lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(O)c1ccc(CCc2cccc3c2N(Cc2cccc(F)c2)CC3)cc1
InChIKey: IAWBENGNIVXQLE-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: FLIPR Assay: Cells were seeded at a density of 5×104 cells per well in Biocoat® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 mM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 ml in each well. Plates were re-equilibrated to 37° C. for a few minutes.Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR™, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration. The peak increase in fluorescence intensity was recorded for each well. On each plate, four wells each served as negative (HBSS-HEPES buffer) and positive controls (standard agonists: BW245C (hDP); PGE2 (hEP1; hEP2/Gqs5; hEP3A/Gqi5; hEP4/Gqs5); PGF2a (hFP); carbacyclin (hIP); U-46619 (hTP), depending on receptor). The peak fluorescence change in each drug-containing well was then expressed relative to the controls.
Measurement: Ki = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 375.44, ALogP 4.87, HBA 2, HBD 1, PSA 40.54, QED 0.67
Compound (SMILES): Cc1ccc(Cn2c(=O)n([C@H](C)CC(=O)O)c(=O)[nH]/c2=N\c2ccc(Oc3ccc(F)cn3)cc2)cc1
InChIKey: RYVNPNYPCWSDAC-QGZVFWFLSA-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 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 505.51, ALogP 3.29, HBA 8, HBD 2, PSA 131.57, QED 0.38
Compound (SMILES): NC(=O)C1CCN(Cc2ccc(-c3cccc(S(N)(=O)=O)c3-c3nnn[nH]3)cc2)C1
InChIKey: HNAJKBXKEPHBRZ-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 4.38 nM (pChEMBL 8.359999656677246)
Descriptors: MW 427.49, ALogP 0.49, HBA 7, HBD 3, PSA 160.95, QED 0.52
Compound (SMILES): Cn1cc(-c2cn3ncc(C#N)c3c(-c3ccc(N4C[C@H]5C[C@@H](C4)C5N)nc3)n2)cn1
InChIKey: WOCTZUJHJGILAO-DBIVSCPCSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TKOPEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1× TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TKOPEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 57.4 nM (pChEMBL 7.239999771118164)
Descriptors: MW 411.47, ALogP 1.85, HBA 9, HBD 1, PSA 113.95, QED 0.55
Compound (SMILES): CN(C1COC1)C(C)(C)C=C(C#N)C(=O)N1CCC[C@H]1Cc1nc(-c2ccc(Oc3ccccc3)cc2F)c2c(N)nccn12
InChIKey: XIFJGSDTBKHODT-QHCPKHFHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzymatic Activity Assay: A Caliper-based kinase assay (Caliper Life Sciences, Hopkinton, Mass.) was used to measure inhibition of BTK kinase activity of a compound of the present disclosure. Serial dilutions of test compounds were incubated with human recombinant BTK (0.5 nM), ATP (16 μM) and a phosphoacceptor peptide substrate FAM-GEEPLYWSFPAKKK-NH2 (1 μM) at room temperature for 3 h. The reaction was then terminated with EDTA, final concentration 20 mM and the phosphorylated reaction product was quantified on a Caliper Desktop Profiler (Caliper LabChip 3000). Percent inhibition was calculated for each compound dilution and the concentration that produced 50% inhibition was calculated.
