lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(Nc2ncnc(-c3ccc(OC4CCOCC4)c(C#N)c3)n2)ccc1N1CCN(CC(F)F)CC1
InChIKey: ISBIOLPSKNYCJM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 and IKKE Biological Assays: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phosphorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2×Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 551.6, ALogP 4.11, HBA 10, HBD 1, PSA 108.66, QED 0.42
Compound (SMILES): C[C@@H]1C[C@H](N)CN(c2ccncc2NCc2ccc(F)c(-c3ccc4sccc4c3)n2)C1
InChIKey: QZOISWBCYKUCLX-APWZRJJASA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05M Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA,) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light. Pim-2 kinase assay20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05M Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 447.58, ALogP 5.28, HBA 6, HBD 2, PSA 67.07, QED 0.44
Compound (SMILES): CCCN(C)C(=O)c1ccc2c(C)cn([C@H](C)C(=O)Nc3cc(Cn4cc(C)cn4)ccc3CCCC(=O)O)c2c1
InChIKey: SDGGVGVPIFRAKD-HSZRJFAPSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: EP4 Antagonistic Activity Measurement: CHO cells expressing human EP4 receptor subtypes were prepared according to the methods of Nishigaki et al. (Non-Patent Literature 4), and used for experiment. Cells cultured to subconfluent were detached, and suspended in an assay medium (MEM containing 1 mmol/L IBMX, 1% HSA) such that a concentration became 1×106 cells/mL. For reaction, PGE2 was added to the cell suspension (25 μL) in a final concentration of 10 nmol/L, either alone or as a 25-μL PGE2 solution containing the test compound. After 30 minutes of reaction at room temperature, the amount of cAMP in the cells was quantified according to the method in the descriptions of the cAMP assay kit (CISBIO).Note here that the antagonistic effect (IC50 value) of the test compound was calculated as a value that represents an inhibition rate against a reaction with PGE2 alone at 10 nM, a concentration that produces a submaximal cAMP producing effect.
Measurement: IC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 557.7, ALogP 5.59, HBA 6, HBD 2, PSA 109.46, QED 0.24
Compound (SMILES): CC(C)(C)OC(=O)N1CC(S(=O)(=O)c2cc(-c3cnc(N)cn3)cc(N3CCOCC3)c2)C1
InChIKey: VVNBBGRJUHXRIN-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition of PI3K alpha: Quantification of ATP to ADP conversion as a measure of PI3Kα activity. Active PI3Kα (Life Technologies), in the presence or absence of PI3Kα inhibitor, was reacted with PIP2:PS (Life Technologies), a substrate specifically optimized for use with Class I PI3 kinases, and ultrapure ATP (Promega). The conversion of ATP to ADP by PI3Kα was measured as luminescence signal via Promega ADP-Glo kinase activity assay. Assay was validated using published PI3Kα inhibitors LY294002, PI-103, BYL719, GDC0198 and also DMSO vehicle control.Compounds were prepared at 100× final concentration as a 12-point, 1:3 serial-dilution in DMSO series, with DMSO control as 12th point. Compound was then diluted in (25 mM HEPES pH 7.5, 1 mM EGTA, 0.3% CHAPS) prior to addition to PI3Kα. Active PI3Kα diluted to 0.24 ng/μL (1.1 nM) in (50 mM HEPES pH 7.5, 6 mM MgCl2, 1 mM EGTA, 200 mM NaCl, 0.03% CHAPS, 8 mM DTT) was incubated with compound for 0 hr and 3 hr prior to the start of the reaction. 25 μM PIP2:PS and 60 μM ATP were diluted from stock in (25 mM HEPES pH 7.5, 1 mM EGTA, 0.3% CHAPS) and added to initiate the PI3Kα reaction. Reaction time was 30 minutes. ATP to ADP conversion was measured in Luminescence Counts on DTX880 Plate Reader (Beckman Coulter). Compound IC50s were reported using GraphPad Prism software. Analytical method was non-linear regression, 4-parameter curve fit with bottom fit to validated PI3Kα inhibitor reference controls and no top fit (floating top).
