lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(-c2cc(-c3cccc(F)c3)cnc2N)ccc1O
InChIKey: LZUQLFFUIVGESG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Fyn (SINGLE PROTEIN), Homo sapiens
UniProt: P06241
Assay: ADP-Glo Kinase Assay: The assay procedure determines the IC50 of each potential FYN kinase inhibitor by measuring the enzyme catalyzed ATP-dependent phosphorylation of the FYN substrate peptide. The ADP-Glo Kinase Assay is specifically designed to quantify kinase activity by measuring the ADP produced in the reaction The reaction buffer comprises 40 mM Tris.HCl (pH 7.5), 20 mM MgCl2, 0.1 mg/ml BSA and 5 microM DTT. Individual compounds within the chemical library were dissolved in DMSO at known concentrations. Briefly, 2.5 microliters of reaction buffer containing 3 picograms of FYN kinase and serial dilutions of each potential FYN kinase inhibitor (9 3-fold dilutions of a 10 micromolar top sample) are placed into each well of a 384 well microtiter plate and incubated at room temperature for 30 minutes. An additional 2.5 microliters of reaction buffer containing 100 picograms of the FYN substrate and 12.5 picomoles of ATP are then added to each well and the reaction carried out for 90 minutes at room temperature.
Measurement: IC50 = 2027 nM (pChEMBL 5.690000057220459)
Descriptors: MW 310.33, ALogP 3.85, HBA 4, HBD 2, PSA 68.37, QED 0.77
Compound (SMILES): Cc1ncc2c(-c3ccc(Nc4ccc(N5CCC(O)CC5)cn4)c4c3CNC4=O)cccn12
InChIKey: CYIMZOSKQUIIBY-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 l/well termination buffer (Carna Biosciences) and signal determination using aCaliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 454.53, ALogP 3.65, HBA 7, HBD 3, PSA 94.79, QED 0.44
Compound (SMILES): C=Cc1nc(-c2nccc([C@@]34CC[C@@H](c5cc(-c6c(F)cccc6F)nnc53)C4(C)C)n2)no1
InChIKey: UPYOGRXQNJVWCU-SXBQZSJRSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Ligand Binding Assay: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present. Assay plates were 96-well polypropylene V-bottom plates. 10 uL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 uL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 uL of 5 uM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 uL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 uL of 1.5 ug/mL RORc receptor was added to wells (or 40 uL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25° C. Filtration
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 458.47, ALogP 5.11, HBA 7, HBD 0, PSA 90.48, QED 0.42
Compound (SMILES): CC(C)c1cnc2c(c1)cc(C(=O)N[C@H](C)c1ccc(C#N)cc1)c(=O)n2Cc1ccc(F)cc1
InChIKey: LYHOHPKCNSYSFJ-GOSISDBHSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: CB1 and CB2 Receptor Binding Assay: CB1R binding protocol involves the use of the same solution buffer used for both incubation and washing reaction (Tris-HCl, 50 mM; EDTA, 2.5 mM; MgCl2, 2.5 mM; BSA, 0.5 mg/mL at pH 7.4), 0.4 nM for [3H]CP-55,940, test compounds (concentrations from 0.001 to 10 μM), and finally 8 μg/sample membrane in a total volume of 200 μL. CB2R binding assays were carried out with two different buffers: incubation buffer (Tris-HCl, 50 mM; MgCl2, 5 mM; CaCl2 1 mM; BSA, 0.2% at pH 7.4) and washing buffer (Tris-HCl, 50 mM; NaCl 500 mM; BSA, 0.1% at pH 7.4). The assay mixture contained incubation buffer, 0.4 nM [3H]CP-55,940, test substances (concentrations from 0.001 to 10 μM), and 4 μg/sample membrane in a total assay volume of 200 μL. Assays were performed in duplicate and incubated for 120 mM at 37° C. After the incubation, the assay mixture is filtered through 96 GF/C filter plates (Perkin Elmer #6005174) using Perkin Elmer Filtermate Harvester, and then washed four times with ice-cold washing buffer.