Measurement: IC50 = 4.3 nM (pChEMBL 8.369999885559082)
Descriptors: MW 609.71, ALogP 5, HBA 9, HBD 1, PSA 122.01, QED 0.21
Compound (SMILES): CC(=O)C1CCC(c2cc(C(F)(F)F)ccc2CN2CCC3(CCN(C(=O)OC(C(F)(F)F)C(F)(F)F)CC3)C2)CC1
InChIKey: IIMHTZQWVOVKCN-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Competitive Activity-Based Protein Profiling (Human): Proteomes (human prefrontal cortex or cell membrane fractions) (50 μL, 1.0-2.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP¿Rh or JW912 (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at room temperature. Reactions were quenched with SDS loading buffer (15 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL using ImageJ 1.49k software.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 616.57, ALogP 7.49, HBA 4, HBD 0, PSA 49.85, QED 0.32
Compound (SMILES): O=C(NCc1cccnc1)Nc1ccc(S(=O)(=O)c2ccc(F)cc2Cl)cc1
InChIKey: CWTLELBVSTVYSW-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100× stock. Buffer A (89 μL) containing 33 nM of NAMPT protein was added to 1 μL of 100× compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 μL of 10× substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 μM NAM, 100 μM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5′-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 μL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 μM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 419.87, ALogP 4.03, HBA 4, HBD 2, PSA 88.16, QED 0.61
Compound (SMILES): CC(C)c1cccc(-c2nc3nc(C(=O)O)nc(N[C@H](C)C4CC4)c3n2Cc2ccc(C(F)(F)F)cc2)c1
InChIKey: PRMUDUBEKQEVHU-MRXNPFEDSA-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 = 122.9 nM (pChEMBL 6.909999847412109)
Descriptors: MW 523.56, ALogP 6.59, HBA 6, HBD 2, PSA 92.93, QED 0.27
Compound (SMILES): O=c1[nH]c2cc(CN3CCN(c4ccc(Cl)nn4)CC3)ccc2c2c1CCCN2
InChIKey: DRPXVULWOOOZDV-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Inhibition Assay: The PARP-1 enzyme inhibitory activities of the compounds of the present invention were assayed in the following manner by use of a kit (cat. 80551) purchased from BPS Bioscience.The 96-well plate provided in BPS Bioscience kit was coated with histone and incubated at 4° C. for 16 hours. Then, the plate was washed four times with PBST (7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 145 mM NaCl, 0.05% Tween 20, pH 7.4), and blocking buffer (provided in BPS Bioscience kit) was added thereto in order to block nonspecific reaction, and was then incubated at 25° C. for 1 hour. After incubation for 1 hour, the plate was washed four times with PBST, and varying concentrations of each of the compounds of the Examples were added to a reaction solution containing PARP-1 enzyme (50 ng/well), an assay mixture and activated DNA, and allowed to react at 25° C. for 1 hour. After 1 hour, each well was washed four times with PBST, and in order to measure the level of ribosylation by PARP enzyme, streptavidin-linked peroxidase (Strep-HRP, 1:50 dilution) was added and allowed to react at 25° C. for 30 minutes. The plate was washed four times with PBST, and then finally an HRP chemiluminescent substrate was added and allowed to react. The level of histone ribosylation formed by each enzyme was quantified using Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., USA).
Measurement: IC50 = 3.25 nM (pChEMBL 8.489999771118164)
Descriptors: MW 410.91, ALogP 2.65, HBA 6, HBD 2, PSA 77.15, QED 0.69
Compound (SMILES): CN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(c1cnc(-c3cc(F)c(F)c(F)c3)nc1)C(=O)N2
InChIKey: CSSLVBHCLDECEY-WMPKNSHKSA-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 β-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 = 335 nM (pChEMBL 6.46999979019165)
Descriptors: MW 481.52, ALogP 4.86, HBA 4, HBD 1, PSA 61.36, QED 0.54
Compound (SMILES): CC(C)Sc1cccc(-c2ccc3nc(CCC(=O)O)ccc3c2)n1
InChIKey: CAESUTOHFPMLHR-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Measurement of Activity of GPR120 Agonist (Cell-Based Assay): CHO-K1 cells expressing Ga16 and hGPR120 were dispensed into each well of a 96-well plate (3×104 cells/100 μl/well) and then incubated in 5% CO2, 37° C. incubator for 18 hours. Each well was treated with 100 μl of Calcium 5 dye (Molecular Devices) solution including 2% DMSO and then incubated in 5% CO2, 37° C. incubator for 1 hour. Serially diluted GPR120 agonists were prepared to a final concentration of 0.5% DMSO in a 96-well plate. Each well was treated with 50 μl of the agonist compounds using Plexstation II, and then fluorescence was measured at Ex 485 nm and Em 525 nm.Fluorescence increased by the serially diluted GPR120 agonists is calculated as a relative percent (%) value based on the fluorescence represented by the treatment of 1% DMSO only. EC50 refers to the concentration of agonist which shows 50% of maximum fluorescence increased by the treatment of agonist. The calculation of measurement was carried out by using statistical software (Prizm).
