentration of 0.1 U/mL in 150 mM NaCl, 5 mM KCl, 1 mg/mL PEG6000, 50 mM HEPES-NaOH (pH7.4) with 300 μM S-2366 (pyroGlu-Pro-Arg-pNA, Chromogenix).
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 547, ALogP 4.41, HBA 10, HBD 2, PSA 132.61, QED 0.33
Compound (SMILES): CS(=O)(=O)NC(=O)c1cc(F)c(COc2cccc(-c3ccc(OCC4CC4)nc3)c2)cc1F
InChIKey: SCMJMGADJDCWPT-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: EFS-FRET Assay: This screen is used to determine the effects of compounds on human Nav1.7 channels, utilizing electrical field stimulation (EFS) system in 96-well plate format on FDSS (Hamamatsu Photonics) platform. The changes of membrane potential are monitored with FRET dye pair, DiSBAC2(3) and PTS 18.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 488.51, ALogP 4.08, HBA 6, HBD 1, PSA 94.59, QED 0.49
Compound (SMILES): COc1cc(CCc2cnc3[nH]c(-c4ccc(N5CCN(C)CC5=O)cc4)cc3n2)cc(OC)c1
InChIKey: KWUHYQMCPAEOHH-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; added fresh 30 mM EDTA and Perkin Elmer Lance Reagents for HTRF at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader.FGFR1 and FGFR2 were measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 μM, respectively and FGFR2, 0.01 nM and 100 μM, respectively.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 471.56, ALogP 3.71, HBA 6, HBD 1, PSA 83.58, QED 0.44
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3ccc(OCCCS(C)(=O)=O)cc3)ncc2F)c1
InChIKey: UKDRGYMUMOVHQD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Omnia Assay Protocol for Potency Assessment Against BTK: Briefly, 10× stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13×ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM 3-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 27° C. with a 0.5 μL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 μL of the ATP/Tyr-Sox peptide substrate mix and monitored every 30-90 seconds for 60 minutes at λex360/λem485 in a Synergy4 plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 485.54, ALogP 4.04, HBA 8, HBD 3, PSA 122.31, QED 0.27
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@H](C(=O)N[C@@H](C)C(F)=C(C)C)C[C@@]3(C)C[C@@H]23)c2c(C)cc(-c3cnc(C)nc3)cc12
InChIKey: JQCQMKBVBSIHSM-ZHDAISQTSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBz and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 546.65, ALogP 4.46, HBA 7, HBD 1, PSA 110.08, QED 0.44
Compound (SMILES): Cc1cccc2c(=O)[nH]c(CS[C@H]3CC[C@H](CN(C)C)CC3)nc12
InChIKey: IFILIFYLYRJDSG-SHTZXODSSA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Inhibition 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 345.51, ALogP 3.59, HBA 4, HBD 1, PSA 48.99, QED 0.9
Compound (SMILES): Cn1cnc(S(=O)(=O)N2C[C@@H](Nc3cccc(OC(F)(F)F)c3)[C@H](c3ccccc3)C2)c1
InChIKey: NGSBRTBPUWROLG-RBUKOAKNSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 466.49, ALogP 3.59, HBA 6, HBD 1, PSA 76.46, QED 0.6
Compound (SMILES): CCOc1ncccc1CNc1cc(-c2cccnc2OCC)nc2c(C)nn(C(C)C)c12
InChIKey: RPFGIRQWMRKNBU-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P54750
Assay: PDE1 Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays was performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 hr at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 hr in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 446.56, ALogP 5.19, HBA 8, HBD 1, PSA 86.98, QED 0.38
Compound (SMILES): CC(C)Oc1cc(Nc2ncnn3ccc(CN4CCC(N)CC4)c23)cc(-c2nnn(C(C)C)n2)c1
InChIKey: VBPUWVVGXJGULI-UHFFFAOYSA-N
Target: AP2-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q2M2I8
Assay: Kinase 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 (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2 and ATP) and test compounds in assay buffer (10 mM Tris-HCL pH 7.4, 10 mM MgCl2, 0.01% Tween-20 and 1.0 mM DTT). The reactions were initiated by the combination of bacterially expressed, GST-Xa-hAAK1 with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA buffer to each sample. The reactions were 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 EDTA quenched control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assays are ATP, 22 μM; (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2, 1.5 μM; GST-Xa-hAAK1, 3.5 nM; and DMSO, 1.6%. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 490.62, ALogP 3.42, HBA 11, HBD 2, PSA 124.31, QED 0.38
Compound (SMILES): O=C(Nc1cc(C(F)(F)F)ccc1N1CCN(Cc2ccoc2)CC1)c1ccc(-c2ccncc2)o1
InChIKey: ZMOOQKNWRLWWGL-UHFFFAOYSA-N
Target: SRSF protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96SB4
Assay: Inhibition Assay: Screening of a series of molecules identified compounds (Compounds 5, 8, 16 to 18 and 43 of Table 3) that had improved potency against SRPK1 against a series of analogous compounds in which the R3 group of the respective compounds of the present invention (as defined by Formula I) was replaced with a pyridyl ring (FIGS. 1 and 2, in which the reference compounds had an IC50 of 6.00 nM and a ΔTm of 12.8° C. (FIG. 1); an IC50 of 38.8 nM (FIG. 2a ) and an IC50 of 3.8 nM (FIG. 2b )) as determined by kinase assay or differential scanning fluorimetry (temperature difference, ΔTm is inversely proportional to the log Kd, i.e. an increased temperature difference indicates a higher affinity and therefore a more potent inhibitor). These included furan, oxazole, pyrazole, thiazole, methyl-pyrazole, and oxadiazole moieties. Replacement of the pyridine ring of indolyl compound 38 in Table 3 with a furan ring resulted in a drop in potency (23.9 nM for compound 38 vs. 65 nM for the resulting furan analogue).
