lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CCC[C@@]2(C)c2nc3c(C)c(C)c(Br)cc3[nH]2)c1
InChIKey: MCOBUUIPLMSSOE-DEOSSOPVSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 493.41, ALogP 4.98, HBA 5, HBD 1, PSA 79.7, QED 0.44
Compound (SMILES): CN(C)/C=C(/Oc1ccc(Cl)cc1)C(=O)c1ccc(C(F)(F)F)cc1
InChIKey: ZLGSPZIJIGOYHK-LFIBNONCSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibition Assay: G-402 cells can be obtained from ATCC (CRL-1440) and were originally derived from a renal leiomyoblastoma.The expression plasmids contains the ORF for either human/cyno CYP11B1 or CYP11B2 under the control of a suitable promoter (CMV-promoter) and a suitable resistance marker (neomycin). Using standard techniques the expression plasmid is transfected into G-402 cells and these cells are then selected for expressing the given resistance markers. Individual cell-clones are then selected and assessed for displaying the desired enzymatic activity using 11-Deoxycorticosterone (Cyp11B2) or 11-Deoxycortisol (Cyp11B1) as a substrate.G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 μg/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 2.03 nM (pChEMBL 8.6899995803833)
Descriptors: MW 369.77, ALogP 5.02, HBA 3, HBD 0, PSA 29.54, QED 0.42
Compound (SMILES): COc1ncc(-c2cc(NC(=O)c3ccnc(C(C)(F)F)c3)cnc2C)cc1C1(C#N)COC1
InChIKey: MMCBOZOQHXYGBR-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity Determination: Assay Materials: Assay buffer is 50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% Bovine Serum Albumin (BSA) and 1 mM dithiothreitol (DTT); Stop buffer is 60 mM ethylenediaminetetraacetic acid (EDTA) and 0.01% Tween® 20: b-Raf (V600E), active; biotinylated Mek, kinase dead; Alpha Screen detection kit (available from PerkinElmer, #6760617R); Anti phospho-MEK1/2 (available from Cell Signaling Technology, Inc. #9121); 384 well low volume assay plates (White Greiner® plates).Assay conditions: b-Raf (V600E) approximately 4 μM; c-Raf approximately 4 nM; biotinylated Mek, kinase dead approximately 10 nM; ATP 10 μM for BRAF (V600E) and 1 μM for CRAF; Pre-incubation time with compounds 60 minutes at room temperature; Reaction time 1 or 3 hours at room temperature.Assay protocol: Raf and biotinylated Mek (kinase dead) were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, after which the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 1248 nM (pChEMBL 5.900000095367432)
Descriptors: MW 465.46, ALogP 4.01, HBA 7, HBD 1, PSA 110.02, QED 0.59
Compound (SMILES): CN(C)[C@H](CNC(=O)NCCc1cn(CCSSCCN=[N+]=[N-])nn1)Cc1ccc(O)cc1
InChIKey: RBURBNOCQBNJEY-SFHVURJKSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Radioligand Binding Assay: Each compound was initially tested at 20 μM and was incubated with 3H-DPN at a concentration equal to the Kd (0.4 nM) of the radioligand in μOR containing Sf9 insect cell membranes. The reaction contained 40 fmol of μOR and was incubated in a buffer of 20 mM HEPES pH 7.5, 100 mM sodium chloride, and 0.1% bovine serum albumin for 1 hour at 25° C. To separate free from bound radioligand, reactions were rapidly filtered over Whatman GF/B filters with the aid of a Brandel harvester and 3H-DPN counts were measured by liquid scintillation. Compounds with more than 25% of 3H-DPN radioactivity were further tested in full dose-response to determine the affinity (Ki) in HEK293 membranes. Subsequently, the 15 analogs were tested in full dose-response for affinity at the μOR and the κOR by the National Institutes of Mental Health Psychoactive Drug Screen Program (PDSP), as were the affinities of compounds 12, PZM21, and their stereoisomers at the μOR, δOR, κOR and nociception receptor.