Measurement: IC50 = 540 nM (pChEMBL 6.269999980926514)
Descriptors: MW 475.57, ALogP 1.96, HBA 9, HBD 1, PSA 127.95, QED 0.71
Compound (SMILES): Nc1cc(I)c2cnc(-n3ccc4ccncc43)cc2c1
InChIKey: XUCVQYARAFUTCJ-UHFFFAOYSA-N
Target: Microtubule-associated protein tau (SINGLE PROTEIN), Homo sapiens
UniProt: P10636
Assay: Tau binding assay: For displacement tau binding assay, unlabeled test compounds were dissolved in DMSO at 1 mM. Dilutions of tests compounds to various concentrations were made in 100% DMSO at 1000× final assay concentration and 0.225 μl aliquots were dispensed into assay plates. Brain homogenates were diluted to 0.5 mg/mL from original 30 mg/mL volume in Assay Buffer, and 200 μl were added to the assay plate for a final concentration of 100 μg wet weight/assay tube. [3H]-6 was prepared at 100× final concentration in Assay Buffer plus 20% DMSO and 25 μl was added to the assay plate for final assay concentration of 0.25 nM. The assay plate was incubated at room temperature (25° C.) for 90 minutes. Unbound and bound ligand were separated by filtration of bound onto GF/C filter plates (pre-treated for 30 min with 0.2% polyethyleninamine) using a Packard UniFilter Harvester and washing away unbound with 2.5 ml per well ice cold 5 mM Tris at pH 7.4. Filter plates were dried for 1 hour in a vacuum oven and 50 μl/well MicroScint-20 were added. Plates were counted 1 min per well by Packard Topcount. Total amount of radioligand used in the assay was determined by counting 25 μl of 10× ligand stock. Data was analyzed using Activity Base software to generate 4 p fit of dose responses; and, Ki values were calculated.
Measurement: Ki = 18.6 nM (pChEMBL 7.730000019073486)
Descriptors: MW 386.2, ALogP 3.76, HBA 4, HBD 1, PSA 56.73, QED 0.4
Compound (SMILES): Cc1c(C(=O)CN2CCC(CC(=O)O)CC2)c2ncc(Br)cc2n1-c1ccc(Cl)cc1
InChIKey: NUWZXVYNEGGQTR-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: P54578
Assay: USP14 Inhibition Assay: Using previously described methodology [B. H. Lee et al. Nature 2010, 467 (9), 179, the contents of which are expressly incorporated by reference herein], select compounds described herein were found to inhibit USP14.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 504.81, ALogP 5.12, HBA 5, HBD 1, PSA 75.43, QED 0.47
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)NC1CCN(C(=O)C2(C(F)(F)F)CC2)CC1
InChIKey: MVSICWOXCOWMCB-UHFFFAOYSA-N
Target: Bifunctional epoxide hydrolase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34913
Assay: Assays for Epoxide Hydrolase Activity: Any of a number of standard assays for determining epoxide hydrolase activity can be used to determine inhibition of sEH. For example, suitable assays are described in Gill, et al., Anal Biochem 131:273-282 (1983); and Borhan, et al., Analytical Biochemistry 231:188-200 (1995)). Suitable in vitro assays are described in Zeldin et al., J Biol. Chem. 268:6402-6407 (1993). Suitable in vivo assays are described in Zeldin et al., Arch Biochem Biophys 330:87-96 (1996). Assays for epoxide hydrolase using both putative natural substrates and surrogate substrates have been reviewed (see, Hammock, et al. In: Methods in Enzymology, Volume III, Steroids and Isoprenoids, Part B, (Law, J. H. and H. C. Rilling, eds. 1985), Academic Press, Orlando, Fla., pp. 303-311 and Wixtrom et al., In: Biochemical Pharmacology and Toxicology, Vol. 1: Methodological Aspects of Drug Metabolizing Enzymes, (Zakim, D. and D. A. Vessey, eds. 1985), John Wiley & Sons, Inc., New York, pp. 1-93. Several spectral based assays exist based on the reactivity or tendency of the resulting diol product to hydrogen bond (see, e.g., Wixtrom, supra, and Hammock. Anal. Biochem. 174:291-299 (1985) and Dietze, et al. Anal. Biochem. 216:176-187 (1994)).The enzyme also can be detected based on the binding of specific ligands to the catalytic site which either immobilize the enzyme or label it with a probe such as dansyl, fluoracein, luciferase, green fluorescent protein or other reagent. The enzyme can be assayed by its hydration of EETs, its hydrolysis of an epoxide to give a colored product as described by Dietze et al., 1994, supra, or its hydrolysis of a radioactive surrogate substrate (Borhan et al., 1995, supra). The enzyme also can be detected based on the generation of fluorescent products following the hydrolysis of the epoxide. Numerous methods of epoxide hydrolase detection have been described (see, e.g., Wixtrom, supra).The assays are normally carried out with a recombinant enzyme following affinity purification. They can be carried out in crude tissue homogenates, cell culture or even in vivo, as known in the art and described in the references cited above.