Measurement: IC50 = 223 nM (pChEMBL 6.650000095367432)
Descriptors: MW 496.49, ALogP 5.53, HBA 6, HBD 1, PSA 74.75, QED 0.38
Compound (SMILES): Cn1c(SCCCN2CCOC(c3ccc(C(F)(F)F)cc3)C2)nnc1-c1ccc(C(N)=O)cn1
InChIKey: BBKTWQKIEOQFST-UHFFFAOYSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: [3H]-Spiperone Binding Assay at hD3: [3H]-Spiperone Binding Assay at hD3 and hD4 recombinant receptors CHO cells transiently transfected with human dopamine type 3 or 4 receptors (CHO-hD3 or CHO-hD4, respectively), were re-suspended in 20 mM HEPES, 2 mM EDTA (pH 7.4), homogenised and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellet was re-suspended in 20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4) and aliquots were kept at −80° C. [3H]-Spiperone Binding experiments were performed in 96 deep-well polypropylene plates in 50 mM Tris/HCl, 120 mM NaCl, 5 mM KCl, 5 mM MgCl2 (pH 7.4). Compounds of invention were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final in the assay). Displacement was performed in the presence of 0.3 nM [3H]-Spiperone. The reaction was initiated by the addition of membrane suspension (4 μg and 12 μg of protein for CHO-hD3- and CHO-hD4 membranes, respectively) and lasted for 90 or 100 min (for hD3 or hD4 membranes, respectively) at 23° C. in a final volume of 500 μl. Non specific binding (NSB) was determined in the presence of 1 μM Spiperone. The binding reaction was stopped by rapid filtration through GF/B filterplates pre-soaked in 0.5% polyetylenimmine (PEI) using a Packard cell harvester. After washing with ice-cold 0.9% NaCl, the plate was left to dry before the addition of Microscint 20 (50 l/well, PerkinElmer). Radioactivity was counted with a TopCount (PerkinElmer). Data were analysed by non-linear regression analysis using GraphPad Prism 5.0 (GraphPad Software). Saturation binding experiments were performed similar to the competition binding experiments using a radioligand concentrations ranging from 0.015 to 4.0 nM. Ref: Mackenzie R. G. et al. (1994). Characterization of the human dopamine D3 receptor expressed in transfected cell lines. Eur. J. Pharmacol., 266:79-8.
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 506.55, ALogP 3.55, HBA 8, HBD 1, PSA 99.16, QED 0.37
Compound (SMILES): O=C(O)c1cn(-c2ccc(N3CCOC3=O)cc2)c(=O)n(C2CCCc3c(Cl)cccc32)c1=O
InChIKey: JNCWZSVHPGNCEI-UHFFFAOYSA-N
Target: Chymase (SINGLE PROTEIN), Homo sapiens
UniProt: P23946
Assay: Enzymatic Assay : The enzyme source used is recombinant human chymase (expressed in HEK293 cells) or chymase purified from hamsters' tongues. The substrate used for chymase is Abz-HPFHL-Lys(Dnp)-NH2. For the assay, 1 μl of a 50-fold concentrated solution of test substance in DMSO, 24 al of enzyme solution (dilution 1:80 000 human or 1:4000 hamster) and 25 al of substrate solution (final concentration 10 μM) in assay buffer (Tris 50 mM (pH 7.5), sodium chloride 150 mM, BSA 0.10%, Chaps 0.10%, glutathione 1 mM, EDTA 1 mM) are combined in a white 384-hole microtiter plate (Greiner Bio-One, Frickenhausen, Germany). The reaction is incubated at 32 degrees for 60 min and the fluorescence emission at 465 nm after excitation at 340 nm is measured in a fluorescence reader, for example Tecan Ultra (Tecan, Männedorf, Switzerland).