Measurement: EC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 352.46, ALogP 4.81, HBA 4, HBD 1, PSA 63.08, QED 0.64
Compound (SMILES): CNc1cc(Nc2cccn([C@@H]3CCC[C@@H]3O)c2=O)nc2c(C(=O)N[C@H]3CCC3(F)F)cnn12
InChIKey: IHEZPEDUFRDIRE-PMPSAXMXSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 473.48, ALogP 2.29, HBA 9, HBD 4, PSA 125.58, QED 0.43
Compound (SMILES): Cc1c(C#N)ccnc1O[C@@H]1CC[C@@H](C)N(C(=O)c2cc(F)ncc2-c2cncs2)C1
InChIKey: OQYWTLSOGJZWJK-CZUORRHYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand binding assay: Radioligand binding assay (described in Bergman et. al. Bioorg. Med. Chem. Lett. 2008, 18, 1425-1430) in which the inhibition constant (Ki) is determined in membranes prepared from CHO cells expressing either the OX1 or OX2 receptor. The intrinsic orexin receptor antagonist activity of a compound which may be used in the present invention may be determined by these assays.
Measurement: Ki = 6.1 nM (pChEMBL 8.210000038146973)
Descriptors: MW 437.5, ALogP 3.99, HBA 7, HBD 0, PSA 92, QED 0.57
Compound (SMILES): Cc1cc(C(=O)NCc2ccc(CN)c(F)c2)c(C)n1Cc1csc(-c2ccccc2)n1
InChIKey: ICSBZJSGYBSVLI-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Plasma kallikrein assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; 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 = 135 nM (pChEMBL 6.869999885559082)
Descriptors: MW 448.57, ALogP 4.8, HBA 5, HBD 2, PSA 72.94, QED 0.43
Compound (SMILES): C=CC(=O)N1CCN(C(=O)N(C)[C@H](C(=O)N[C@H]2Cc3cc(O)cc(c3)-c3ccc4c(c3)c(c(-c3ccccc3CCOC)n4CC)CC(C)(C)COC(=O)[C@@H]3CCCN(N3)C2=O)C(C)C)CC1
InChIKey: SGIGSPVNEPOEBZ-MDGKGBRESA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 918.15, ALogP 5.94, HBA 10, HBD 3, PSA 165.99, QED 0.13
Compound (SMILES): CN(C(=O)c1ccc(Cl)cc1)C1CCN(C(=O)[C@@](C)(O)C(F)(F)F)CC1
InChIKey: QITIRBFSMNKZPF-MRXNPFEDSA-N
Target: [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q15119
Assay: Biochemical Activity Assay: The PDC inactivation assay is performed in Greiner 384-well microtiter plates and is used for high throughput screen. 4 μl of PDHK2 (human, rec, Carna Bioscience, 10 ng/μl-137 nM final concentration) and PDC (isolated from porcine heart, Sigma-Aldrich, 20 mU/ml final concentration) are incubated in the absence or presence of the test compound (10 dilution concentrations) for 30 min at room temperature in kinase buffer (15 mM potassium phosphate buffer, pH 7.0, 60 mM KCl, 1.5 mM DTT, 2.5 mM MgCl2, 0.0125% (w/v) BSA, 0.125% Pluronic F-68). The kinase reaction is started by the addition of 4 μl ATP substrate solution (fc 5 μM in kinase buffer). After 30 min incubation at 37° C. 40 μl of PDC reaction solution (100 mM Tris/HCl, pH 7.8, 0.5 mM EDTA, 1 mM MgCl2, 50 mM NaF, 0.25 mM Coenzyme A, 5 mM pyruvate, 1 mM NAD, 5 mM DTT, 1 mM thiamine pyrophosphate) is added. The first fluorescence measurement is performed on a Perkin Elmer Envision (Exc 340 nm, Em 450 nm). The reaction is incubated for 45 min at room temperature. Afterwards a second fluorescence measurement is performed and the PDC activity is calculated by the difference between both measurements. As full value for the PDHK2 assay the inhibitor-free PDHK2 reaction is used.
