lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCOP(C)(=O)c1ccccc1-c1ccc(N2CC[C@@H](NC(=O)Nc3ccc(C(F)(F)F)cc3F)C2=O)c(F)c1F
InChIKey: WZGCBJZHEPJCEZ-WYTVWHHTSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FPR2 and FPR1 Cyclic Adenosine Monophosphate (cAMP) Assays: A mixture of forskolin (5 μM final for FPR2 or 10 μM final for FPR1) and IBMX (200 μM final) were added to 384-well Proxiplates (Perkin-Elmer) pre-dotted with test compounds in DMSO (1% final) at final concentrations in the range of 0.020 nM to 100 μM. Chinese Hamster Ovary cells (CHO) overexpressing human FPR1 or human FPR2 receptors were cultured in F-12 (Ham's) medium supplemented with 10% qualified FBS, 250 μg/ml zeocin and 300 μg/ml hygromycin (Life Technologies). Reactions were initiated by adding 2,000 human FPR2 cells per well or 4,000 human FPR1 cells per well in Dulbecco's PBS (with calcium and magnesium) (Life Technologies) supplemented with 0.1% BSA (Perkin-Elmer). The reaction mixtures were incubated for 30 min at room temperature. The level of intracellular cAMP was determined using the HTRF HiRange cAMP assay reagent kit (Cisbio) according to manufacturer's instruction. Solutions of cryptate conjugated anti-cAMP and d2 fluorophore-labelled cAMP were made in a supplied lysis buffer separately. Upon completion of the reaction, the cells were lysed with equal volume of the d2-cAMP solution and anti-cAMP solution. After a 1-h room temperature incubation, time-resolved fluorescence intensity was measured using the Envision (Perkin-Elmer) at 400 nm excitation and dual emission at 590 nm and 665 nm. A calibration curve was constructed with an external cAMP standard at concentrations ranging from 1 μM to 0.1 pM by plotting the fluorescent intensity ratio from 665 nm emission to the intensity from the 590 nm emission against cAMP concentrations. The potency and activity of a compound to inhibit cAMP production was then determined by fitting to a 4-parametric logistic equation from a plot of cAMP level versus compound concentrations.
Measurement: EC50 = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 599.47, ALogP 6.29, HBA 4, HBD 2, PSA 87.74, QED 0.25
Compound (SMILES): COc1cc(Nc2nc(=O)c(F)cn2Cc2cc(F)c(F)c(F)c2)c(C)nc1OC
InChIKey: ANUURLHJEDOVHI-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 424.35, ALogP 3.31, HBA 7, HBD 1, PSA 78.27, QED 0.48
Compound (SMILES): N#CN1CCC(NS(=O)(=O)c2ccc(-c3ccccc3)cn2)C1
InChIKey: SIJCHMWGIWVJAH-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase isozyme L1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09936
Assay: UCHL1 Biochemical IC50 Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series to be 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. UCHL1 was diluted in reaction buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.05 μl/well and 10 μl of diluted UCHL1 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2-hour incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 = 55000 nM (pChEMBL 4.260000228881836)
Descriptors: MW 328.4, ALogP 1.58, HBA 5, HBD 1, PSA 86.09, QED 0.86
Compound (SMILES): NCC1CN(c2nc(N)c(-c3cccc(Cl)c3Cl)c(C(N)=O)n2)CC12CCOCC2
InChIKey: QQQNGYGISVPCPI-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Biochemical Assay: The inhibition of SHP2 by compounds of the invention was monitored using the surrogate substrate DiFMUP after protein activation by a peptide bearing two appropriately spaced phosphotyrosine. Full length SHP2 protein (Recombinant HumanSHP-2, E. coli derived Ser2Arg593, N-terminal 6His tag from R&D systems; 0.0.24 nM) was incubated with activating peptide, IRSI_2pY (New England Peptide, 140 nM) and DiFMUP (molecular probes, 80 uM) at RT in buffer (HEPES pH 7.2 60 mM, DDT 5 mM, KCl 75 mM, NaCl 75 mM, EDTA 1 mM, Tween 20 0.05%) in presence of compound (10 concentrations range, top concentration 50 Î¼M) for 60 min. The generation of the DiFMU product by activated SHP2 was monitored through Fluorescence measurement with a PerkinElmer Envision reader.
