lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#Cc1cc(-c2ccnc3[nH]c(-c4ccc(N5CCOCC5)cc4)cc23)ccc1O[C@@H]1CCN(C(=O)CO)C1
InChIKey: ZHQUWWWIBAHOPH-XMMPIXPASA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Biological Assay for TBK1 and IKKs: 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 phopshorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKs 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 = 1.34 nM (pChEMBL 8.869999885559082)
Descriptors: MW 523.59, ALogP 3.58, HBA 7, HBD 2, PSA 114.71, QED 0.4
Compound (SMILES): Cc1nc2ccc(S(=O)(=O)N3CCCC3)c(C#N)c2c(=O)n1CCOc1ccc(Cl)cc1-c1ccnc2c(C(=O)O)csc12
InChIKey: JZGDKNVLGDTVTK-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 19.8 nM (pChEMBL 7.699999809265137)
Descriptors: MW 650.14, ALogP 5.07, HBA 10, HBD 1, PSA 155.48, QED 0.24
Compound (SMILES): CCc1ccc(N(CC(C)C)S(=O)(=O)c2ccc(NCC3(N4CCCC4)COC3)c(S(C)(=N)=O)c2)cc1
InChIKey: GZOMFFUVFNZJSM-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: GAL4-RORgamma Transactivation: The RORγ transactivation model was developed from the line HG5LN, which is a HeLa line that stably expresses a luciferase reporter gene controlled by a pentamer of the GAL4 recognition domain of yeast and of a β-globin promoter. The HG5LN line was stably transfected by the DNA-binding domain (DBD) of GAL4 fused to the ROR gamma ligand-binding domain (LBD). Molecules that inhibit the ROR gamma constitutive activity reduce the luciferase expression, thus leading to a reduction in the emitted luminescence.The cells are seeded in 384-well plates (5000 cells in 45 μL/well of culture medium containing 10% fetal calf serum) and incubated for 4 hours at 37° C., 5% CO2. 5 μL of the test molecules (compounds described in the tables described above) are then added to each well and the plates are incubated for 18 hours at a temperature of 37° C. under 5% of CO2. 20 μL of luciferase substrate (Promega) are added to each well and the luminescence emitted is read by a microplate reader.The luminescence units (RLU) are normalized by positive controls (POS containing a saturated concentration of benzenesulfonamide, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]) and negative controls (NEG containing DMSO): % inhibition=((RLU−NEG)*100)/(POS−NEG). The IC50 values are calculated from a 4-parameter logistic model using the XLFit software (IDBS).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 548.78, ALogP 4.41, HBA 7, HBD 2, PSA 102.8, QED 0.43
Compound (SMILES): CN(C1CCOC1)S(=O)(=O)C[C@H]1C[C@@H](N(C)c2ncnc3[nH]ccc23)C1
InChIKey: OEJSZIRZZNLLAY-XMGJGUJMSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 379.49, ALogP 1.22, HBA 6, HBD 1, PSA 91.42, QED 0.81
Compound (SMILES): CC(C)(N)C=C(C#N)C(=O)NCCn1c(NC(=O)c2ccc(-c3cn[nH]c3)s2)nc2ccccc21
InChIKey: KITXKLPNBSNKMV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Enzymatic Activity Assay: The ability of the compounds of the present disclosure to inhibit ITK was measured using the Caliper assay format, which is an electrophoretic separation of a phosphorylated peptide substrate from unphosphorylated peptide. The enzymatic reaction occurred in a buffer of 100 mM HEPES pH 7.5, 5 mM MgCl2, 0.01% Triton-X 100, 0.1% Bovine Serum Albumin, and 1% DMSO. Three-fold dilutions of compounds were prepared in DMSO. Compounds were added to enzyme and pre-incubated for 15 minutes prior to reaction. The enzymatic reaction was initiated by addition of phospho-acceptor peptide FAM-GEEPLYWSFPAKKK-NH2 (also known as SRCtide) to 1 uM and ATP to its Km value (ITK: 10 uM). The reaction proceeded for 3 hours and was terminated by addition of EDTA. The assay employed an enzyme concentration of 1 nM. The top compound concentration was 5 uM. The amount of phosphorylated substrate was determined by the Caliper instrumentation, and dose-response curves were fit using standard methods to determine the IC50 values.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 488.58, ALogP 3.04, HBA 8, HBD 4, PSA 154.51, QED 0.22