Measurement: IC50 = 390 nM (pChEMBL 6.409999847412109)
Descriptors: MW 392.81, ALogP 2.72, HBA 3, HBD 1, PSA 60.85, QED 0.86
Compound (SMILES): O=C(Nc1ccc(C(F)(F)F)c(F)c1)NC1CCN(C(=O)C2CCOC2)CC1
InChIKey: ZUWZREBWJHJAFL-UHFFFAOYSA-N
Target: Bifunctional epoxide hydrolase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34913
Assay: FRET-displacement assay: FRET assays to determine Ki for the compounds of Table I were carried out as described previously (Lee et al. Analytical Biochemistry 434 (2013) 259-268). In order to prevent leaching of fluorescence impurities from the plastic tube and non-specific binding to sEH inhibitors, the inhibitor stock solution (10 mM, DMSO) was stored in glass vials. In addition, sEH was diluted to desired concentration (20 nM) with sodium phosphate buffer (PB) (100 mM sodium phosphate, pH 7.4, 0.01% gelatin) to avoid loss of protein from non-specific binding to the cuvette surface. All buffer used in this assay was filtered by sterilized filtration unit (Millipore Durapore PVDF Membrane, pore size: 0.22 um).Measurement in 96-Well PlatesAll the measurement for FRET-based displacement assay in 96-well plate format were done in TECAN Infinite M1000 Pro 96 well fluorescence plate reader.Pre-Treatment of 96-Well PlateIn order to prevent non-specific binding of sEH or inhibitor on the 96-well plate, the 96 well plates were pre-incubated with PB with 0.1% gelatin overnight at rt. The gelatin coats the plate and prevents non-specific binding of sEH and sEH inhibitors to the plate. The buffer was discarded and the plate was dried before use.Assay ProcedureThe sEH stock was diluted to the desired concentration (20 nM) by PB (100 mM sodium phosphate, 0.1% gelatin, pH 7.4). ACPU (one equivalent to sEH, 10 mM, Ethanol) was added to the sEH solution and was incubated for 2 h at rt. The sEH-ACPU mixture (20 nM, 100 mM sodium phosphate, 0.1% gelatin, pH 7.4, 150 uL) was added to each well.The baseline fluorescence (F0) (λexcitation at 280 nm, λemission at 450 nm) of the samples was measured after the z-position and gain were optimized automatically by the fluorometers. The z and gain value was noted and will be used for the later fluorescent measurement. Because DMSO has been known to quench fluorescence. 1% DMSO in PB was served as a control (FDMSO). The desired concentration of inhibitors which is the concentration that 100% of sEH was bound to inhibitor, was added at the first well and was further diluted by 2-fold across the rest of the wells. Based on our study, 12 datum points which correspond to 12 different concentrations of the inhibitor, provide significant data to calculate the accurate Ki for the inhibitors. The samples were incubated at 30° C. for 1.5 h. Then, the fluorescence (λexcitation at 280 nm, λemission at 450 nm) of the samples was measured using the z-position and gain values that previously obtained.
Measurement: Ki = 2.09 nM (pChEMBL 8.680000305175781)
Descriptors: MW 403.38, ALogP 2.99, HBA 3, HBD 2, PSA 70.67, QED 0.76
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](NC(=O)c1cn(-c3c(F)ccc(Cl)c3F)cn1)c1cc-2ccn1
InChIKey: ZHTFAOPBJGTZTJ-ZHRRBRCNSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as inhibitors of the coagulation Factors XIa, VIIa, IXa, Xa, XIIa, plasma kallikrein or thrombin, can be determined using a relevant purified serine protease, respectively, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at room temperature or at 37° C. Hydrolysis of the substrate resulted in the release of pNA (para nitroaniline), which was monitored spectrophotometrically by measuring the increase in absorbance at 405 nm, or the release of AMC (amino methylcoumarin), which was monitored spectrofluorometrically by measuring the increase in emission at 460 nm with excitation at 380 nm. A decrease in the rate of absorbance or fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.