Measurement: IC50 = 4700 nM (pChEMBL 5.329999923706055)
Descriptors: MW 451.36, ALogP 2.32, HBA 7, HBD 3, PSA 133.38, QED 0.65
Compound (SMILES): Cn1ncc(CC(=O)N[C@H]2CC[C@](F)(CCN3CCc4nc(OCC(F)(F)F)sc4CC3)CC2)n1
InChIKey: JDGWCJQTEUISIR-GGYDESQDSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Binding Inhibition for Dopamine D3 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl.6HO (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl) (067-31, NAKARAI CHEMICALS, LTD.) Radioligand: (final concentration) 2 nM [H]-Methylspiperone ([H���N-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 uM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma] SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well) Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 0.18 nM (pChEMBL 9.739999771118164)
Descriptors: MW 518.58, ALogP 3.01, HBA 8, HBD 1, PSA 85.17, QED 0.54
Compound (SMILES): Cc1nc(N2CCOC(C)C2)c2[nH]c(N)c(S(=O)(=O)c3cc(F)ccc3F)c2n1
InChIKey: DBOFFBLMKYKHOJ-UHFFFAOYSA-N
Target: Free fatty acid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15552
Assay: Ca-Flux Functional Assay: GPR43 agonist/PAM activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by FLIPR (manufactured by Molecular Devices). GPR43 mediated increases in intracellular Ca2+ concentration were readily detected upon activation with sodium acetate. Prior to the assay (24 hours), CHO-K1 Gα16 cells stably expressing human GPR43 were-seeded in cell culture medium in black, clear-bottom 384-well plates (Corning Inc) and grown overnight at 37° C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (Molecular Devices) diluted in HBSS containing 25 mM HEPES, 2.5 mM Probenecid, 0.1% BSA for 1 hour at 37° C., 5% CO2. 10 point half log concentration response curves of sodium acetate from 10 mM were conducted prior to the testing of compounds to calculate the sodium acetate concentration that produces 20% of the maximal response (EC20). Test compounds (at 10 point half log concentration response curves from 10 μM) were added in the presence of sodium acetate to achieve a final concentration that produces approximately 20% maximal response as calculated from the previous experiment. The changes in fluorescent signal were monitored by FLIPR upon addition of the compound/EC20 sodium acetate mix. The EC50 values were determined from ten point concentration response curves. Curves were generated using the average of two wells for each data point.
Measurement: EC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 423.45, ALogP 2.18, HBA 7, HBD 2, PSA 114.2, QED 0.66
Compound (SMILES): O=C(NC1(c2ccc(C(F)(F)F)cc2)CCC1)c1cc(=O)[nH]c(-c2ncccn2)n1
InChIKey: RHBCJIVQCHESTG-UHFFFAOYSA-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 = 83 nM (pChEMBL 7.079999923706055)
Descriptors: MW 415.38, ALogP 3.05, HBA 5, HBD 2, PSA 100.63, QED 0.68
Compound (SMILES): Nc1nc(Nc2cc(Cl)c(-c3ccc(OCC(=O)O)cc3)c(Cl)c2)n[nH]1
InChIKey: PLDAMLMGPXCFRN-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Pseudotyped HCV particle (HCVpp) reporter assay: Plasmids expressing HCV E1 and E2 envelope proteins of GT1a H77 strain (Proc Natl Acad Sci USA 1997 94:8738-43) or GT1b Con1 strain (Science 1999 285:110-3) were constructed by cloning the nucleic acids encoding the last 60 amino acids of HCV core protein and all of the HCV E1 and E2 proteins into pcDNA3.1(+) vector. Plasmid pVSV-G expressing the glycoprotein G of the vesicular stomatitis virus (VSV G) is from Clontech (cat #631530). The HIV packaging construct expressing the firefly luciferase reporter gene was modified based on the envelope defective pNL.4.3.Luc-Râ¿¿.Eâ¿¿ vector (Virology 1995 206:935-44) by further deleting part of the HIV envelope protein.