Measurement: IC50 = 7.9 nM (pChEMBL 8.100000381469727)
Descriptors: MW 468.53, ALogP 5.07, HBA 5, HBD 1, PSA 87.78, QED 0.43
Compound (SMILES): CCN(CCN1CCC(n2ccc3cc(F)ccc32)CC1)C(=O)C(C)O
InChIKey: CJXRRILKADPIQO-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: Binding Activity Assay: Human 5-HT2A Receptor, Human 5-HT7 Receptor, and Human D2 Receptor: Binding affinity of the present compound for human 5-HT2A receptor, human 5-HT7 receptor, and human D2 receptor was measured by the following procedures.CHO cell membrane fraction in which human 5-HT2A receptor, human 5-HT7 receptor, or human D2 receptor was expressed was purchased from PerkinElmer, Inc. In a test for evaluating binding affinity, a test compound dissolved in dimethylsulfoxide (DMSO) and each receptor membrane sample diluted in buffer were mixed with [3H]Ketanserin, [3H]SB-269970, or [3H]Spiperone (all purchased from PerkinElmer, Inc.) for 5-HT2A receptor, 5-HT7 receptor, or D2 receptor, respectively. Each mixture was incubated at room temperature for 60 minutes. Non specific binding to receptors was obtained from a competitive binding test in the presence of 10 μmol/L 8-OH-DPAT, 10 μmol/L Mianserin, or 10 μmol/L Spiperone, respectively. Radioactivity caused by binding to receptors was measured with a liquid scintillation counter (PerkinElmer, Inc.), and 50% inhibition concentration was calculated.
Measurement: Ki = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 361.46, ALogP 2.65, HBA 4, HBD 1, PSA 48.71, QED 0.86
Compound (SMILES): CC1(C)[C@H]2CC[C@]1(c1cncc(-c3ccc(CNS(C)(=O)=O)nc3)n1)c1nnc(-c3c(F)cccc3F)cc12
InChIKey: PLEJDBNLUCKLNE-VKGTZQKMSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Ligand Binding Assay: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present. Assay plates were 96-well polypropylene V-bottom plates. 10 uL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 uL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 uL of 5 uM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 uL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 uL of 1.5 ug/mL RORc receptor was added to wells (or 40 uL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25° C. Filtration
Measurement: IC50 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 548.62, ALogP 4.53, HBA 7, HBD 1, PSA 110.62, QED 0.38
Compound (SMILES): O=C1CS(=O)(=O)c2nccnc2N1Cc1cccc(C(F)(F)F)c1
InChIKey: UUABYDRZDPBBTM-UHFFFAOYSA-N
Target: Intermediate conductance calcium-activated potassium channel protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15554
Assay: KCa3.1 Assay: HEK293 cells over-expressing human KCa3.1 are used to measure the effects of compounds in inhibiting the KCa3.1 channel function. The assay is based on measuring the influx of Tl+ through KCa3.1 using a FLIPR dye assay (FLIPR Potassium Ion Channel Assay Kit #R8154 by Molecular Devices). The day before the assay HEK293/KCa3.1 cells are seeded on a 384 well plate. On the day of the assay, media is removed and replaced with Molecular Devices Potassium Ion Channel dye then incubated for 1 hour according to manufacturer's recommendations. The cells are then treated with compounds or DMSO control for 15 min, followed by addition of thallium (final concentration=1.5 mM) and ionomycin (final concentration=1 uM). The plate is read on HAMAMATSU 60000 measuring fluorescence signal for 50 seconds using (Em: 565-625 nm). Data analysis: HAMAMATSU 6000 data are exported using max-min value for frame 10 and 40 and corrected by spatial uniformity correction. Percent of control (POC) is calculated as 100×(max-minFlsample−max-minFllow)/(max-minFlhi−max-minFllow). Max-min Flsample is max-min value of each sample well containing test compound. Max-min Fllow is average max-min value of wells containing reference inhibitory compound.