Measurement: Ki = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 493.66, ALogP 2.69, HBA 9, HBD 3, PSA 144.07, QED 0.11
Compound (SMILES): O=C(N[C@H]1CC[C@H](CCN2CCN(c3nsc4ccccc34)CC2)CC1)c1cc2cc([N+](=O)[O-])ccc2[nH]1
InChIKey: GLRWZEISUAIVMI-XUTJKUGGSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Receptor Binding Test: 1. Experimental Materials:(1) D2 receptor Cell Transfection:This experiment utilizes the plasmid vector containing the gene of D2 receptor protein for transfecting HEK293 cells by calcium phosphate transfection method. By culturing the transfected cells in culture medium containing G418 and selecting cell clones followed by radioligand binding test, a stable cell strain capable of expressing the D2 receptor protein stably is finally obtained.(2) Materials for the Receptor Binding Test:Isotopic ligand [3H] Spiperone (113.0 Ci/mmol) purchased from Sigma Corporation; (+) spiperone purchased from RBI Corporation; GF/B glassfiber filter purchased from Whatman Corporation; imported and subpackaged Tris; PPO and POPOP purchased from Shanghai No. 1 Reagent Factory; and fat-soluble scintillation fluid. Beckman LS-6500 multifunction liquid scintillation counter.2. Experimental Methods:    (1) Cells:HEK-293 cells are infected with the recombinant viruses containing various genes. 48-72 h later, receptor proteins are massively expressed on the membrane. The cells are centrifuged at 1000 rpm for 5 min, then the culture supernatant is discarded and the cell pellets are collected, preserved in refrigerator at −20° C., and resuspended with Tris-HCl reaction buffer (pH=7.5) before use.(2) Competitive Receptor Binding Test:The compound to be tested, the radioligand (20 μL for each) and the receptor protein (160 μL) are added to a reaction tube, wherein the compound to be tested and the positive drug both have the final concentration of 10 μmol/L.After being incubated in water bath at 30° C. for 50 min, the tube is immediately transferred to ice bath to stop the reaction. The mixture is subjected to rapid suction filtration through GF/C glassfiber filter on Millipore cell sample collector, washed with eluent (50 mM Tris-HCl, pH 7.5) (3 mL×3) and dried under microwave for 5-6 min. The filter is transferred to a centrifuge tube (0.5 mL), added with 500 μL fat-soluble scintillation fluid, left in the dark for over min, and counted to determine the radioactivity intensity. The percentage inhibition ratio for each compound to inhibit the isotopic ligand binding is calculated with the following equation:inhibition ratio (1%)=(total binding tube CPM−compound CPM)/(total binding tube CPM−non-specific binding tube CPM)×100%Each compound is tested in duplicate, and the tests are carried out independently twice.Compounds with an inhibition ratio of over 95% are subjected to the receptor binding test at a series of concentrations so as to determine half maximal inhibitory concentration (IC50, the concentration of the compound required for inhibiting 50% binding of [3H]-Spiperone to D2 receptor). Each concentration is tested in duplicate, and tests are carried out independently twice for each compound.