Measurement: Ki = 10.4 nM (pChEMBL 7.980000019073486)
Descriptors: MW 609.03, ALogP 6.27, HBA 7, HBD 3, PSA 127.24, QED 0.23
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(-c3ccn4c(C)nnc4c3)nn2-c2ccccc2)[C@H](c2ccnc(F)c2)O1
InChIKey: VUWOVPFCJGYXEF-KCWPFWIISA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: Seven point five μL per well of human TrkA (PV3144, Lifetechnologies, final concentration: 1 nmol/L) suspended in the assay buffer (100 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20 and 1 mmol/L dithiothreitol (DTT)) was applied in a 384 well plate and the plate was pre-incubated for 15 min at room temperature with 0.4 μL of each compound (final concentration: 200 μmol/L-1 pmol/L) dissolved in DMSO. Then, fluorescent substrate (FL-peptide 27, 760424, PerkinElmer, final concentration: 1.5 μmol/L) and ATP (final concentration 500 μmol/L) dissolved in the assay buffer was added in each well. After the incubation of 120 min at 37° C., fifteen μL of termination buffer (100 mmol/L HEPES, 40 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20, 1 mmol/L DTT and 0.16 vol % Coating Reagent 3) was added in each well to stop the enzyme reaction. Fluorescent intensities (FI) of phosphorylated and non-phosphorylated fluorescent substrates were measured by LabChip EZReader II (Caliper LifeSciences, Inc.), and conversion ratio (CR) was calculated by the following formula-1. The CR in the well applied with DMSO alone was used as a negative control and the CR in the well without applying TrkA was used as a positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 571.62, ALogP 3.86, HBA 10, HBD 2, PSA 123.73, QED 0.27
Compound (SMILES): COC(=O)Nc1ccc(C(=O)N[C@@H](CC2CC2)C(=O)N2CC[C@]3(C2)OC(=O)Nc2ccc(Cl)cc23)cc1
InChIKey: JARFYEPFEKTRFV-HFZDXXHNSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Factor XIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIa can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at room temperature or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 40 pM (Sekisui Diagnostics) and he synthetic substrate, Z-Gly-Pro-Arg-AFC, TFA salt (Sigma #C0980) at a concentration of 100 μM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration≤0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (Vo) or presence of inhibitor (Vi) were measured. Assuming competitive inhibition, and that unity is negligible compared Km/[S], [I]/e, and [I]/e (where [S], [I], and e respectively represent the total concentrations, of substrate, inhibitor and enzyme), the equilibrium constant (Ki) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of Vo/Vi on [I] shown in the following equation. V o /V i=1+[I]/K iThe activities shown by this assay indicate that the compounds of the invention may be therapeutically useful for treating or preventing various cardiovascular and/or cerebrovascular thromboembolic conditions in patients suffering from unstable angina, acute coronary syndrome, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, stroke such as thrombotic stroke or embolic stroke, venous thrombosis, coronary and cerebral arterial thrombosis, cerebral and pulmonary embolism, atherosclerosis, deep vein thrombosis, disseminated intravascular coagulation, and reocclusion or restenosis of recanalized vessels.