Measurement: IC50 = 2600 nM (pChEMBL 5.579999923706055)
Descriptors: MW 357.31, ALogP 1.82, HBA 5, HBD 0, PSA 80.23, QED 0.82
Compound (SMILES): Cc1cc(-c2nnc(N)nc2-c2ccc(OC(F)F)cc2)cc(C)c1C#N
InChIKey: WQMTZOQDYGNJHK-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Binding Assay: All test compounds were prepared as a stock solution of 10 mM in 100% DMSO.Inhibition binding assays were performed using 2.5 μg of membranes prepared from HEK293 cells transiently transfected with human adenosine A2a receptor or 10 μg of membranes prepared from CHO cells stably transfected with human adenosine A1 receptor. Membranes were incubated in 50 mM Tris-HCl (HEK293-hA2a; pH 7.4) or 20 mM HEPES, 100 mM NaCl, 10 mM MgCl2 (CHO-hA1; pH 7.4) in the presence of varying concentrations of test compound and 1 nM [3H]ZM241385 (HEK293-hA2a) or [3H]DPCPX (CHO-hA1) at 25° C. for 1 h. The assay was then terminated by rapid filtration onto GF/B grade Unifilter plates using a TomTec cell harvester, followed by 5×0.5 ml washes with double distilled H2O. Nonspecific binding was defined in the presence of 1 μM CGS15943 (HEK293-hA2a) or 1 μM DPCPX (CHO-hA1). Bound radioactivity was determined by liquid scintillation counting (Trilux Microbeta® Counter) and inhibition curves were analysed using a four-parameter logistic equation.
Measurement: Ki = 490 nM (pChEMBL 6.309999942779541)
Descriptors: MW 367.36, ALogP 3.88, HBA 6, HBD 1, PSA 97.71, QED 0.75
Compound (SMILES): O=C1Nc2cc(OC(F)(F)F)ccc2Oc2ccc(-c3ccncc3)cc21
InChIKey: JBOXXVXBHZWJPW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: TBD: TBD
Measurement: IC50 = 3722 nM (pChEMBL 5.429999828338623)
Descriptors: MW 372.3, ALogP 5.01, HBA 4, HBD 1, PSA 60.45, QED 0.69
Compound (SMILES): C[C@@H]1CN(c2cc(=O)[nH]c(N3CCC[C@@H]3Cc3ccc(F)cc3)n2)CCO1
InChIKey: KWIXFWNTCQAVMZ-RHSMWYFYSA-N
Target: Serine-protein kinase ATM (SINGLE PROTEIN), Homo sapiens
UniProt: Q13315
Assay: Biological Assay: ATM: Recombinant, full length human FLAG-tagged ATM activity (Eurofins 14-933) was measured in an ELISA for p53 S15 phosphorylation, in a 384 well V-bottom polypropylene plate (Greiner Bio One 781280). Reactions contained 0.75 nM ATM, 25 nM full length myc tagged p53 (Eurofins 23-034), 10 μM UltraPure ATP (Promega V915B) and serial dilutions of inhibitors (0.1% DMSO final) in 25 mM HEPES, pH 7.5. 5 mM MgCl2, 2.5 mM MnCl2, 0.006% Brij-35, 0.5% glycerol, 0.1 mg/ml BSA, 0.5 mM DTT and were allowed to proceed for 30 min at 20° C. 70 mM EDTA final terminated the kinase reactions. An aliquot of the terminated reaction (25 μl) was transferred to a 384-well, high binding microplate (Greiner Bio One 781061), precoated with anti-myc capture antibody overnight (Millipore 05-724) diluted in 1:1000 in PBS, then blocked with Odyssey blocking buffer (LI-COR Biosciences 92740000) for 60 min at 20° C. The terminated reaction was allowed to capture for 2 h at 20° C. Phosphorylation was measured using an antibody specific to p53 S15 phosphorylation (1:5000 Abcam ab38497), anti-rabbit HRP (1:1000 Cell Signalling Technology, 7074) and TMB (ab171522). Antibodies were diluted in Odyssey blocking buffer and incubated for 60 min at 20° C. Three washes with PBS containing 0.05% (v/v) Tween 20 (PBS-T) were performed between each incubation. The TMB reaction was stopped with an equal volume of 0.2 M sulfuric acid and absorbance read at 450 nm on the Perkin Elmer Envision within 30 min of terminating the reaction. The percentage of inhibition was calculated for each concentration of compound using 0.1% DMSO control wells as 0% inhibition and 1 μM KU60019 as 100% inhibition.