Measurement: Ki = 21.3 nM (pChEMBL 7.670000076293945)
Descriptors: MW 532.67, ALogP 5.19, HBA 7, HBD 2, PSA 107.4, QED 0.25
Compound (SMILES): C[C@@H](CO[C@@H]1CCCN(C2CCN(c3ncc(C(F)(F)F)cn3)CC2)C1=O)Nc1cn[nH]c(=O)c1C(F)(F)F
InChIKey: ZOLBTJKZSZKZMS-SUMWQHHRSA-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 μL 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 platereader 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 is delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe Ain each well. Control wells containing a negative control of 0.25% DMSO vehicle or a positive control of 100 μM 5-(5-(piperidin-4-yloxy)isoindolin-2-yl)-4-(trifluoromethyl)pyridazin-3(2H)-one were used to calculate the % inhibition.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 563.5, ALogP 3.07, HBA 8, HBD 2, PSA 116.34, QED 0.49
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(C(C)OC)cc4n3CC3CC3)n(Cc3cnn(C)c3)c12
InChIKey: GSYVVXPNVSDLFQ-JWUZOTJJSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 107 nM (pChEMBL 6.96999979019165)
Descriptors: MW 607.76, ALogP 5.13, HBA 9, HBD 1, PSA 105.36, QED 0.25
Compound (SMILES): COc1cccc(Cn2ncn(-c3ccc(-c4cn[nH]c4)cc3)c2=O)c1
InChIKey: IONAYADDLWQYEJ-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL, assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 347.38, ALogP 2.48, HBA 6, HBD 1, PSA 77.73, QED 0.6
Compound (SMILES): CCCC[S+]([O-])c1sc2nc(-c3cccs3)cc(-c3ccc(Br)cc3)c2c1N
InChIKey: OXAFQVHZGFYWIM-UHFFFAOYSA-N
Target: 15-hydroxyprostaglandin dehydrogenase [NAD(+)] (SINGLE PROTEIN), Homo sapiens
UniProt: P15428
Assay: In Vitro Assay: Each compound for inhibiting enzymatic activity of recombinant 15-PGDH in an in vitro assay.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 491.5, ALogP 6.94, HBA 5, HBD 1, PSA 61.97, QED 0.3
Compound (SMILES): COCCN1C(=O)N(CCC(=O)NCCO)C[C@]12CC[C@](c1ccccc1)(N(C)C)CC2
InChIKey: QMPRWIDOMJJAAF-PSWAGMNNSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Receptor Binding Assay: MOP: The hMOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl (pH 7.4) supplemented with 0.052 mg/ml bovine serum albumin (Sigma-Aldrich Co. St. Louis. Mo.). The final assay volume (250 μl/well) included 1 nM of [N-allyl-2.3-3H]naloxone as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 25 μM unlabelled naloxone 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 90 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]naloxone-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation.
Measurement: Ki = 510 nM (pChEMBL 6.289999961853027)
Descriptors: MW 446.59, ALogP 1.64, HBA 5, HBD 2, PSA 85.35, QED 0.57
Compound (SMILES): COc1cc(OCc2cccc(-c3ccccc3)c2C)cc(OC)c1CNCC(O)c1ccncc1
InChIKey: MMFLNKBJUGWDQC-UHFFFAOYSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Homogenous Time-Resolved Fluorescence (HTRF) Binding Assay: All binding studies were performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (withv) bovine serum albumin and 0.05% (v/v) Tween-20. For the PD-1-Ig/PD-L1-His binding assay, inhibitors were pre-incubated with PD-L1-His (10 nM final) for 15 m in 4 μl of assay buffer, followed by addition of PD-1-Ig (20 nM final) in 1 μl of assay buffer and further incubation for 15 m. PD-L1 from either human, cyno, or mouse were used. HTRF detection was achieved using europium crypate-labeled anti-Ig (1 nM final) and allophycocyanin (APC) labeled anti-His (20 nM final). Antibodies were diluted in HTRF detection buffer and 5 μl was dispensed on top of binding reaction. The reaction mixture was allowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer. Additional binding assays were established between PD-1-Ig/PD-L2-His (20 & 5 nM, respectively), CD80-His/PD-L1-Ig (100 & 10 nM, respectively) and CD80-His/CTLA4-Ig (10 & 5 nM, respectively). Competition studies between biotinylated SEQ ID NO:71 and human PD-L1-His were performed as follows. Inhibitors were pre-incubated with PD-L1-His (10 nM final) for 60 m in 4 μl of assay buffer followed by addition of biotinylated SEQ ID NO:71 (0.5 nM final) in 1 μl of assay buffer. Binding was allowed to equilibrate for 30 m followed by addition of europium crypated labeled Strepatavidin (2.5 pM final) and APC-labeled anti-His (20 nM final) in 5 μl of HTRF buffer. The reaction was allowed to equilibrate for 30 m and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer.