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 481.89, ALogP 3.23, HBA 7, HBD 1, PSA 110.84, QED 0.61
Compound (SMILES): CN1C(=N)N[C@@]2(COCc3ccc(NC(=O)c4ccc(Cl)cn4)cc32)CS1(=O)=O
InChIKey: KZHBDZCQOITHGE-SFHVURJKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window Inhibitor IC50 values are derived from non-linear regression analysis of concentration response curves.
Measurement: Ki = 272 nM (pChEMBL 6.570000171661377)
Descriptors: MW 435.89, ALogP 1.51, HBA 6, HBD 3, PSA 124.48, QED 0.66
Compound (SMILES): CC(C)(C)c1nc(N2CC[C@H](O)C2)c2ncn(Cc3c[nH]nc3C(F)(F)F)c2n1
InChIKey: RSELOMPDFHGKMG-NSHDSACASA-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), 1x HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 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% NaN3) 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 as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 uM to 0.13 nM cAMP. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature. The compounds of the invention are CB2 receptor agonists with EC50 below 1 uM and selectivity versus CB1 in the corresponding assay of at least 10 fold. Particular compound of the invention are CB2 receptor agonists with EC50 below 0.05 uM and selectivity versus CB1 in the corresponding assay of at least 500 fold.
Measurement: EC50 = 22.3 nM (pChEMBL 7.650000095367432)
Descriptors: MW 409.42, ALogP 2.49, HBA 7, HBD 2, PSA 95.75, QED 0.69
Compound (SMILES): O=C(c1cnc(NC2(c3cnccn3)CC2)nc1)N1CCC2(CCOCC2)C1
InChIKey: UPRUVVBRLBNFMW-UHFFFAOYSA-N
Target: Pantetheinase (SINGLE PROTEIN), Homo sapiens
UniProt: O95497
Assay: Enzyme Assay: The test inhibitors were solubilized in DMSO to a stock concentration of 30 mM. On the day of the assay, dose response plates were prepared by diluting the inhibitors in DMSO at compound concentration 200-fold the final in-assay concentration. Intermediate concentrations were prepared by diluting in DMSO in a four-fold series for a total of 11 data points.To prepare a working solution of human vanin-1, the enzyme was diluted to 33.3 pM in the assay buffer consisting of 50 mM Tris-HCl pH=8.0, 50 mM KCl, 0.005% Brij-35 and 1.6 mM cysteamine. To begin the assay 100 nL was transferred from the compound plate to the assay plate. Next, 15 μL of the vanin-1 working solution were transferred to the assay plate. The inhibitor and enzyme were incubated at room temperature for 30 minutes. The enzyme reaction was then initiated by the addition of 5 μL of 200 μM pantetheine 7-amino-4-trifluoromethylcoumarin prepared in assay buffer. The final concentrations in the assay were 25 pM human vanin-1 and 50 uM substrate. The final concentration of DMSO was 0.5%. The assay plates were incubated for 60 minutes and before they were read on a Perkin Elmer EnVision Model 2103 using a 405 nm excitation wavelength and a 510 nm emission wavelength for detection.