Measurement: IC50 = 34.9 nM (pChEMBL 7.460000038146973)
Descriptors: MW 372.44, ALogP 2.35, HBA 5, HBD 1, PSA 61.46, QED 0.89
Compound (SMILES): O=C(Nc1cn2nc(-c3c(-c4ccc(F)cc4)ncn3C3CCNCC3)ccc2n1)c1ccnc2ccccc12
InChIKey: YJDWEUUAJDICLP-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 1.03 nM (pChEMBL 8.989999771118164)
Descriptors: MW 532.58, ALogP 5.12, HBA 8, HBD 2, PSA 102.03, QED 0.32
Compound (SMILES): CN1CC[C@H](c2nc(-c3ccc(C(=O)Nc4cc(C(F)(F)F)ccn4)cc3)c3c(N)nccn23)C[C@@H]1C(=O)O
InChIKey: ADDSNSWMLACXKM-FUHWJXTLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated A5 peptide (Biotin-EQEDEPEGDYFEWLE-NH2) (SEQ.ID.NO.: 1), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10)) compound concentrations.
Measurement: IC50 = 10.2 nM (pChEMBL 7.989999771118164)
Descriptors: MW 539.52, ALogP 3.91, HBA 8, HBD 3, PSA 138.74, QED 0.35
Compound (SMILES): C=CC(=O)N1C[C@@H](n2nc(C#Cc3cc4ncn(C5CC5)c4cc3F)c(C(N)=O)c2NC)C[C@H]1C
InChIKey: VQTWPNLKVTURHJ-PBHICJAKSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: FGFR2 Kinase Assay: Small molecule inhibition of FGFR2 kinase activity was evaluated using a fluorescence-based microfluidic mobility shift assay. FGFR2 catalyzes the production of ADP from ATP during phosphoryl transfer to the substrate peptide, FLPeptide30 (5-FAM-KKKKEEIYFFFCONH2) (Perkin Elmer, 760430). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product were measured and the ratio of these values used to generate % conversion of substrate to product by the LabChip EZ reader (Perkin Elmer). Wild type FGFR2 (Carna Bioscience, 08-134) at 0.06 nM was prepared with 1.5 μM substrate, 10 mM MgCl and 100 μM ATP in a butler containing 50 nM HEPES, 1 mM EGTA, 0.01% Brij-35, 0.05% BSA, and 2 mM DTT prior to addition of compounds in DMSO and incubation for 80 minutes at room temperature. The reaction was terminated by addition of 0.5 MEDIA. IC50 values were calculated using the inhibition of conversion ratio using Dotniatics Knowledge Solutions Studies curve fitting (Dotmatics, Bishops Stortford, UK, CM23).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 475.53, ALogP 2.6, HBA 7, HBD 2, PSA 111.07, QED 0.44
Compound (SMILES): CN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(CC(=O)N1C[C@H](O)[C@@H](O)C1)C(=O)N2CC1CCC1
InChIKey: LWZIGIZBASNRBO-PHIRODEFSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Human Nociceptin/Orphanin FQ Peptide (hNOP) Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4). The final assay volume (250 μl/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences, Inc. Boston, Mass., USA), and either test compound in dilution series or 1 μM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration, which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd., Buckinghamshire, UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences, Inc. Boston, Mass., USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ß-counter (PerkinElmer Life Sciences/Wallac, Turku, Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]nociceptin-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation, (Cheng and Prusoff, 1973).