Measurement: EC50 = 4967 nM (pChEMBL 5.300000190734863)
Descriptors: MW 394.22, ALogP 3.57, HBA 6, HBD 4, PSA 126.15, QED 0.5
Compound (SMILES): COc1ncc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)Nc1ncc(Cc2ccccc2)s1
InChIKey: JIJWWHNUODVFHM-UHFFFAOYSA-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 = 26000 nM (pChEMBL 4.579999923706055)
Descriptors: MW 525.52, ALogP 4.7, HBA 9, HBD 2, PSA 120.32, QED 0.33
Compound (SMILES): C#CCN(C)Cc1nnc(-c2ccc3[nH]c(-c4ccc(OC)c(OC)c4)c(C(C)C)c3c2)o1
InChIKey: LOJZKRLTEPTDOF-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: 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-κc Band 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 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-Bue 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 = 703 nM (pChEMBL 6.150000095367432)
Descriptors: MW 444.54, ALogP 5.09, HBA 6, HBD 1, PSA 76.41, QED 0.38
Compound (SMILES): O=C(NC1CCN(c2cccc(C(F)(F)F)c2)C1)c1ccc(Oc2cc3c(cc2Cl)C(C(=O)O)CCO3)cc1
InChIKey: HMHRQXJIORJXGT-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Binding Inhibition Assay (4% HSA): The coding sequence of human DP2 was introduced into the human Leukemic cell line K562 by electroporation and stable clones expressing DP2 were obtained by limiting dilution followed by cell surface staining with a rat monoclonal antibody specific for human DP2. Membranes were prepared from one of these DP2 expressing clones and used to determine the ability of compounds of the present invention to inhibit binding of prostaglandin D2 (PGD2) to its receptor DP2 in the presence of one or more of the following serum protein concentrations, 4% HSA, by the following procedure. Membranes (1.25 μg/well for 4% HSA) were mixed with 3H-labeled PGD2 and various concentrations of test compounds in 150 μL of binding buffer (50 mM Tris-HCl, pH 7.4, 40 mM MgCl2, 0.1% bovine serum albumin, 0.1% NaN3) in 96-well U-bottom polypropylene plates. After incubation for 60 minutes at room temperature, the assay was transferred to a filtration plate (#MAFB; Millipore Corporation, Bedford, Mass.), and washed three times with binding buffer. Radioactivity was measured by a scintillation counter (TopCount; PerkinElmer Life Sciences, Boston, Mass.). Nonspecific binding was determined by incubations in the presence of 1 μM unlabeled PGD2 or 5 μM of a known DP2 antagonist. EC50 values for inhibition of binding were determined for each compound tested from the inflexion point of a standard 4-parameter logistical curve fitted to the values obtained. Compounds of the invention had EC50 values less than 5 micromolar in one or more of the binding assays.
Measurement: EC50 = 242 nM (pChEMBL 6.619999885559082)
Descriptors: MW 560.96, ALogP 6.11, HBA 5, HBD 2, PSA 88.1, QED 0.38
Compound (SMILES): O=C(c1ccccc1Cl)n1nc(-c2cccn(Cc3ccsc3)c2=O)cc1NCc1ccc(Cl)s1
InChIKey: JFWOZEXCXHNMKU-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: KLKB1 Activity Assay: Human KLKB1 protein was obtained from Enzyme Research Labs. The chromogenic substrate S-2302 was obtained from DiaPharma. KLKB1 was assayed in buffer containing 0.05 M Tris (pH 7.4), 0.01 M NaCl and 0.2% w/v PEG-8000. The final concentration of enzyme used was 3 nM KLKB1. The final concentration of substrate used was 250 μM S-2302 for KLKB1. All assays were performed in 96-well microtiter plates at room temperature (RT). The enzyme and inhibitor were pre-incubated for 10 minutes then substrate was added and read at 405 nm in a SpectraMax Plus Spectrophotometer (Molecular Devices). Inhibitor IC50 values were determined by adding test compound as ten point, three-fold serial dilutions in buffer solution, as known in the art. The plate was read at 10 minutes after substrate addition.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 541.49, ALogP 6.49, HBA 8, HBD 1, PSA 68.92, QED 0.26
Compound (SMILES): CCCSc1cccc(-c2cc(F)c(N3CCC(CC(=O)O)CC3)c(F)c2)n1
InChIKey: UMQLSCJGOWZJSI-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Measurement of Activity of GPR120 Agonist (Cell-Based Assay): CHO-K1 cells expressing Ga16 and hGPR120 were dispensed into each well of a 96-well plate (3×104 cells/100 μl/well) and then incubated in 5% CO2, 37° C. incubator for 18 hours. Each well was treated with 100 μl of Calcium 5 dye (Molecular Devices) solution including 2% DMSO and then incubated in 5% CO2, 37° C. incubator for 1 hour. Serially diluted GPR120 agonists were prepared to a final concentration of 0.5% DMSO in a 96-well plate. Each well was treated with 50 μl of the agonist compounds using Plexstation II, and then fluorescence was measured at Ex 485 nm and Em 525 nm.Fluorescence increased by the serially diluted GPR120 agonists is calculated as a relative percent (%) value based on the fluorescence represented by the treatment of 1% DMSO only. EC50 refers to the concentration of agonist which shows 50% of maximum fluorescence increased by the treatment of agonist. The calculation of measurement was carried out by using statistical software (Prizm).