Measurement: IC50 < 0.041 nM (pChEMBL 10.38700008392334)
Descriptors: MW 380.45, ALogP 2.01, HBA 7, HBD 1, PSA 93.13, QED 0.87
Compound (SMILES): COc1ccc(F)c2[nH]c(C(=O)N[C@@H](CC(C)(C)C)C(=O)N[C@H](C#N)C[C@@H]3CCNC3=O)cc12
InChIKey: CJXNCBSSADROTD-DEYYWGMASA-N
Target: Replicase polyprotein 1ab (SINGLE PROTEIN), Severe acute respiratory syndrome coronavirus 2
UniProt: P0DTD1
Assay: SARS-CoV-2 Coronavirus 3C Protease FRET Assay: The proteolytic activity of the main protease, 3CLpro, of SARS-CoV-2 was monitored using a continuous fluorescence resonance energy transfer (FRET) assay. The SARS-CoV-2 3CLpro assay measures the activity of full-length SARS-CoV-2 3CL protease to cleave a synthetic fluorogenic substrate peptide with the following sequence: Dabcyl-KTSAVLQ-SGFRKME-Edans modelled on a consensus peptide (V. Grum-Tokars et al. Evaluating the 3C-like protease activity of SARS-coronavirus: recommendations for standardized assays for drug discovery. Virus Research 133 (2008) 63-73). The fluorescence of the cleaved Edans peptide (excitation 340 nm/emission 490 nm) is measured using a fluorescence intensity protocol on a Flexstation reader (Molecular Devices). The fluorescent signal is reduced in the present of PF-835231, a potent inhibitor of SARS-CoV-2 3CLpro. The assay reaction buffer contained 20 mM Tris-HCl (pH 7.3), 100 nM NaCl, 1 mM EDTA and 25 μM peptide substrate. Enzyme reactions were initiated with the addition of 15 nM SARS-CoV-2 3CL protease and allowed to proceed for 60 minutes at 23° C. Percent inhibition or activity was calculated based on control wells containing no compound (0% inhibition/100% activity) and a control compound (100% inhibition/0% activity). IC50 values were generated using a four-parameter fit model using ABASE software (IDBS). Ki values were fit to the Morrison equation with the enzyme concentration parameter fixed to 15 nM, the Km parameter fixed to 14 μM and the substrate concentration parameter fixed to 25 μM using ABASE software (IDBS).
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 471.53, ALogP 2.38, HBA 5, HBD 4, PSA 136.11, QED 0.47
Compound (SMILES): COC1(c2ccnc(C(N)=O)c2)C2CCCC1CN([C@@H]1CC[C@@H]1c1ccccc1)C2
InChIKey: ZQHVIWMWIKGMDD-ZTMBSTQPSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Competitive Displacement Assay: Membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed one type of the cloned human opioid receptor were incubated with 12 different concentrations of the compound in the presence of 0.25 nM [3H]DAMGO, 0.2 nM [3H]naltrindole or 1 nM [3H]U69,593 in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO and [3H]U69,593. Because of a slower association of [3H]naltrindole with the receptor, a 3 h incubation was used with this radioligand. Samples incubated with [3H]naltrindole also contained 10 mM MgCl2 and 0.5 mM phenylmethylsulfonyl fluoride. Nonspecific binding was measured by inclusion of 10 μM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber filters using a Brandel 48-well cell harvester. The filters were subsequently washed three times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL Ecoscint A scintillation fluid. For [3H]naltrindole and [3H]U69,593 binding, the filters were soaked in 0.1% polyethylenimine for at least 60 min before use.
Measurement: Ki > 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 405.54, ALogP 3.7, HBA 4, HBD 1, PSA 68.45, QED 0.82
Compound (SMILES): O=COCCc1cs/c(=N\C2CCCCC2)n1/N=C/c1ccc(O)c(O)c1O
InChIKey: WFGISUVWIISGLF-LELOSUNBSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 homolog (SINGLE PROTEIN), Mus musculus
UniProt: P97287
Assay: High-Throughput Competitive Fluorescence Polarization (FP) Screening Assay: A high-throughput competitive fluorescence polarization (FP) screening assay (Z-factor, 0.62) was developed based on the direct binding interaction between FITC-MCL-1 SAHBA and MCL-1ΔNΔC (EC50, 14 nM) (FIGS. 1A, 2A, and 3A). A compilation of 71,296 small molecules was screened for the capacity to displace FITC-MCL-1 SAHBA from recombinant MCL-1ΔNΔC (aa 172-327) (FIG. 4). To enrich for MCL-1-selective molecules by detecting binding activity for the BCL-XL subclass of anti-apoptotic proteins, the library was also counterscreened using a competitive FP assay (Z-factor, 0.71) developed based on the direct and selective interaction between FITC-BAD BH3 and BCL-XLΔC (EC50, 26 nM) (FIGS. 1B, 2B, and 3B). Small molecules with an apparent preference for MCL-1ΔNΔC (208 compounds, 0.3% hit rate), as defined both by >50% displacement of the FITC-MCL-1 SAHBA/MCL-1ΔNΔC interaction and a >45% difference in peptide displacement from MCL-1ΔNΔC vs. BCL-XLΔC, were advanced to increasingly stringent confirmatory in vitro binding assays including: (1) repeat single-dose testing of 208 molecules in the differential competitive FP screen; (2) alternative single-dose selectivity screen for 130 confirmed MCL-1-directed antagonists comparing relative displacement of FITC-BID BH3, a dual binder,35 from MCL-1 ΔNΔC vs. BCL-XLΔC; and then (3) dose-responsive competitive binding of the 64 most selective molecules against the FITC-MCL-1 SAHBA/MCL-1 ΔNΔC complex (FIG. 4).