Measurement: Ki = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 439.36, ALogP 4.04, HBA 3, HBD 2, PSA 70.67, QED 0.7
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3cccc(C)c3)nn2CCCO)C1
InChIKey: OZTOVYXNMHQXIF-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (6×His disclosed as SEO ID NO: 3) (Perkin Elmer AD0110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 328.42, ALogP 2.22, HBA 5, HBD 2, PSA 70.39, QED 0.88
Compound (SMILES): Cn1c(=O)c(C(=O)NCC(=O)O)c(O)c2ccc(Oc3ccccc3F)cc21
InChIKey: JRJDZFSTCZSDLQ-UHFFFAOYSA-N
Target: Hypoxia-inducible factor 1-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q16665
Assay: Erythropoietin (EPO) Biological Assay: The activities of compounds of the invention in inducing erythropoietin (EPO) production were assessed by using human live cancer cell strain Hep3B (ATCC: American type culture collection, Manassas, Va.). Hep3B cells seeded in a 96-well plate with a density of 2.5×104 cells/well were cultivated overnight in DMEM culture medium (Dulbecco's Modified Eagle's Medium) at 37° C. in the presence of 10% fetal bovine serum (FBS). The next day, the supernatant liquid of culture medium was abandoned by suction, and to the residue was added fresh DMEM (containing 0.5% DMSO, 0.5% FBS) containing compound of the invention with a series of concentrations (0.31160.00 μM) or containing solvent used for comparison. The cells were cultivated at 37° C. for 24 h. The supernatant liquid was collected and the EPO concentration of the supernatant was quantified by using human EPO ELISA kit (Abcam). The activity of each compound in inducing erythropoietin (EPO) production was represented by half maximal effective concentration (EC50).
Measurement: EC50 = 6020 nM (pChEMBL 5.21999979019165)
Descriptors: MW 386.34, ALogP 1.99, HBA 6, HBD 3, PSA 117.86, QED 0.62
Compound (SMILES): C=CC(=O)N1CC(Oc2cc3c(Nc4ccc(Cl)c(-c5cnco5)c4)ncnc3cc2OC)C1
InChIKey: ACQGRWSJSVUXTG-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Cell Viability Assay and IC50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described. Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone at a final volume of 40 μL per well. After 3 days hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes, and bioluminescence was determined using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO treated cells, and normalized values were plotted in GraphPad Prism using non-linear regression fit to normalized data with a variable slope. IC50 values were calculated by GraphPad Prism at 50% inhibition.
Measurement: IC50 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 477.91, ALogP 4.47, HBA 8, HBD 1, PSA 102.61, QED 0.39
Compound (SMILES): CC(C)NCc1cc(F)c(-c2cc3c(-c4cnn(C)c4)n[nH]c3cn2)c(F)c1
InChIKey: NVITVKIPYWGYDR-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μl of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 382.42, ALogP 3.8, HBA 5, HBD 2, PSA 71.42, QED 0.55
Compound (SMILES): Cc1cc(OCCCc2c3n(c4c(-c5c(C)nn(C)c5C)c(Cl)ccc24)[C@H](C)CN(c2cc(N(C)C)cc4cc(C(=O)O)n(C)c24)C3=O)cc(C)c1Cl
InChIKey: YSITXFTUUTVLLN-HSZRJFAPSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: TR-FRET Binding Assay: Measurements were carried out in OptiPlate-384 White Opaque 384-well plates (Perkin Elmer. Shelton, Conn.). The assay was run using a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) that was purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification, 300 nM FITC-Bak peptide, 1 nM recombinant Maltose Binding Protein (MBP) tagged Mcl-1 (residues 172-327) and 1 nM terbium conjugated-MBP antibody were added to assay buffer (4.5 mM Monobasic potassium phosphate, 15.5 mM Dibasic potassium phosphate, 1 mM Sodium EDTA, 50 mM NaCl, 1 mM DTT, 0.05% Pluronic F-68, pH 7.5). Compounds are diluted in DMSO in a 16-point, semilog serial dilution scheme. For the BAK FITC TR-FRET assay 50 nL compound is added to 20 μL of assay buffer containing Bak-FITC. MBP tagged Mcl-1 protein and antibody, for a final DMSO concentration of 0.25% and a top concentration of 10 μM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 3 hours at room temperature. TR-FRET signal (Delta F) is measured on a Biotek Cytation 3 multimode plate reader equipped with a filter cube containing an Ex 340/30 Em 620/10 nm filter and an Ex 340/30 Em 520 filter.