Measurement: EC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 406.5, ALogP 5.22, HBA 4, HBD 1, PSA 53.43, QED 0.64
Compound (SMILES): N#Cc1cnn2cc(F)c(N3C[C@@H](F)C[C@@H]3c3cc(F)cc(C(N)=O)c3)nc12
InChIKey: ZILGMLTVSJMUDM-DZGCQCFKSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Enzyme Assay: In each well of a 384-well plate, 1 nM-1.5 nM of wild type NTRK1 enzyme (BPS Bioscience; 40280) was incubated in a total of 12.5 μL of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1-2 μM CSKtide (Tuft's University or Anaspec; FITC-AHA-KKKKD DIYFFFG-NH2) and 1 mM ATP at 25° C. for 60 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 μL of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.7 psi, upstream voltage −500, downstream voltage −3000, post sample sip 35 s). Data was normalized to 0% and 100% inhibition controls and the IC50 calculated using a 4-parameter fit in the CORE LIMS.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 386.34, ALogP 2.27, HBA 6, HBD 1, PSA 100.31, QED 0.74
Compound (SMILES): O=C1[C@@H](F)CCN1C[C@H](O)Cn1c2ccccc2c2ccccc21
InChIKey: URLWXAVERPIENG-BBRMVZONSA-N
Target: Period circadian protein homolog 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15055
Assay: Per2 Assay: Compounds were screened by using a high-throughput circadian assay system as previously described in Zhang, E. E. et al. Cell, 2009, 139, 199-210. In brief, stable U20S reporter cells harboring Per2-dLuc were plated at a density of 30,000 cells/well in Corning 96-well, solid white, flat bottom, TC-treated microplates (Corning), and incubated for 48 hours at 37° C. in the presence of 5% CO2 in a medium of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 units/mL)-streptomycin (100 μg/mL). Compounds of formula I are solubilized in dimethylsulfoxide (DMSO), typically at a concentration of 2 mg/mL. DMSO stocks are then serially diluted in DMSO, typically diluting 3-fold for each dilution step. Following the 48 h period, cell culture medium is removed from plated cells and cells are synchronized with 200 μL/well of complete cell culture medium (described above), supplemented with 5 μM forskolin (Tocris) and 1 mM beetle luciferin (Promega). Immediately following synchronization, 1 μL of compound dilution is added to each well. Plates are sealed, shaken briefly, and gene expression was monitored by measuring luminescence (Tecan Infinite M200 or Tecan Infinite M200 Pro) continuously for a minimum of 3 days at 35° C. Raw luminescence data (counts) are first analyzed using Multicycle software (Actimetrics, Inc.) to determine the amplitude (amp), period, and phase (phz) for each compound concentration. The period length for control wells (i.e. no compound, DMSO only) should be 26-30 h. Amp data are then plotted against the logarithm compound concentration (M) and analyzed by nonlinear regression analysis to determine the EC50.