Measurement: IC50 = 9.5 nM (pChEMBL 8.020000457763672)
Descriptors: MW 405.48, ALogP 2.5, HBA 9, HBD 3, PSA 116.64, QED 0.28
Compound (SMILES): O=C(C[C@@H]1CCc2cc(C(O)(C(F)(F)F)C(F)(F)F)ccc2N1S(=O)(=O)c1ccc(F)cc1)N[C@@H]1CCCC[C@@H]1CO
InChIKey: IBGVAQXRLZNIFR-PIPMEXSNSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol 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 chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 23.2 nM (pChEMBL 7.630000114440918)
Descriptors: MW 626.59, ALogP 4.71, HBA 5, HBD 3, PSA 106.94, QED 0.39
Compound (SMILES): CCn1c2ccccc2c2cc(Cc3cncc4cc(OC)c(OC)cc34)ccc21
InChIKey: OZEYXJABOHBCFJ-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Homogenous Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Technology: The phosphodiesterase assay was developed using the LANCE® cAMP kit (PerkinElmer). The assay buffer contained HBSS with 5 mM HEPES, 0.1% BSA, and 1.5 mM MgCl2, pH 7.4. PDE10A (BPS Bioscience) was used at 200 pg/well (with a specific activity of 3200 pmole/min/μg with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 μM cAMP, 2.5 kU 5′ nucleotidase, 37° C., 20 min) and PDE4D3 (BPS Bioscience) was used at 100 pg/well (with a specific activity of 32713 pmole/min/μg with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 μM cAMP, 2.5 kU 5′ nucleotidase, 37° C., 20 min). The Biotin-cAMP tracer, supplied in 10 mmol/L Tris-HCl buffered (pH 8.0) salt solution with 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1% bovine serum albumin (BSA), and 0.05% sodium azide, is used at a dilution of 1/375. The assay detection mixture contained the LANCE Eu-W8044 labeled streptavidin 1/2250 (supplied in 50 mmol/L Tris-HCl buffered (pH 7.8) salt solution with 0.9% sodium chloride (NaCl), 0.1% BSA, and 0.05% sodium azide) and the Alexa Fluor® 647-anti cAMP antibody 1/200 (supplied in 50 mmol/L Tris-HCl buffered (pH 7.8) salt solution with 0.9% NaCl, 0.1% BSA, and 0.05% sodium azide). Chemical compounds were dissolved in DMSO (final concentration 2% (v/v)).
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 396.49, ALogP 5.97, HBA 4, HBD 0, PSA 36.28, QED 0.37
Compound (SMILES): CC1(C)Oc2ncnc(N)c2N=C1c1ccc([C@H]2CC[C@H](OP(=O)(O)c3ccccc3)CC2)cc1
InChIKey: KPJKBUDXBSKPAJ-KESTWPANSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Inhibition Assay: The inhibition of DGAT-1 activity can be assessed in intestinal microsome preparations by monitoring the incorporation of radiolabeled fatty acyl-CoA into DAG.Methods: Commercial microsomal preparations containing 60 ug of protein are incubated in assay buffer [20 uM 1,2-didecanloyl glycerol, 5 uM 14C decanoyl-CoA, 5 mM MgCl2, 0.4% BSA, 0.1% dimethyl sulfoxide (DMSO), 50 mM HEPES-pH 7.5] in the presence of varying concentrations of inhibitors that are dispensed from 100% DMSO stock solutions. Final assay volumes are 200 uL. Reactions are carried out for 45 minutes in 96-well polystyrene microtiter plates at ambient temperature. Following the ambient temperature incubation, assay mixtures are applied to a 96-well filter plate (Catalog # MSHVN4510, Millipore Inc.; Billerica, Mass.) under vacuum. The filter plate is pre-equilibrated with 100 uL of 70% ethanol followed by 200 uL of assay buffer. Filters are dried, removed and placed in scintillation vials with 4 mL of scintillation cocktail (Catalog #6013329; PerkinElmer Inc.; Waltham, Mass.). De novo 14C-TAG formed in the assay and trapped on the filters is quantified with use of a liquid scintillation counter (Model # LS6500 Beckman Coulter, Inc.; Fullerton, Calif.). The IC50 is defined as the concentration of compound that results in a 50% reduction in TAG synthesis.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 492.52, ALogP 4.9, HBA 7, HBD 2, PSA 119.92, QED 0.49