Measurement: IC50 = 53 nM (pChEMBL 7.28000020980835)
Descriptors: MW 484.6, ALogP 5.48, HBA 6, HBD 2, PSA 72.84, QED 0.29
Compound (SMILES): COc1ncc(CN2C3CC2CN(c2ccc(-c4cc(OCC(C)(C)O)cn5ncc(Cl)c45)cn2)C3)cc1F
InChIKey: JWLHRAFETUJCIS-UHFFFAOYSA-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. 62TK0PEC) 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. 62TK0PEC). 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 = 42.5 nM (pChEMBL 7.369999885559082)
Descriptors: MW 553.04, ALogP 4.21, HBA 9, HBD 1, PSA 88.25, QED 0.35
Compound (SMILES): Cc1nc2cc(C(F)(F)F)cc(C#N)c2c(=O)n1CCOc1ccc(Cl)cc1N1CCC(CC(=O)O)CC1
InChIKey: NQJKVVGRTJRDBR-UHFFFAOYSA-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 = 1320 nM (pChEMBL 5.880000114440918)
Descriptors: MW 548.95, ALogP 5.02, HBA 7, HBD 1, PSA 108.45, QED 0.44
Compound (SMILES): CC#CC(=O)N1CC[C@H](c2cn(-c3ccc(Oc4c(F)cccc4F)cc3)c3c(N)n[nH]c(=O)c23)C1
InChIKey: LMSZKRSNARUUMF-HNNXBMFYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Cell Proliferation Assays: MTS testing kit was purchased from Promega. The DMEM, Fetal bovine serum and Penicillin-Streptomycin were purchased from Gibco. Dimethyl sulfoxide (DMSO) was purchased from Sigma.To investigate whether a compound is able to inhibit the activity of BTK in cells, a mechanism-based assay using DOHH2 (DSMZ catalog#: ACC47) and Mino (ATCC® Number CRL-3000) cells was developed. In this assay, inhibition of BTK was detected by the inhibition of DOHH2 and Mino cell proliferations. DOHH2 or Mino cells were cultured in culture flasks to 40-80% confluence in RPMI-1640 plus 10% fetal bovine serum. C ells were collected and plated onto 96-well plates at desired cell density (DOHH2: 5000 cells/well; Mino: 10000 cells/well). Plates were incubated overnight at 37° C., with 5% CO2 to adhere. Compounds were added to the plates, and the final compound concentrations were 10000, 3333, 1111, 270, 124, 41, 14, 4.6 and 1.5 nM. Plates were placed at 37° C., with 5% CO2 for 120 h (DOHH2) or 72 h (Mino). 20 μl MTS/100 μl medium mixture solution were added to each well and the plates were incubated for exactly 2 hours. The reaction was stopped by adding 25 μl 10% SDS per well. Absorbance at 490 nm and 650 nm (reference wavelength) were measured and IC50 was calculated using GraphPad Prism 5.0.
Measurement: IC50 = 369 nM (pChEMBL 6.429999828338623)
Descriptors: MW 489.48, ALogP 3.71, HBA 6, HBD 2, PSA 106.24, QED 0.43
Compound (SMILES): Cc1cc(-n2cccn2)ccc1S(=O)(=O)c1ccc(NC(=O)NCc2cccnc2)cc1
InChIKey: MHPLHELTHRIVCN-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 447.52, ALogP 3.73, HBA 6, HBD 2, PSA 105.98, QED 0.47
Compound (SMILES): Cc1c(C(=O)N(C)c2ccc(O)cc2)cc(-c2cc3c(cc2C(=O)N2Cc4ccccc4C[C@H]2C)CN(C(=O)COc2ccccc2)C3)n1C
InChIKey: DJUGIRCZRVLYJS-AREMUKBSSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, labelled (histag-Bcl-2 such that Bcl-2 corresponds to the UniProtKB® primary accession number: P10415), at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 3.6 nM (pChEMBL 8.4399995803833)
Descriptors: MW 668.79, ALogP 6.49, HBA 6, HBD 1, PSA 95.32, QED 0.21
Compound (SMILES): Fc1ccc(-c2scc(C#CCc3ccccc3)c2-c2ccncc2)cc1