Measurement: Ki = 7.4 nM (pChEMBL 8.130000114440918)
Descriptors: MW 484.64, ALogP 1.86, HBA 5, HBD 2, PSA 87.56, QED 0.64
Compound (SMILES): COC(=O)c1ccc2c(N(Cc3ccc(-c4ccc(Cl)c(C#N)c4)cc3)C(=O)CC(C)(C)C)nccc2c1
InChIKey: KBBGUMCXXDOPPI-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: In Vitro FXR Assay (TK): SeedingCV-1 cells were seeded at a density of 2,000,000 cells in a T175 flask with DMEM+10% charcoal double-stripped FBS and incubated at 37° C. in 5% CO2 for 18 h (O/N).TransfectionAfter 18 h of incubation, the medium in the T175 flask was changed with fresh DMEM+10% charcoal super-stripped serum. In a polypropylene tube, 2500 μL OptiMEM (Life Technologies, Cat #31985-062) was combined with expression plasmids for hFXR, hRXR, TK-ECRE-luc and pCMX-YFP. The tube was then briefly vortexed and incubated at room temperature for 5 minutes. Transfection reagent (X-tremeGENE HP from Roche, Cat #06 366 236 001) was added to the OptiMEM/plasmid mixture vortexed and incubated at room temperature for 20 minutes. Following incubation, the transfection reagent/DNA mixture complex was added to cells in the T175 flask and the cells were incubated at 37° C. in 5% CO2 for 18 h (O/N).Test CompoundsCompounds were serially diluted in DMSO and added to transfected CV-1 cells. The cells were then incubated for 18 hrs. The next day cells were lysed and examined for luminescence.
Measurement: EC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 526.04, ALogP 7.18, HBA 5, HBD 0, PSA 83.29, QED 0.25
Compound (SMILES): Cc1ccc(F)c([C@@H](C)[C@H](NS(=O)(=O)c2ncc(Br)cc2C(N)=O)c2n[nH]c(=O)o2)c1C
InChIKey: SUDQOGCFXPWVPJ-BMIGLBTASA-N
Target: Ribonucleotide reductase (PROTEIN COMPLEX), Homo sapiens
UniProt: P23921
Assay: Human RNR Inhibition Effect: The inhibitory activity against the ribonucleotide reduction reaction (hereinafter referred to as RNR reaction) of the test compound was determined by measuring the formation of deoxycytidine diphosphate (hereinafter referred to as dCDP) from cytidine diphosphate (hereinafter referred to as CDP) by the following method.Human M1 subunit and human M2 subunit (mutant lacking amino terminal 59 amino acids), which are fused a histidine tag at the amino terminus, are overexpressed in Escherichia coli and are solubilized after collection, and histidine tagged human M1 and M2 proteins were purified on a nickel chelate column.For measuring the inhibitory activity of the test compound against the RNR reaction, the method described in the document [CANCER RESEARCH 64, 1-6, 2004] was referred to.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 528.36, ALogP 2.2, HBA 7, HBD 3, PSA 161.04, QED 0.42
Compound (SMILES): CC1CCCCN1c1nc2nc(C(=O)O)nc(N[C@H](C)C3CCC3)c2n1C[C@H]1CC[C@H](C)CC1
InChIKey: YQHNCNOHGNUECN-CEBOQPEMSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 2.231 nM (pChEMBL 8.649999618530273)
Descriptors: MW 468.65, ALogP 5.33, HBA 7, HBD 2, PSA 96.17, QED 0.57
Compound (SMILES): NC(=O)c1nn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cccc(-c3ccccc3Cl)n2)c2ccc(C#Cc3ncccn3)cc12
InChIKey: NJXNLTUKQTWENR-IBVKSMDESA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition 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 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 623.05, ALogP 3.62, HBA 8, HBD 2, PSA 148.99, QED 0.27
Compound (SMILES): COc1cccc(CN2CCC(Oc3ccc(S(=O)(=O)Nc4nccs4)cc3Cl)CC2)c1
InChIKey: HWOQCTUXWXDBJF-UHFFFAOYSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS ρCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 494.04, ALogP 4.65, HBA 7, HBD 1, PSA 80.76, QED 0.49
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(N)c(C)c3N(Cc3cnn(C)c3)C2=O)c1F
InChIKey: XJDWLJXIEVLSPK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR Enzymatic Assay: The inhibitor potency of the exemplified 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; 30 mM EDTA with Perkin Elmer Lance Reagents 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 (BMG Labtech).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. The enzymes were purchased from Millipore or Invitrogen.GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active.The compounds of the invention were found to be inhibitors of one or more of FGFR1, FGFR2, and FGFR3 according to the above-described assay.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 444.44, ALogP 3.15, HBA 7, HBD 1, PSA 98.74, QED 0.65