Measurement: EC50 = 263 nM (pChEMBL 6.579999923706055)
Descriptors: MW 326.37, ALogP 2.73, HBA 3, HBD 1, PSA 45.47, QED 0.8
Compound (SMILES): O=c1nc(NCC2CC2)c2ccc(C3CC3)cc2n1-c1cccc2nccn12
InChIKey: GLHQHGSFOMHRJE-UHFFFAOYSA-N
Target: S-adenosylmethionine synthase isoform type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31153
Assay: Phosphate Sensor Fluorescence Assay : MAT2A enzyme is incubated with a test compound in DMSO or DMSO and its substrates (L-methionine and ATP) in a microtiter plate. The enzymatic reaction is stopped by the addition of Working Phosphate Sensor Mixture. The plate is analyzed for fluorescence at 450 nm. The high control (DMSO with enzyme and its substrates) gives high fluorescence which represents no inhibition of enzymatic activity while the low control (DMSO with MAT2A substrates and no enzyme) gives low fluorescence which represents full inhibition of enzymatic activity.Materials:Human MAT2A: Cepter, amino acids 1-395Tris, pH 7.5: Invitrogen cat #15567-027KCl: Ambion cat #AM9640GMgCl2: Ambion cat #AM9530GBrij-35: Sigma cat B4184-10MLDTT: Goldbio cat #DTT100BGG: Sigma cat #G5009-25GPNP: Novus Biologicals cat #NBP1-508727-MEG: Cayman Chemical cat #15988L-Methionine: Alfa Aesar cat #J61904ATP: Alfa Aesar cat #J60336Phosphate Sensor: Thermo Fisher cat #PV4407EDTA: Life Tech cat #15575-038Assay plate: 384-well black polypropylene plate: Thomas Scientific cat #1149Q35Final Assay Conditions:Assay Buffer: 50 mM Tris, pH 7.5/50 mM KCl/10 mM MgCl2/0.01% Brij-35/1 mM DTT/0.1% BGG/40 nM PNP/6 uM 7-MEGMAT2A: 10 nM for Cepter clone ID 329, lot 00023-123 before the addition of Working Phosphate Sensor Mixture    5 nM for Cepter clone ID 334, lot 00023-148 before the addition of Working Phosphate Sensor MixtureL-methionine: 500 uM before the addition of Working Phosphate Sensor MixtureATP: 500 uM before the addition of Working Phosphate Sensor MixtureProcedure:For the assay, a mixture of 1 mM L-methionine/1 mM ATP (2× final pre-stopped concentration) in assay buffer; MAT2A (2× final pre-stopped concentration) in Assay Buffer and Working Phosphate Sensor Mixture (1.5 uM Phosphate Sensor/30 mM EDTA in Assay Buffer, which is 3× final concentrations) were prepared. Test compounds or DMSO were added to the appropriate well suing D300e digital dispenser. 5 μl/well of Assay Buffer was added to the wells corresponding to the negative control and 5 μl/well of MAT2A was added to all the wells except for those corresponding to the negative control. After incubating the plate at room temperature for 15 minutes, 5 μl/well of the 1 mM L-methionine/1 mM ATP mixture was added to all wells. The plate was centrifuged at 1000 rpm for 1 minute and then incubated at room temperature for 1 hour. 5 μl of the Working Phosphate Sensor Mixture was added to all wells and the plate was centrifuged at 1000 rpm for 1 minute. The plate was read for fluorescence at 450 nm after exciting at 430 nm.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 371.44, ALogP 3.73, HBA 6, HBD 1, PSA 64.22, QED 0.58
Compound (SMILES): CCCOc1ccc(C)cc1C(=O)N1CC2CN(c3cc(N(C)C)nc(C)n3)CC2C1
InChIKey: DGRKWZZERBPHFU-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Receptor Radioligand Binding Assay: Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 10 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and vortexed for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-SB674042 (Moravek Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM (1-(6,8-difluoro-2-methylquinolin-4-yl)-3-[4-(dimethylamino)phenyl]urea, CAS Registry #288150-92-5). The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-5B674042 diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 423.56, ALogP 3.16, HBA 6, HBD 0, PSA 61.8, QED 0.71
Compound (SMILES): O=C1NCc2nc(-c3c(F)cccc3F)nc(Nc3ccc(CC(=O)N4CC5(COC5)C4)cc3)c21
InChIKey: HNWGWZAGRUXXDW-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Tyk2 & JAK2 Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 477.47, ALogP 2.81, HBA 6, HBD 2, PSA 96.45, QED 0.59
Compound (SMILES): Cc1ncc(NC(=O)c2ccnc(C(F)(F)F)c2)cc1-c1cnc(Cl)c(N2CCOCC2)c1
InChIKey: LMDLKOPJSLEMAM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x 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 40x 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 2xATP 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 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 477.87, ALogP 4.61, HBA 6, HBD 1, PSA 80.24, QED 0.55
Compound (SMILES): O=C(NC(CO)(CO)CO)c1ccc2c(-c3ccccc3Cl)nn(-c3ccc(F)cc3)c2c1
InChIKey: HGFZAVZHQAIJQK-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: Enzymatic Activity Assay: DGAT2 activity was determined by measuring the amount of enzymatic product triolein (1,2,3-Tri(cis-9-octadecenoyl)glycerol) using the membrane prep mentioned above. The assay was carried out in deep well 384 plates in a final volume of 40 μL at room temperature. The assay mixture contained the following: assay buffer (100 mM Tris.Cl, pH 7.0, 20 mM MgCl2, 5% ethanol), 25 μM of diolein, 10 μM of oleoyl-CoA and 10 ng/μL of DGAT2 membrane.