InChIKey: DBZANHAZLXFEKX-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: Fluorescence polarisation (FP) assay for inactive p38alpha MARK: FP signals were measured with a PHERAstar microplate reader (BMG Labtech) using black, low-binding half-area 96 well plates (Corning). Each binding data point was carried out in duplicate or triplicate. Data were recorded in millipolarisation (mP) units and measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Tris(hydroxymethyl)aminomethane (Tris) assay buffer was used in all binding experiments and consisted of 10 mM Tris, 50 nM potassium chloride, and 3.5 mM 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate (Chaps) at pH 8.0. The recombinant inactive His-tag p38α MAPK used in the ligand binding assays were prepared as described by Bukhtiyarova et al.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 369.46, ALogP 6.21, HBA 2, HBD 0, PSA 12.89, QED 0.39
Compound (SMILES): Cc1sc(=O)n(C)c1-c1cnc2c3ccc(C(C)(C)O)cc3n(C(c3ccccc3)C3CCOCC3)c2c1
InChIKey: WXUZUWNNARJAQC-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: All assay components were dissolved in buffer composition 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM DTT, 0.005% Tween 20, and 100 ug/ml BSA for BRD4 (1-477 and 44-460). The final concentrations of the bromodomain proteins are 1.6 nM BRD4(44-168), 1 nM BRD4(333-460), and 1 nM BRD4(1-477 or 44-460), and the fluorescent probe molecule is 100 nM, 50 nM, and 7.5 nM respectively. All proteins were biotinylated. A streptavidin labeled with terbium cryptate (Cisbio SA-Tb) was used as detection, and pre-mixed with the bromodomain protein at a final concentration of 0.2 nM. In some instances for BRD4 (44-460), anti-His terbium cryptate was used as a detection. 7.5 nl of dose-responsed test compound or dmso vehicle (0.0375%) was pre-spotted in a black Corning 384 well plate and 10 ul each of bromodomain/detection reagent and fluorescent small molecule solution were added to the plate, and the reaction incubated for 60 min at room temperature. Plates were then read on EnVision plate reader, (λex=340 nm, acceptor λEm=520 nm, and donor λEm=615 nm, LANCE D400 mirror). Time resolved fluorescence intensity measurements were made at both emissions, and the ratio of acceptor/donor was calculated and used for data analysis.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 527.69, ALogP 6.17, HBA 7, HBD 1, PSA 69.28, QED 0.3
Compound (SMILES): CCc1c(F)cc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)C2CCC(F)(F)CC2)C[C@@H]1F
InChIKey: CRFRNMGXBKYWQL-VQTJNVASSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 13000 nM (pChEMBL 4.889999866485596)
Descriptors: MW 600.54, ALogP 4.8, HBA 6, HBD 2, PSA 105.62, QED 0.41
Compound (SMILES): CC(C)[C@@H](NC(=O)c1cc(=O)[nH]c(-c2ncccn2)n1)c1ccc(OC(F)(F)F)cc1
InChIKey: JUPCHRWQQKBGIK-MRXNPFEDSA-N
Target: GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (SINGLE PROTEIN), Macaca mulatta
UniProt: B3VTJ2
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 433.39, ALogP 3.25, HBA 6, HBD 2, PSA 109.86, QED 0.62
Compound (SMILES): C=CC(=O)N1CC(NC(=O)Cn2c(C)c(C(=O)N3CCc4ccncc4C3)c3cc(Br)ccc32)C1
InChIKey: HTSALKJSTPHCKN-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay (2 hours): Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 2 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 17400 nM (pChEMBL 4.760000228881836)
Descriptors: MW 536.43, ALogP 2.82, HBA 5, HBD 1, PSA 87.54, QED 0.51
Compound (SMILES): NC(=O)c1cnc(N[C@@H]2CCCC[C@@H]2N)nc1Nc1ccc2c(c1)CN(C(=O)OCc1ccccc1)CC2
InChIKey: WJJILRQLHCVIFQ-BJKOFHAPSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: ADP-Glo Kinase Assay: The Promega ADP-Glo Kinase Assay is a luminescent ADP detection assay that provides a universal, homogeneous, high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during a kinase reaction.