Compound (SMILES): C=CC(=O)N1CCCC(n2nc(-c3ccc(Oc4c(F)c(F)cc(F)c4F)cc3F)c3c(N)ncnc32)C1
InChIKey: JUXVTKDUICDETA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition Assay of BTK Kinase Activity: The enzyme reaction mixture of BTK wild type standard HTRF assay contained 1 nM BTK wild type, 1 μM biotin-TK1 peptide, and 30 μM ATP in a buffer. The enzyme reaction were carried out at room temperature for 60 minutes. 5 μl of 0.2 M EDTA were added to quench the reaction and then the inhibitors (5 μl) were added at final concentrations of 2 nM antibody and 62.5 nM XL665. The plates were incubated at room temperature for 60 minutes and then read in the Envision plate reader. The readouts were transformed into inhibition rate % by the equation of (Min Ratio)/(Max−Min)*100%. Hence the IC50 data of test compounds were generated by using four parameters curve fitting.
Measurement: IC50 = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 530.46, ALogP 4.91, HBA 7, HBD 1, PSA 99.16, QED 0.22
Compound (SMILES): N#Cc1cncc2ncn([C@H]3CCC[C@H]3Oc3ccc(Cl)cc3-c3cccc(C(=O)O)c3)c(=O)c12
InChIKey: DIYUDWUTEANGPU-JTHBVZDNSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 486.92, ALogP 4.85, HBA 7, HBD 1, PSA 118.1, QED 0.43
Compound (SMILES): O=C(O)c1ccc(N2C(=O)/C(=C\c3c(Cl)cccc3C3CC3)c3c(F)cccc32)cc1O
InChIKey: UWABLRUEXSUBJP-PDGQHHTCSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: TR-FRET Screening: The compounds of the present application can regulate (inhibit) the biological activity of the nuclear receptor RORγ, and the intensity of such a regulation (inhibition) can be evaluated by TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) screening system. Nuclear receptor cofactors (co-activators and co-repressors) can regulate the transcription of target genes through interactions with nuclear receptors. If a ligand (test compound) interferes with the interaction between a nuclear receptor and a cofactor, such a ligand (test compound) can regulate the transcription of the corresponding gene.The method employs LanthaScreen TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) technique of Life Technologies Co. to test the compounds for their abilities to regulate (agonize or inversely agonize) the interaction between RORγ and its co-activator. RORγ-LBD is indirectly labeled by binding a terbium(Tb)-labeled anti-GST antibody (Life Technologies #PV3550) onto the GST tag of RORγ-LBD (Life Technologies #PV5887). When no ligand is present, RORγ can continuously bind to a fluorescein-labeled co-activator, and the binding of an agonist to RORγ can enhance the interaction between RORγ and the fluorescein-labeled co-activator; while the binding of an inverse agonist to RORγ can inhibit the interaction between RORγ and the fluorescein-labeled co-activator. When the fluorescein-labeled co-activator and the terbium-labeled anti-GST antibody-RORγ-LBD composite are close to each other to a certain distance, an energy transfer can occur, generating a TR-FRET signal. The co-activators employed in the method are not limited to D22 (Life Technologies #PV4386).(1) The final concentrations in the reaction system and the reaction conditions in the method are shown in the table below:TABLE 2 Concentration of Total Concentration of Fluorescein-labeled Concentration of Tb-labeled volume Reaction ROR γ-LBD co-activator activator peptide anti-GST antibody (μL) conditions 2 nM D22 150 nM 2 nM 20 22° C., 5 hr Fluorescein-D22
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 449.87, ALogP 5.98, HBA 3, HBD 2, PSA 77.84, QED 0.48
Compound (SMILES): CC1(C)OB(c2cnc(N)c(-c3ccc(NC(=O)Nc4ccccc4)cc3)c2)OC1(C)C
InChIKey: MPBUNHUNYNCBLY-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: Biochemical Assay: Biochemical PDGFRβ kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 36 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 μl per well of kinase buffer containing 200-300 ng purified cytoplasmic domain PDGFR-b protein (Millipore). Following a 60 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm. IC50 values for compound inhibition were calculated directly from graphs of optical density (arbitrary units) versus compound concentration following subtraction of blank values.