Measurement: IC50 = 432 nM (pChEMBL 6.369999885559082)
Descriptors: MW 469.9, ALogP 2.93, HBA 6, HBD 4, PSA 107.61, QED 0.33
Compound (SMILES): CC(C)Nc1cc(-c2cnc3cc(C#N)cnn23)ncc1C(=O)NC[C@H](F)C(C)(C)O
InChIKey: IEBJIQFRGCQIQM-SFHVURJKSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 425.47, ALogP 2.32, HBA 8, HBD 3, PSA 128.23, QED 0.53
Compound (SMILES): C=CC(=O)N1C[C@@H](C)N(C(=O)N(C)[C@H](C(=O)N[C@H]2Cc3nc(cs3)-c3ccc4c(c3)c(c(-c3cccnc3[C@H](C)OC)n4CC)CC(C)(C)COC(=O)[C@@H]3CCCN(N3)C2=O)C(C)C)C[C@@H]1C
InChIKey: HQUHXXXTDVJWQB-YLAIFOTQSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 938.21, ALogP 6.39, HBA 12, HBD 2, PSA 171.54, QED 0.14
Compound (SMILES): COc1c(N=S(C)(C)=O)cc2ncnc(Nc3ccc(F)cc3O[C@H]3COC[C@@H]3O)c2c1C
InChIKey: RCEKLIVCBVRHEU-HKUYNNGSSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: MNK2a In Vitro Kinase Assay (Assay 1): The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled elF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.In the 384-well plates 3 μl of test compound from the intermediate dilution is mixed with 4 μl of the activated MNK2 enzyme (final concentration of 10 nM) and 4 μl of the peptide (final concentration of 25 μM)/ultra pure ATP (final concentration of 20 μM), all dissolved in assay buffer. This step is followed by an incubation time of 90 min, then 10 μl of ADP Glo reagent are added, followed by 40 min of incubation. Then 20 μl of kinase detection reagent are admixed. The plates are sealed and after an incubation period of 30 min, the luminescence signal is measured in an Envision reader to determine the amount of produced ADP. All incubation steps are performed at room temperature.The assay buffer consists of 20 mM HEPES, 2 mM DTT, 0.01% BSA, 20 mM MgCl2 and 0.1% Pluronic F-127.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 476.53, ALogP 3.33, HBA 9, HBD 2, PSA 115.16, QED 0.56
Compound (SMILES): Cc1cc(-c2n[nH]c3cc4c(cc23)CN([C@@H]2CCCN(Cc3cc(F)ccc3F)C2)C(=O)N4)ccn1
InChIKey: XPMNLWOHLPNLPX-OAQYLSRUSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Z′-LYTE Kinase Assay: The inhibition of ERK activity by the compounds disclosed herein was determined using the Z′-LYTE kinase assay kit (Life Technologies) with a Ser/Thr 3 peptide substrate (Life Technologies) according to manufacturers' instructions. The assay was run with an ERK2 enzyme (Life Technologies) concentration of 0.47 ng/μL at 100 μM ATP (approximately the ATP Km for ERK2). The IC50 values for the compounds were determined with 3-fold serial dilutions in duplicate. The compounds were first diluted in 1:3 dilutions in 100% DMSO at 100× the desired concentration, and then further diluted (1:25) in 20 mM HEPES buffer (Invitrogen) to make 4× solutions prior to adding to the enzyme solution. The final DMSO concentration in the assay was 1%. Final reaction volume was 20 μL/well in 384-well plates. Kinase reactions were conducted or 1 hour followed by the assay development reaction (1 hour) in a 20 ul/well in a 384 well plate format.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 488.54, ALogP 5.22, HBA 4, HBD 2, PSA 77.15, QED 0.41
Compound (SMILES): Cn1cc(-n2ccc3c(Oc4cc(CN)ccn4)cccc32)cn1
InChIKey: XNRPIWBMJFMHEK-UHFFFAOYSA-N
Target: Lysyl oxidase homolog 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y4K0