Measurement: IC50 = 22.2 nM (pChEMBL 7.650000095367432)
Descriptors: MW 515.62, ALogP 3.7, HBA 8, HBD 4, PSA 148.49, QED 0.37
Compound (SMILES): CCC[C@H](N)c1cccc(-c2cc(N3CC(F)(F)C3)c3cnn(-c4cccc(CO)n4)c3c2)n1
InChIKey: ZELLWHHAZYEEGP-IBGZPJMESA-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, 1 mM 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 < 100 nM (pChEMBL 7)
Descriptors: MW 464.52, ALogP 4.23, HBA 7, HBD 2, PSA 93.09, QED 0.43
Compound (SMILES): Cc1cccc2cc(Cn3cnc4c(N)ncnc43)c(-c3ccccc3CO)nc12
InChIKey: OYMGZDXGGHWOOD-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Luciferase-Coupled Chemiluminescence Assay: Kinase activity is measured as the percent of ATP consumed following the kinase reaction using luciferase-luciferin-coupled chemiluminescence. Reactions were conducted in 384 or 1536-well white medium binding microtiter plates (Greiner). Kinase reactions were initiated by combining test compounds, kinase, and ATP in a 20 μL volume (6 μL volume for 1536-well plate). The reaction mixture was incubated at ambient temperature for 2 h. Following the kinase reaction, a 20 μL (or 3 μL for 1536-well plate) aliquot of KinaseGlo (Promega) was added and the chemiluminescence signal measured using an EnVision plate reader (Perkin Elmer). Total ATP consumption was limited to 25-60% and the IC50 values correlate well with those determined by radiometric assays.
Measurement: IC50 = 375 nM (pChEMBL 6.429999828338623)
Descriptors: MW 396.45, ALogP 3.47, HBA 7, HBD 2, PSA 102.74, QED 0.48
Compound (SMILES): Cc1cc(-c2cc(Cl)ccc2OCCn2c(C)nc3cnc(N4CCc5ccccc54)c(C#N)c3c2=O)c2scc(C(=O)O)c2n1
InChIKey: PUWVJEIRVPZSAW-UHFFFAOYSA-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 = 20.4 nM (pChEMBL 7.690000057220459)
Descriptors: MW 649.13, ALogP 6.68, HBA 10, HBD 1, PSA 134.23, QED 0.2
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCN(C(=O)NCCN)CC4)cc3c2C(C)C)cn2ncnc2c1C
InChIKey: DKRTWQVFUMGFRN-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 473.63, ALogP 4.47, HBA 5, HBD 3, PSA 104.34, QED 0.4
Compound (SMILES): CN1CCOc2c1c(N1CCCC3(CCCN3C)CC1)nn1c(-c3cccc(OC(F)(F)F)c3)nnc21
InChIKey: FAWXEROBGIJQQG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM-1 Biochemical Assay: The biochemical assay to measure PIM-1 activity relies on the ADP Hunter assay kit (DiscoveRx Corp., Cat. #90-0077), that determines the amount of ADP as direct product of the kinase enzyme activity.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 517.56, ALogP 3.97, HBA 9, HBD 0, PSA 71.26, QED 0.52
Compound (SMILES): O[C@@H]1[C@H](O)CS[C@H]1c1nnc2c(NC3CC3)nccn12
InChIKey: VNTWJBPODQBJTA-IWSPIJDZSA-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: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 293.35, ALogP 0.21, HBA 8, HBD 3, PSA 95.57, QED 0.74
Compound (SMILES): Cn1cnc(C[C@H](NC(=O)C2CC2)C(=O)Nc2cccc(-c3ccc(Oc4ccc(F)cc4)cc3)n2)c1
InChIKey: ZECFTVLPBBHBSU-VWLOTQADSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: In vitro assays were performed in a recombinant cell line expressing cDNA encoding the alpha subunit (Nav1.7, SCN9a, PN1, NE) of human Nav1.7 (Accession No. NM_002977). The cell line was provided by investigators at Yale University (Cummins et al, J. Neurosci. 18(23): 9607-9619 (1998)). For dominant selection of the Nav1.7-expressing clones, the expression plasmid co-expressed the neomycin resistance gene. The cell line was constructed in the human embryonic kidney cell line, HEK293, under the influence of the CMV major late promoter, and stable clones were selected using limiting dilution cloning and antibiotic selection using the neomycin analogue, G418. Recombinant beta and gamma subunits were not introduced into this cell line. Additional cell lines expressing recombinant Nav1.7 cloned from other species can also be used, alone or in combination with various beta subunits, gamma subunits or chaperones.