Measurement: Ki = 0.21 nM (pChEMBL 9.680000305175781)
Descriptors: MW 755.75, ALogP 9.07, HBA 8, HBD 1, PSA 97.76, QED 0.15
Compound (SMILES): CCCC[C@@H](CC(=O)O)Nc1nc(N)nc(C)c1Cc1ccccc1OC
InChIKey: OYDQVZSFWZPAFP-HNNXBMFYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: HEK TLR7 Assay: HEK-Blue TLR7 cells were purchased from Invivogen (San Diego, Calif.). The following description was taken from the product information sheet."HEK-Blue hTLR7 cells are designed for studying the stimulation of human TLR7 (hTLR7) by monitoring the activation of NF-kB. HEK-Blue hTLR7 cells were obtained by co-transfection of the hTLR7 gene and an optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene into HEK293 cells. The SEAP reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-kB and AP-1-binding sites. Stimulation with a TLR7 ligand activates NF-kB and AP-1 which induce the production of SEAP, which is detected by the HEK-Blue Detection cell culture medium."A typical assay protocol involved the following steps:1. Cells were cultured according to the product information sheet.2. 10 mM compound stock in DMSO were first diluted to 3 mM and then 3-fold serially diluted using DMSO to afford a 10-pt dilution.3. 3 μl of the diluted DMSO were added to 57 μl HEK-Blue Detection media for a further 20-fold dilution.4. 10 μl of the diluted compound in assay media were added into 40 μl cell culture (in HEK-Blue Detection media) in 384-well plate. Final cell concentration=8,000 cells per well.5. The plates were incubated at 37° C. in 5% CO2 for 16 h. SEAP was determined using a spectrophotometer at 620-655 nm.
Measurement: EC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 372.47, ALogP 3.41, HBA 6, HBD 3, PSA 110.36, QED 0.59
Compound (SMILES): CCC1(c2ccccc2)NC(=O)N(NC(=O)Nc2ccc(Br)cc2)C1=O
InChIKey: LLOSQGDYZOFGRP-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FLIPR: HEK-Gα16 cells stably expressing the human FPR2 receptor was utilized. Cells were plated into 384-well poly-D-lysine coated plates at a density of 18,000 cells per well one day prior to use. The growth media was DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic, 50 μg/ml hygromycin, and 400 μg/ml geneticin. On the day of the experiment, the cells were washed twice with Hank's Balanced Salt Solution supplemented with 20 mM HEPES (HBSS/hepes buffer). The cells were then dye loaded with 2 μM Fluo-4 diluted in the HBSS/Hepes buffer and incubated at 37° C. for 40 minutes. Extracellular dye was removed by washing the cell plates four times prior to placing the plates in the FLIPR (Fluorometric Imaging Plate Reader, Molecular Devices). Ligands were diluted in HBSS/Hepes buffer and prepared in 384-well microplates. Data for Ca+2 responses were obtained in relative fluorescence units.
Measurement: EC50 = 523 nM (pChEMBL 6.28000020980835)
Descriptors: MW 417.26, ALogP 3.34, HBA 3, HBD 3, PSA 90.54, QED 0.67
Compound (SMILES): COC(C)(C)[C@H]1CN(C2CCN(c3nnc(N)[nH]3)CC2)[C@@H](Cc2ccc(Cl)cc2)CO1
InChIKey: ITISUZUNOJIUPF-RBUKOAKNSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: ERG Channel Binding Assay: Fluorescence polarization assay based on the principle of fluorescence polarization where a red-shifted fluorescent tracer is displaced from the hERG channel by compounds that bind to the channel was used to establish the binding of the compounds to hERG channel. Briefly, the compounds in serial dilutions were incubated with a membrane fraction containing hERG channel protein and a high-affinity red fluorescent hERG channel ligand in black 384-well plate. The reaction was incubated for 4 h at room temperature followed by the measurement of the fluorescence polarization using Tecan Spark multimode plate reader at excitation wavelength of 535 nm and emission wavelength 590 nm. The IC50 values were calculated using GraphPad Prism.