Assay: Human LOXL2 Amine Oxidase Activity Assay: LOXL2 amine oxidase activity is evaluated by measuring Amplex Red fluorescence using 10-20× concentrated conditioned media from CHO cells stably expressing human LOXL2. To assay for amine oxidase activity, 10 μL of the concentrated conditioned media is incubated with 2 μL of test compound in DMSO and 73 μL Assay Buffer (50 mM Borate Buffer, pH8) for 2 h at 37° C. After the 2 h incubation, 5 μl of 10 mM 1,5-Diaminopentane (DAP) diluted in Assay Buffer and 10 μl of Amplex Red Mix (8.5 μl Assay Buffer+0.5 μl of 10 mM Amplex Red+1 μl of 500 U/ml Horseradish Peroxidase) are added and the plate mixed and immediately placed on the FlexStaion for fluorescence measurements. Fluorescence is read in kinetic mode every 2 min for 1 hour at excitation=544 and emission=590. The amine oxidase activity is calculated from the slope of the linear portion of the curve
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 319.37, ALogP 3.01, HBA 6, HBD 1, PSA 70.89, QED 0.63
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)CCc2ncc(Cl)cn2)nnc1[C@@H]1COCCO1
InChIKey: QSDKCYUMVHXPRU-INIZCTEOSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.).
Measurement: EC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 510.96, ALogP 1.8, HBA 11, HBD 1, PSA 139.58, QED 0.45
Compound (SMILES): COc1ncc(-c2cc(CNCCO)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(CC(O)C(F)(F)F)C[C@@H]1F
InChIKey: VOISIWQGCYXPGE-SWQDIRLTSA-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 = 60000 nM (pChEMBL 4.21999979019165)
Descriptors: MW 556.52, ALogP 0.14, HBA 11, HBD 5, PSA 163.16, QED 0.17
Compound (SMILES): C=CC(=O)N1CC(CCNc2ncnc(N)c2-c2ccc(Oc3ccccc3)cc2)C1
InChIKey: YULIYRJBXMSHOD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzyme Assay: The following describes a microfluidic, off-chip mobility shift kinase assay used to measure inherent potency of compounds against BTK enzyme. Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent BTK Enzyme Assay IC50. These IC50 values are reported in ranges wherein: A<100 nM, B<1 uM, and C>1 uM.2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM Hepes, pH 7.5, and 10 mM DTT.5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27 C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM Hepes, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorylation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel, Switzerland). Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent PBMC BTK Enzyme Assay IC50.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 415.5, ALogP 3.96, HBA 6, HBD 2, PSA 93.37, QED 0.54
Compound (SMILES): COc1cnc(-n2cnc(CCS(C)(=O)=O)n2)nc1[C@@]12CC[C@@H](c3cc(-c4c(F)cccc4F)nnc31)C2(C)C
InChIKey: JWQPTRMODDXXFM-RKOGDMNLSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Ligand Binding Assay: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present. Assay plates were 96-well polypropylene V-bottom plates. 10 uL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 uL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 uL of 5 uM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 uL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 uL of 1.5 ug/mL RORc receptor was added to wells (or 40 uL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25° C. Filtration
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 567.62, ALogP 3.59, HBA 10, HBD 0, PSA 125.64, QED 0.33
Compound (SMILES): Cn1cc(-c2nc3c(-c4ccc(CNC(=O)c5nc(C(C)(C)C)no5)cc4CO)ccnc3[nH]2)cn1
InChIKey: VBANFKKWZYDAEQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Microfluidic Off-Chip Mobility Shift Assay Protocol for Potency Assessment Against BTK Enzyme: Briefly, 2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2; developed in-house) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM HEPES, pH 7.5, and 10 mM DTT. 5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27° C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM HEPES, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorilation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel. Switzerland).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 486.54, ALogP 3.13, HBA 9, HBD 3, PSA 147.64, QED 0.33