Measurement: IC50 = 111 nM (pChEMBL 6.960000038146973)
Descriptors: MW 499.55, ALogP 4.49, HBA 6, HBD 2, PSA 98.14, QED 0.35
Compound (SMILES): COc1cc(N2CCNCC2)cc(OC)c1-c1cc(Nc2cnc(C#N)cn2)n[nH]1
InChIKey: SNXKDWJPOICZDW-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Kinase Inhibiting Activity Assay: Chk-1 Kinase Inhibiting ActivityThe compounds of the invention were tested for activity against Chk-1 kinase using the materials and protocols set out below.Reaction Buffer:Base Reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO*Required cofactors are added individually to each kinase reactionReaction Procedure:(i) Prepare indicated substrate in freshly prepared Base Reaction Buffer(ii) Deliver any required cofactors to the substrate solution above(iii) Deliver indicated kinase into the substrate solution and gently mix(iv) Deliver compounds in DMSO into the kinase reaction mixture(v) Deliver 33P-ATP (specific activity 0.01 μCi/μl final) into the reaction mixture to initiate the reaction.(vi) Incubate kinase reaction for 120 minutes at room temperature(vii) Reactions are spotted onto P81 ion exchange paper (Whatman #3698-915)(viii) Wash filters extensively in 0.1% phosphoric acid.(ix) Dry filters and measure counts in scintillation counterKinase Information:CHK-1 Genbank Accession # AF016582Recombinant full length construct, N-terminal GST tagged, purified from insect cells.No special measures were taken to activate this kinase.Final concentration in assay=0.5 nMSubstrate: CHKtidePeptide sequence: [KKKVSRSGLYRSPSMPENLNRPR]Final concentration in assay=20 μM
Measurement: IC50 = 0.15 nM (pChEMBL 9.819999694824219)
Descriptors: MW 406.45, ALogP 1.91, HBA 9, HBD 3, PSA 124.01, QED 0.56
Compound (SMILES): N#Cc1cc(S(=O)(=O)N2CC[C@@H]2C(=O)N(Cc2ccc(C3CCCCC3)cc2)c2ccc(C(=O)O)c(O)c2)ccc1F
InChIKey: SWXZOBDDOYMISI-MUUNZHRXSA-N
Target: Signal transducer and activator of transcription 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P40763
Assay: In Vitro Assay: Nuclear extract preparations and DNA-binding activity/electrophoretic mobility shift assay (EMSA) were carried out as previously described (Zhang X, et al., (2010) Biochem Pharmacol 79:1398-409; Zhang X, et al., (2012) Proc Natl Acad Sci USA 109:9623-8). The 32P-labeled oligonucleotide probes used were hSIE (high affinity sis-inducible element from the c-fos gene, m67 variant, 5′-AGCTTCATTTCCCGTAAATCCCTA) (SEQ ID NO: 1) that binds Stat1 and Stat3 and MGFe (mammary gland factor element from the bovine β-casein gene promoter, 5′-AGATTTCTAGGAATTCAA) (SEQ ID NO: 2) for Stat1 and Stat5 binding. Except where indicated, nuclear extracts were pre-incubated with compound for 30 min at room temperature prior to incubation with the radiolabeled probe for 30 min at 30° C. before subjecting to EMSA analysis. Where appropriate, bands corresponding to Stat3:DNA complexes were scanned and quantified for each concentration of compound using ImageJ and plotted as percent of control (DMSO) against concentration of compound, from which the IC50 values were derived.