Measurement: IC50 = 206 nM (pChEMBL 6.690000057220459)
Descriptors: MW 430.32
Compound (SMILES): CCN1CCCCCCCCNc2cc(ccn2)-c2nc(cs2)C1=O
InChIKey: RAMHQEFXQIHDJE-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: The biochemical assay is in a AlphaScreen format. The kinase reaction is based on the IRAK-4 phosphorylation of a biotin labeled peptide. The phosphopeptide is incubated with anti-phosphothreonine antibody as well as streptavidin- and protein A-coated beads. Binding of the protein-A coated beads to the antibody and the streptavidin beads to the peptide, leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.Generally, the kinase reaction is carried out at 1 nM IRAK4, 1.6 μM peptide, 10 μM ATP in reaction buffer 50 mM Hepes, 60 mM NaCl, 5 mM MgCl2, 0.25 mM MnCl2, 2 mM DTT, 0.01% BSA, 0.01% Tween-20) for 3.5 h at RT.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 358.51, ALogP 4.43, HBA 5, HBD 1, PSA 58.12, QED 0.82
Compound (SMILES): COc1ccccc1Cc1cncc(C(=O)NCc2ccc3c(N)nccc3c2)c1
InChIKey: RJBICVKPFVWOFZ-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Plasma kallikrein inhibitory activity: 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; Stürzebecher 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 = 20200 nM (pChEMBL 4.699999809265137)
Descriptors: MW 398.47, ALogP 3.74, HBA 5, HBD 2, PSA 90.13, QED 0.52
Compound (SMILES): COc1ncc(-c2ccnc(NC(C)=O)c2)cc1NS(=O)(=O)N(C)C
InChIKey: VJYPPVBMJWHGNO-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: Enzyme Assay : 100 nL compounds in DMSO are added to wells of a 384 well microtitre plate (Greiner 780076). At room temperature: 5 ul VPS34 reaction buffer (Invitrogen Assay Buffer Q (diluted 1 in 5 with nanopure water) plus 2 mM DTT and 2 mM MnCl2) containing ATP (20 uM, Promega) and 200 uM PI-PS substrate (Invitrogen PV5122) is added followed immediately by 5 ul VPS34 reaction buffer (as above) containing VPS34 (5 nM, Millennium Protein Sciences Group) and the mixture is incubated with shaking at room temperature for 1 hour. Then 5 ul VPS34 stop-detect mix (as per Invitrogen Adapta Assay kit (PV5009) instructions (contains kinase quench buffer, TR-FRET buffer, Adapta Eu anti-ADP antibody and Alexa Fluor 647 ADP tracer)) is added to quench the reaction. The plates are then incubated for 30 minutes at room temperature with shaking and then read on a BMG PheraStar Plus reader.For the assay methods described above, test compound percent inhibition, at various concentrations, is calculated relative to control (DMSO and EDTA) treated samples. Compound concentration versus percent inhibition curves are fitted to generate IC50 values. One skilled in the art will appreciate that the values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 365.42, ALogP 1.33, HBA 6, HBD 2, PSA 113.52, QED 0.8
Compound (SMILES): CCOc1cnc(NCc2ccc(-c3ccc4ncnc(N)c4c3)s2)c(C(=O)NCc2ccc(F)c(F)c2)c1
InChIKey: DKTWLILWYMWFQW-UHFFFAOYSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 546.6, ALogP 5.55, HBA 8, HBD 3, PSA 115.05, QED 0.22
Compound (SMILES): C[C@@H]1CC[C@@H](c2nc(C(C)(C)O)cs2)CN1C(=O)c1ccsc1-n1nccn1