Measurement: Ki = 726 nM (pChEMBL 6.139999866485596)
Descriptors: MW 526.98, ALogP 4.11, HBA 6, HBD 3, PSA 126.07, QED 0.52
Compound (SMILES): CCCNC(=O)N1CCc2c(-c3cccc(C(F)(F)F)c3)ccc(CNC(=O)[C@@H]3C[C@H]3c3nc(C)no3)c2C1
InChIKey: SLPZLEZPEUCFKI-DHIUTWEWSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: Enzymatic Activity Assay: DGAT2 activity was determined by measuring the amount of enzymatic product triolein (1,2,3-Tri(cis-9-octadecenoyl)glycerol) using the membrane prep mentioned above. The assay was carried out in deep well 384 plates in a final volume of 40 μL at room temperature. The assay mixture contained the following: assay buffer (100 mM Tris.Cl, pH 7.0, 20 mM MgCl2, 5% ethanol), 25 μM of diolein, 10 μM of oleoyl-CoA and 10 ng/μL of DGAT2 membrane.
Measurement: IC50 = 11.5 nM (pChEMBL 7.940000057220459)
Descriptors: MW 541.57, ALogP 4.96, HBA 5, HBD 2, PSA 100.36, QED 0.44
Compound (SMILES): O=C(CCNC[C@H]1c2ccccc2CN1c1cn[nH]c(=O)c1C(F)(F)F)N1CCN(c2ncc(C(F)(F)F)cn2)CC1
InChIKey: YBLKRMGMSMVFQD-IBGZPJMESA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 596.54, ALogP 2.99, HBA 8, HBD 2, PSA 110.35, QED 0.32
Compound (SMILES): COC1(CCN2C(=O)N(c3cnc(C)nc3)C[C@]23CC[C@](c2ccccc2)(N(C)C)CC3)CCC1
InChIKey: BXQOTDKKLBGWIX-DBAYTGFXSA-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 β-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 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 477.65, ALogP 4.76, HBA 5, HBD 0, PSA 61.8, QED 0.58
Compound (SMILES): Cc1ccc(F)c(Oc2nc(NS(=O)(=O)C(C)C)cc(-c3ccc(=O)n(C)c3)n2)c1Cl
InChIKey: VIMNTGURPDAEFU-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro Enzyme Inhibition Assay-CREBBP Inhibition: Determination of the IC50 for the CREBBP inhibitors disclosed herein was performed as follows: CREBBP was cloned and expressed in E. Coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CREBBP binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CREBBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20-minute incubation at room temperature, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After two hours of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.The ability of the compounds disclosed herein to inhibit CBP activity was quantified and the respective IC50 value was determined.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 466.92, ALogP 3.89, HBA 7, HBD 1, PSA 103.18, QED 0.59
Compound (SMILES): Cc1ccc(N2C[C@]3(CC[C@](c4ccccc4)(N(C)C)CC3)NC2=O)cc1
InChIKey: HAMMACHMSPLPLY-ZRZAMGCNSA-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 β-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 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 363.51, ALogP 4.29, HBA 2, HBD 1, PSA 35.58, QED 0.88
Compound (SMILES): Cc1cc(-c2ccc3nc(NC(=O)[C@@H]4C[C@@H]4F)sc3c2)sn1
InChIKey: QYALQSNXLUMARL-ZJUUUORDSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: c-abl Kinase Assay: ADP-Glo assay kit was purchased from Promega. Magnesium chloride (MgCl2), bovine serum albumin (BSA), ethylene glycol-bis(β-aminoethyl ether)-N,N′,N′-tetraacetic acid (EGTA), tween-20, 1,4-dithiothreitol (DTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. HEPES buffer was purchased from Gibco. ABL1 kinase and Abltide were purchased from Signalchem.c-abl kinase activity was measured by Promega's ADP-Glo Assay. In this assay, His-tagged recombinant human ABL1 (0.25 ng/μl) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of Abltide (0.01 μg/μl) and 5 μL of ATP (25 μM) in buffer (50 mM HEPES, 7.5; 10 mM MgCl2; 1 mM EGTA; 0.05% BSA; 0.01% Tween-20; 2 mM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30° C. for 30-min. After the incubation, 25 μL ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 μL per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software or SigmaPlot 13.0.