Measurement: IC50 = 17500 nM (pChEMBL 4.760000228881836)
Descriptors: MW 449, ALogP 2.75, HBA 7, HBD 2, PSA 92.53, QED 0.7
Compound (SMILES): C=CC(=O)N(C)c1ccccc1Nc1nc(Nc2cc(C(N)=O)ccc2C#N)ncc1Cl
InChIKey: XHUGUWSOZKNKKA-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P27361
Assay: Omnia Assay: The protocol below describes continuous-read kinase assays to measure potency of compounds against activated ERK1 enzyme. The mechanics of the assay platform are best described by the vendor (Invitrogen, Carlsbad, Calif.) on their website at the following URL: invitrogen.com/site/us/en/home.html.Briefly, a 1.25× stock of ERK1 enzyme (14-439-K) from Millipore (Billerica, Mass.), 5×ATP (AS001A) and ST17-Sox conjugated peptide substrate (KNZ1171C) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT. 10 μL of ATP/ST17-sox peptide substrate mix was combined with 0.5 μL volume of 100% DMSO and serially diluted compounds were prepared in 100% DMSO in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.). Kinase reactions were started with the addition of 40 μL of ERK1 solution and monitored every 71 seconds for 30-240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30+ minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes or seconds) and then plotted against inhibitor concentration to estimate AppIC50 from log [Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 447.89, ALogP 3.74, HBA 7, HBD 3, PSA 137.03, QED 0.47
Compound (SMILES): Cc1cs/c(=N\C2CCCc3ccccc32)n1/N=C/c1ccc(O)c(O)c1O
InChIKey: OTAZSMORWYVMPJ-VPEKAMMISA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 homolog (SINGLE PROTEIN), Mus musculus
UniProt: P97287
Assay: High-Throughput Competitive Fluorescence Polarization (FP) Screening Assay: A high-throughput competitive fluorescence polarization (FP) screening assay (Z-factor, 0.62) was developed based on the direct binding interaction between FITC-MCL-1 SAHBA and MCL-1ΔNΔC (EC50, 14 nM) (FIGS. 1A, 2A, and 3A). A compilation of 71,296 small molecules was screened for the capacity to displace FITC-MCL-1 SAHBA from recombinant MCL-1ΔNΔC (aa 172-327) (FIG. 4). To enrich for MCL-1-selective molecules by detecting binding activity for the BCL-XL subclass of anti-apoptotic proteins, the library was also counterscreened using a competitive FP assay (Z-factor, 0.71) developed based on the direct and selective interaction between FITC-BAD BH3 and BCL-XLΔC (EC50, 26 nM) (FIGS. 1B, 2B, and 3B). Small molecules with an apparent preference for MCL-1ΔNΔC (208 compounds, 0.3% hit rate), as defined both by >50% displacement of the FITC-MCL-1 SAHBA/MCL-1ΔNΔC interaction and a >45% difference in peptide displacement from MCL-1ΔNΔC vs. BCL-XLΔC, were advanced to increasingly stringent confirmatory in vitro binding assays including: (1) repeat single-dose testing of 208 molecules in the differential competitive FP screen; (2) alternative single-dose selectivity screen for 130 confirmed MCL-1-directed antagonists comparing relative displacement of FITC-BID BH3, a dual binder,35 from MCL-1 ΔNΔC vs. BCL-XLΔC; and then (3) dose-responsive competitive binding of the 64 most selective molecules against the FITC-MCL-1 SAHBA/MCL-1 ΔNΔC complex (FIG. 4).
Measurement: IC50 = 4.36 nM (pChEMBL 8.359999656677246)
Descriptors: MW 395.48, ALogP 3.84, HBA 7, HBD 3, PSA 90.34, QED 0.46
Compound (SMILES): Cc1cccc(Cc2ccc3ncc(-c4ccncc4)n3n2)c1
InChIKey: HWYUVMUWVBZZDM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Fyn (SINGLE PROTEIN), Homo sapiens
UniProt: P06241
Assay: ADP-Glo Kinase Assay: The assay procedure determines the IC50 of each potential FYN kinase inhibitor by measuring the enzyme catalyzed ATP-dependent phosphorylation of the FYN substrate peptide. The ADP-Glo Kinase Assay is specifically designed to quantify kinase activity by measuring the ADP produced in the reaction The reaction buffer comprises 40 mM Tris.HCl (pH 7.5), 20 mM MgCl2, 0.1 mg/ml BSA and 5 microM DTT. Individual compounds within the chemical library were dissolved in DMSO at known concentrations. Briefly, 2.5 microliters of reaction buffer containing 3 picograms of FYN kinase and serial dilutions of each potential FYN kinase inhibitor (9 3-fold dilutions of a 10 micromolar top sample) are placed into each well of a 384 well microtiter plate and incubated at room temperature for 30 minutes. An additional 2.5 microliters of reaction buffer containing 100 picograms of the FYN substrate and 12.5 picomoles of ATP are then added to each well and the reaction carried out for 90 minutes at room temperature.