Measurement: IC50 = 21900 nM (pChEMBL 4.659999847412109)
Descriptors: MW 591.66, ALogP 5.15, HBA 6, HBD 2, PSA 139.01, QED 0.37
Compound (SMILES): O=C(NO)C(Cc1ccccc1)n1nc2c(-c3ccncc3)cccc2c1C#CC1CC1
InChIKey: NDSIQRFNFIYMSR-UHFFFAOYSA-N
Target: Histone deacetylase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BY41
Assay: Enzymatic Assay: HDAC-Glo I/II Assay Kit was purchased from Promega Corporation (Madison, Wis.), including the following components: HDAC-Glo I/II Buffer (25 mM Tris buffer, pH 8.0, supplemented with 137 mM: NaCl, 2.7 mM KCl, 1% v/v Triton X-100 and 1 mM: MgCl2), luminogenic substrates Boc-GAK(Ac)-aminoluciferin (HDAC-Glo I/II substrate), proprietary developer reagent (containing trypsin). Assay:The HDAC8 reaction was performed at room temperature in white 384-well polystyrene, flat-bottom microtiter plate (Greiner Bio-one, Monroe, N.C.) in a final volume of 20 μL. Test compounds were first diluted serially in DMSO and 100 nL were transferred to the plate wells by Echo 550 (Labcyte, Sunnyvale, Calif.) before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The HDAC-Glo I/II assay reagent was prepared by rehydration of lyophilized HDAC-Glo I/II substrate in 10 mL. HDAC-Glo I/II assay buffer followed with the addition of 10 μL of developer reagent. 10 uL of 3 nM HDAC8 in the HDAC-Glo I/II assay buffer were first dispensed into the microliter plate containing the compounds and incubated at room temperature for 30 min. The reactions were initiated with the addition of 10 uL HDAC-Glo I/II assay reagent with Multidrop.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 422.49, ALogP 4.15, HBA 5, HBD 2, PSA 80.04, QED 0.29
Compound (SMILES): CC(C)c1cc(Nc2nc(N3CCCC[C@@H](N)C3)ncc2C(N)=O)cc(C(C)C)n1
InChIKey: CTRCDYXUFDYSHB-OAHLLOKOSA-N
Target: Calcium/calmodulin-dependent protein kinase type 1D (SINGLE PROTEIN), Homo sapiens
UniProt: Q8IU85
Assay: Biochemical CaMK1D Enzymatic Activity Assay: The following describes an ADP-Glo Kinase Assay, which measures the ADP formed from a kinase reaction; the ADP generated is converted into ATP and is used to generate light in a luciferase reaction. The assay is used to assess the effect of compounds on the activity of purified CaMK1 D.Materials and Solutions:All reagents are from Sigma-Aldrich unless otherwise specified. ADP-Glo Kinase Assay (Promega, V9102). His-tagged CaMK1D_1-385 (Fisher Scientific, PR6770A). Autocamtide-2 (SignalChem, A15-58). Calmodulin (Merck, 208694). 1M Tris-HCl pH7.5 (Fisher Scientific, 10123722). 1M DTT (Fisher Scientific, 10674545). Calcium chloride (C1016). Magnesium Chloride (M8266). DMSO (D8418). Autocamtide-2 provided as a lyophilised powder and prepared as a 10 mM stock in MilliQ water. RB: 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 CaCl2), just prior to use 1M DTT was added to a final concentration of 2 mM.Assay Protocol: 7.88 μL reaction mixture (including: calmodulin, Autocamtide-2, CaMK1 D in RB) was incubated with 12 μL test compound in 100% DMSO. To start the reaction 4 μL of ATP mixture were added. Final assay concentrations: 3 nM CaMK1D, 1 μM Calmodulin, 125 μM Autocamtide-2 and 10 μM ATP. Plates were incubated at 25C for 2 hours prior to the 1:1 addition of ADP-Glo reagent. Plates were incubated for a further 1 hour prior to the 1:1 addition of ADP-Glo substrate. After 30 minutes plates read with the EnVision® Multilabel Plate Reader, using Luminescence 700. Compound IC50 was determined using a 4-parameter equation and are reported in nM.
Measurement: IC50 = 147 nM (pChEMBL 6.829999923706055)
Descriptors: MW 411.55, ALogP 3.28, HBA 7, HBD 3, PSA 123.05, QED 0.67