Measurement: IC50 = 11.5 nM (pChEMBL 7.940000057220459)
Descriptors: MW 333.41, ALogP 4.02, HBA 5, HBD 1, PSA 54.88, QED 0.79
Compound (SMILES): [C-]#[N+]c1c(Cl)cc(C(C)n2nc(C)c3c(N)ncnc32)c(OC)c1C1CN(C(=O)NC(C)(C)C)C1
InChIKey: JICXNLXOZGPECN-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3K delta Scintillation Proximity Assay: [γ-33P]ATP (10 mCi/mL) was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) was purchased from Millipore (Bedford, Mass.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.). Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from GE healthcare life sciences (Piscataway, N.J.). The kinase reaction was conducted in polystyrene 384-well matrix white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The PI3K assays were carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi [γ-33P] ATP, 4 nM PI3Kδ. Reactions were incubated for 210 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 3.0 software.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 497, ALogP 4.45, HBA 7, HBD 2, PSA 115.55, QED 0.52
Compound (SMILES): Clc1ccc(Sc2cccc3[nH]ccc23)c(Cl)c1
InChIKey: FSHJAKGFYCXRKF-UHFFFAOYSA-N
Target: GTP-binding protein Rheb (SINGLE PROTEIN), Homo sapiens
UniProt: Q15382
Assay: In Vitro Kinase Assay: For the Rheb-dependent in vitro kinase assay, HA-GST-Rheb was first loaded with GTP-γ-S (Millipore #20-176) by incubating 600 ng/μL Rheb with 10 mM EDTA and 0.4 mM GTP-γ-S at 30° C. for 10 minutes. MgCl2 was added to a final concentration of 20 mM to stop the reaction. This mixture was used at 20× in the kinase assay.Loaded HA-GST-Rheb was then incubated with compound (final DMSO concentration 1%) for 30 minutes at room temperature. mTORC1, GFP-4E-BP1 substrate (Life Technologies #PV4759), and kinase assay buffer (HEPES pH 7.4, KCl, MgCl2) were added to the Rheb-compound mixture and incubated at room temperature for 20 minutes. ATP was then added, and the kinase reaction proceeded at 30° C. for 45 min before being stopped by the addition of SDS-PAGE loading buffer for Westerns or 10 mM EDTA for the LanthaScreen readout. During the kinase reaction, the final concentration of reagents is as follows: Rheb30 ng/μL, compoundvariable, GTPγS0.02 mM, EDTA0.5 mM, MgCl21 mM, mTORC1fixed volume of prep (molarity unknown), GFP-4E-BP1 substrate15 ng/μL, ATP0.5 mM. For LanthaScreen analysis (Thermo Fisher #PV4757), the manufacturer's instructions were followed and conditions were optimized for the amount of Rheb and mTORC1. The final concentration of reagents was as follows: 100 nM loaded HA-GST-Rheb, compound (variable), 0.5 mM EDTA, 10 mM MgCl2, 25 mM HEPES pH 7.4, 50 mM KCl, mTORC1 (fixed volume0.1 μL in 10 μL total volume), 0.4 μM GFP-4E-BP1 substrate, 0.5 mM ATP. Optimization is summarized in FIG. 1. Results were read on an EnVision reader (PerkinElmer) using 495ex/520em filters (Life Technologies, #PV00315).
Measurement: IC50 = 51500 nM (pChEMBL 4.289999961853027)
Descriptors: MW 294.21, ALogP 5.63, HBA 1, HBD 1, PSA 15.79, QED 0.65
Compound (SMILES): O=C(O)c1cc(CNc2nccn3c([C@@H]4SC[C@@H](O)[C@H]4O)cnc23)ccc1Cl
InChIKey: GBYNZKDYQCRIOF-KFWWJZLASA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Assay for Binding Affinity for Adenosine Receptor A3: HEK-293 cell membrane homogenates (32 μg protein), in which A3 adenosine receptors were expressed, are incubated for 120 min at 22° C. with 0.15 nM [1251]AB-MECA in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA and 2 UI/ml ADA. Nonspecific binding is determined in the presence of 1 μM IB-MECA. Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is IB-MECA, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 420.88, ALogP 2.2, HBA 8, HBD 4, PSA 119.98, QED 0.5