Measurement: IC50 = 977 nM (pChEMBL 6.010000228881836)
Descriptors: MW 300.37, ALogP 3.69, HBA 4, HBD 0, PSA 43.08, QED 0.58
Compound (SMILES): COc1cc(F)c(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)CC(F)(F)F)C[C@@H]1F
InChIKey: LNDBBXBJXKQGCW-UONOGXRCSA-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 = 12000 nM (pChEMBL 4.920000076293945)
Descriptors: MW 566.41, ALogP 3.38, HBA 7, HBD 2, PSA 114.85, QED 0.46
Compound (SMILES): COC(=O)c1c(O)cc(O)c(Cl)c1CCc1ccc(OC)cc1Cc1ccccc1
InChIKey: MGNDUXSLXVZBIM-UHFFFAOYSA-N
Target: Endoplasmin (SINGLE PROTEIN), Canis lupus familiaris
UniProt: P41148
Assay: Fluorescence Polarization Assay: The assay was performed in 96-well black, flat-bottom plates with a final volume of 100 μL. 25 μL of assay buffer (20 mM HEPES, pH 7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, 2 mM DTT, 0.1 mg/mL BGG, and 0.01% NP-40) were added, followed by 25 μL of assay buffer containing 6 nM FITC-GDA (fluorescent tracer, stock in DMSO, diluted in assay buffer) and 50 μL of assay buffer containing 10 nM of either Grp94 or Hsp90α were added to each well. For each plate, wells containing buffer only (background), tracer in buffer only (low polarization control) and protein, tracer, and 1% DMSO (final concentration, high polarization control) were included. Compounds were then added with a final concentration of DMSO=1%. Plates were incubated at 4° C. with rocking for 24 h. Polarization values (in mP units) were measured at 37° C. with an excitation filter at 485 nm and an emission filter at 528 nm. Polarization values were correlated to % tracer bound and compound concentrations. The concentration at which the tracer was 50% displaced by compound of interested were calculated and reported as apparent Kd's.
Measurement: Kd = 680 nM (pChEMBL 6.170000076293945)
Descriptors: MW 426.9, ALogP 4.92, HBA 5, HBD 2, PSA 75.99, QED 0.52
Compound (SMILES): CS(=O)(=O)c1ccc(CNC(=O)c2ccc3c(c2)nc(C2CC2)n3Cc2cccc3c2OC(F)(F)O3)cc1
InChIKey: GJKQADRYWICFSS-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : Compounds described herein were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 ag of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. #T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 539.56, ALogP 4.62, HBA 7, HBD 1, PSA 99.52, QED 0.37
Compound (SMILES): Cc1ncc(NC(=O)c2cccc(C(F)F)c2)cc1-c1cc(N2CCOCC2)c2nccn2n1
InChIKey: TUIBXKTXNDTECM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Raf Activity: 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, then 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 = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 464.48, ALogP 4.13, HBA 7, HBD 1, PSA 84.65, QED 0.48
Compound (SMILES): COc1ccc(-c2cc(NCCc3ccccn3)c3ccccc3n2)cc1
InChIKey: ZYZBNFTUBXNIOG-UHFFFAOYSA-N
Target: Low molecular weight phosphotyrosine protein phosphatase (SINGLE PROTEIN), Homo sapiens
UniProt: P24666
Assay: LMPTP Primary Screening Protocol: A listing of materials is provided:    Item, source, catalog no.    LMPTP-A Enzyme Stock Solution (4.22 mg/ml or 206.8 μM), SBMRI Protein Facility,    N/A    OMFP, Sigma, M2629-100 MG    Bis-Tris pH 6.0, Fisher Sci, BP301-100    Triton-X 100, Sigma, T9284    DTT, Sigma, D9779    Mol. Grade Water, Mediatech, Inc., 46-000-CM    1536-well black High base opaque bottom plate, Nexus Biosystems, 00019120Final assay conditions are:    0.625 nM LMPTP-A Enzyme    40 μM OMFP    50 mM Bis-Tris pH 6.0    1 mM DTT    0.01% Triton-X 100    20 μM test compound    3% DMSO (2% from substrate and 1% from compounds)    6 μL reaction volume    50 minutes incubation at room tempAssay Procedure    1. Prepare Reagents as described in section F. Recipe.    2. Using LabCyte Echo, transfer 60 nL from 2 mM test compound source plate into assay plate Col. 5-48 (final concentration of test compounds is 20 μM). 60 nL of DMSO should be transferred to col. 1-4 for positive and negative control wells.    3. Spin plates at 1000 rpm for 1 minute in centrifuge.    4. Using the Beckman Coulter Bioraptr, add 3 μL/well of control buffer to columns 1 and 2.    5. Using the Bioraptr, add 3 μL/well of enzyme solution to col. 3-48.    6. Using the Bioraptr, add 3 μL/well of substrate solution to col. 1-48.    7. Spin plates at 1000 rpm for 1 minute in centrifuge.    8. Incubate plates in the dark at room temperature for 50 minutes.    9. Read plates on PerkinElmer Viewlux using a FI protocol.