InChIKey: JAGZPDABOCYXRW-CHWSQXEVSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: Briefly, for intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the orexin-1 receptor (e.g., rat or human) or the orexin-2 receptor (e.g., rat or human), are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at approximately 20,000 cells per well into 384-well clear bottom sterile plates coated with poly-D-lysine. The seeded plates are incubated overnight at 37° C. and 5% CO2. Human ala-6,12 orexin-A can be used as the agonist and prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then in assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 minutes (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4 AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 minutes, and then 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM of agonist ala-6,12 orexin-A with buffer in place of test compound.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 417.56, ALogP 3.42, HBA 8, HBD 1, PSA 84.14, QED 0.7
Compound (SMILES): CCn1nc(CC(C)(C)COC(=O)c2ccc(S(=O)(=O)C(C)C)cc2)c2c1C(=O)NCC1(CCOCC1)C2
InChIKey: QVMJXTTWAHKBNE-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: PDE4 Assay: The human PDE4D catalytic domain (UniProt no. Q08499 [S380-L740]) was incubated with a mixture of non-labelled cAMP (cyclic adenosine monophosphate) and fluorescein amidite (FAM) conjugated cAMP and titrated test or reference Compound. Following brief incubation the enzymatic reaction was stopped by addition of binding buffer containing nanoparticles with immobilized trivalent metal ions capable of binding 1) AMP phospho groups and 2) terbium (Tb) donor fluorophores. Subsequent excitation of the Tb donor triggers time-resolved FRET to adjacent FAM acceptor molecules resulting in light emission. In the presence of a PDE4 inhibitor, AMP generation was reduced resulting in a lower fluorescence signal. The cAMP phosphodiester is not bound by the detection system.The results were calculated as the molar concentrations resulting in 50% inhibition of the substrate cleavage compared to controls samples, and are expressed as a range of IC50 (nM).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 545.7, ALogP 3.59, HBA 8, HBD 1, PSA 116.59, QED 0.5
Compound (SMILES): C[C@H](Nc1nccc(N2CCOC2=O)n1)c1cc2cc(Cl)ccc2[nH]c1=O
InChIKey: BTUGZKWGQKVBMC-JTQLQIEISA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: IDH1-R132H and IDH1-R132C Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 M Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 385.81, ALogP 3.1, HBA 6, HBD 2, PSA 100.21, QED 0.72
Compound (SMILES): O=c1[nH]nc(Cc2ccc(F)c(P3(=O)CCN(c4ncccn4)CC3)c2)c2ccccc12
InChIKey: XJZZZXBGQBLBGP-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Enzyme Assay: Inhibition of PARP-1 enzymatic activities was measured using an HT Universal Colorimetric PARP Assay Kit (Trevigen, catalogue #4677-096-K) following the standard procedures. Serial dilutions of PARP compounds were added to appropriate wells. The compound was incubated with PARP enzyme at RT for 10 min, and the reaction was initiated by addition of the PARP substrate cocktail, Each concentration of compound was tested in triplicate wells. After 15 min reaction, the reaction was stopped, and the Strep-HRP was added into each well and incubated for 1 h at RT. After addition of TACS-Sapphire colorimetric substrate and incubated in dark for 15 min, the OD450 was read out with an EnVision instrument. IC50 values (the concentration at which 50% of the enzyme activity is inhibited) were calculated, which are determined over a range of different concentrations, normally from 10 uM to 0.1 nM.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 449.43, ALogP 2.95, HBA 6, HBD 1, PSA 91.84, QED 0.48