Measurement: IC50 = 2900 nM (pChEMBL 5.539999961853027)
Descriptors: MW 355.44, ALogP 4.96, HBA 4, HBD 1, PSA 47.04, QED 0.53
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1Cn1c(=O)[nH]c2cc(CN3CCN(C4CCOCC4)CC3)c(Br)cc2c1=O
InChIKey: IQDPWZJEXQVCAL-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: Binding Activity: DDR1 binding activity was measured using the LanthaScreen(Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen(Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 640, ALogP 3.24, HBA 8, HBD 1, PSA 104.71, QED 0.42
Compound (SMILES): COc1c(F)cccc1-c1ccc2nc(NC(=O)[C@@H]3C[C@@H]3F)cn2c1
InChIKey: CFNYLETUNRYRAW-OCCSQVGLSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Kinase Assay: c-abl: ADP-Glo assay kit was purchased from Promega. Magnesium chloride (MgCl2), bovine serum albumin (BSA), ethylene glycol-bis(p-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), triton X-100, 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% Triton X-100; 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 and Sigma Plot 13.0.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 343.33, ALogP 3.45, HBA 4, HBD 1, PSA 55.63, QED 0.79
Compound (SMILES): FC(F)c1cccc(-c2nc(-n3ccc4cnccc43)c3cccn3n2)n1
InChIKey: SFOSATDEFMLXAI-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 362.34, ALogP 4.07, HBA 6, HBD 0, PSA 60.9, QED 0.49
Compound (SMILES): Cc1nc(N)ccc1CNC(=O)[C@H](C)NC(=O)[C@H]1C[C@H](Cc2ccc(Cl)c(Br)c2)CN1
InChIKey: WTOJJMPCDMBSPF-ZUEVXXBESA-N
Target: Mannan-binding lectin serine protease 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00187
Assay: Enzymatic Assay for MASP-2: The assay was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50 values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed., John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 508.85, ALogP 2.73, HBA 5, HBD 4, PSA 109.14, QED 0.46
Compound (SMILES): Cc1nc2nonc2c(N)c1Cc1nccc(C#N)n1
InChIKey: RHULLGSACCVQQN-UHFFFAOYSA-N
Target: Membrane-bound ghrelin O-acyltransferase MBOAT4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96T53
Assay: hGOAT Activity in HEK293 Cells after Incubation: Cells are plated with a density of 5000 cells/well in 384-well poly-D-lysin plates and incubated for 1 day at 37° C., 5% CO2 in DMEM medium, 10% FCS, 1×NEAA, Puromycin (0.5 μg/ml) and G418 (1 mg/ml). Then the medium is changed to a identical medium without FCS and containing Octanoate-BSA (final concentration 100 μM each) and compound in DMSO (final DMSO concentration 0.3%). After incubation for 5 hours acylghrelin in the medium is measured by ELISAThe medium sample is diluted 1:25 in Elisa buffer, A 25 ul aliquot is transferred to a 384-well ELISA plate previously washed 4 times with 100 μL wash buffer, and 25 μl tracer-solution is added. After incubation overnight (20 h) at 4° C. temperature the plate is washed 4 times with 100 μl wash-buffer per well. Finally 50 μl Ellman's reagent is added to each well and the plate is incubated in the dark for 20 minutes. The absorbance is measured at 405 nm in an Envision multilabel reader and the amount of acylated ghrelin is calculated according to a acylated ghrelin standard curve provided in the same plate.Each assay plate contains wells with vehicle controls (1% DMSO) for the measurement of non-inhibited transfer reaction (=100% Ctl) and wells with 10 μM ([Dap3]-Ghrelin) as controls for fully inhibited GOAT enzymeThe analysis of the data is performed by calculation of the percentage of acyl-ghrelin produced in the presence of test compound compared to the amount of acyl-ghrelin produced in the vehicle control samples. An inhibitor of the GOAT enzyme will give values between 100% CTL (no inhibition) and 0% CTL (complete inhibition). IC50 values are calculated with Assay Explorer or other suited software based on curve fitting of results of 8 different compound concentrations.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 267.25, ALogP 0.76, HBA 8, HBD 1, PSA 127.4, QED 0.72
Compound (SMILES): Cc1ncc(-c2ccc3cnc(NC(=O)c4ccnc(N5CCN(C)CC5)c4)cc3c2)cn1
InChIKey: KBWLQLRFZYIZSN-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Kinase Assay: The DYRK1A kinase assay was run using the Ser/Thr 18 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as a ratio of coumarin emission/fluorescein emission.Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated ("0% Control") and phosphorylated ("100% control") forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (luM top) was run to serve as a positive compound control.
Measurement: EC50 = 158 nM (pChEMBL 6.800000190734863)
Descriptors: MW 439.52, ALogP 3.4, HBA 7, HBD 1, PSA 87.14, QED 0.52
Compound (SMILES): CN(C)CCOc1ccc2[nH]ncc2c1-c1ccc2nc(NC(=O)C3C[C@@H]3F)sc2c1
InChIKey: MDFYPUUSMTWHEH-WUJWULDRSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: ADP-Glo kinase assay: HPK1 kinase activity was measured by Promega's ADP-Glo kinase assay. In this assay, 5 ng of recombinant human HPK1 (signalchem) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of MBP (0.5 μg/μl) and 5 μL of ATP (25 μM) in buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 5 μM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30′ C for 40 minutes. After the incubation, 25 uL 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 uL 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 and SigmaPlot13.0.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 439.52, ALogP 4.08, HBA 6, HBD 2, PSA 83.14, QED 0.45
Compound (SMILES): COc1cc2nccc(Cc3ccc(NC(=O)c4cn(C)c(=O)n(-c5ccc(F)cc5)c4=O)cc3F)c2cc1OC
InChIKey: JQDRLXRKQDUMIA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Kinase Assay: The ability of compounds to inhibit the kinase activity of recombinant human baculovirus-expressed AXL was measured by homogeneous TRF (HTRF) using Cisbio's KinEASE assay system in white 384-well Optiplates. Assay buffer contained 1 mM DTT, 2 mM MnCl2, 2% DMSO, 50 nM supplement enzymatic buffer, and 1× enzymatic buffer. A 2× concentration of tyrosine kinase (TK) substrate-biotin/ATP mixture made in assay buffer was added to plates at 10 μL/well using the Multidrop Combi (Thermo Fisher Scientific, Waltham, Mass.). The final concentrations were 0.3 μM TK substrate-biotin, and 1.3 μM ATP. Compounds (100 nL), diluted in 100% DMSO on the Biomek FX, (Beckman Coulter, Inc., Brea, Calif.), were transferred to the assay plates using the Biomek FX pintool (2.5% final DMSO in assay). A 2× concentration (final=12 ng/mL) of GST-AXL (diluted in assay buffer) was added to plates at 10 uL/well using the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 30 minutes. A 4× stock of Streptavidin-XL665 (final=18.8 nM) and a 1:100 diluted stock of TK antibody-cryptate were made in HTRF detection buffer and mixed together just prior to adding 20 μL/well on the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 1 hour. The fluorescence of the resulting solution was measured using the PerkinElmer EnVision 2102 multi-label plate reader (PerkinElmer, Waltham, Mass.) with an excitation wavelength of 337 nm (laser) and emission wavelengths of 590 and 665 nm. Raw data was expressed as the ratio of 665/590×10,000.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 558.54, ALogP 4.22, HBA 8, HBD 1, PSA 104.45, QED 0.32