Compound (SMILES): C[C@@H](CNc1ncc(SC(F)F)cn1)CNc1nc2ccc(C(=O)N3CCC4C(=O)OCC4C3)cc2s1
InChIKey: VVJXAORXAZMDAQ-KTAKPFMOSA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: Surface Plasmon Resonance (SPR) Assay: Surface plasmon resonance data was collected on a Biacore T200 or 3000 system (GE Healthcare) at 25° C. Streptavidin was immobilized on a CMS (GE Healthcare) or CMD500d sensor chip (XanTec Bioanalytics) using standard amine-coupling chemistry at 25° C. with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated with a 12 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 μL/min. For capture of streptavidin, protein was diluted to 0.2 mg/mL in 10 mM sodium acetate (pH 4.5) and captured by injecting 100 μL onto the activated chip surface. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5). Avi-tagged PCSK9 protein was captured on the streptavidin surface by injection of 150 μL of protein diluted to 16 μg/mL in HBS-N, 0.05% tween-20, 0.1 mM CaCl2. Typical surface densities obtained were 8000-10000 RU. SPR binding data were obtained using an appropriate dilution series of each compound at a flow rate of 30 μL/ min, with a capture time of 100 s and dissociation times of 300 s. Running buffer for compound binding studies was HBS-N, 0.05% tween-20, 0.1 mM CaCl2, 4% DMSO. Data were corrected for DMSO excluded volume effects. All data were double-referenced for blank injections and reference surface using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software).
Measurement: Kd < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 548.64, ALogP 4.2, HBA 10, HBD 2, PSA 109.34, QED 0.3
Compound (SMILES): CC1Oc2cnccc2-c2ccc(C(=O)NCc3cccc(C(=O)O)c3)cc21
InChIKey: JDFVHGBBVAYOEB-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 0.85 nM (pChEMBL 9.069999694824219)
Descriptors: MW 374.4, ALogP 3.83, HBA 4, HBD 2, PSA 88.52, QED 0.72
Compound (SMILES): NC(=O)c1nn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cccc(-c3ccccc3Cl)c2F)c2ccc(Oc3cncnc3)cc12
InChIKey: UPKARXJXTLSKQF-QUGAMOGWSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Enzyme Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 M each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 632.03, ALogP 4.75, HBA 8, HBD 2, PSA 145.33, QED 0.25
Compound (SMILES): B[P@]1(=O)OC[C@H]2O[C@@H](n3cnc4c(N)ncnc43)C(F)[C@H]2O[P@](B)(=O)OC[C@H]2O[C@@H](n3cnc4c(N)ncnc43)[C@@H](F)C2O1
InChIKey: PYYRTETWQZAPJI-FJWQIJGCSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: In Vitro Binding Assay: Fluorescence polarization assay (FP assay) was used to detect the affinity of compounds for human STING proteins. There were a certain amount of fluorescein-labeled c-di-GMP and different concentrations of test compounds in the reaction system. When the C-terminal protein of recombinant human STING was added, two small molecules competitively combined with the protein. The rotation of the fluorescein-labeled c-di-GMP in the bound phase is relatively slow in the liquid phase, and the degree of fluorescence polarization detected is relatively high. The degree of fluorescence polarization is inversely proportional to the concentration and affinity of the compound to be tested. By detecting the size of the polarized light in the reaction system, we can accurately know the affinity of the compound to be tested for human STING.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 654.04
Compound (SMILES): O=C(N[C@H]1CCCC[C@@H]1O)C1=CN(Cc2ccccc2C(F)(F)F)c2ccccc2O1
InChIKey: XHRJIUFZGURRAY-HKUYNNGSSA-N
Target: Muscarinic acetylcholine receptor M1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11229
Assay: Determination of Allosteric Potency EC50 Values for Muscarinic M1 Receptor: A stable CHO cell line expressing recombinant human Muscarinic M1 receptor and pCRE-Luc reporter system was used for cell-based assay. The assay offers a non-radioactive based approach to determine binding of a compound to GPCRs. In this specific assay, the level of intracellular cyclic AMP which is modulated by activation or inhibition of the receptor is measured. The recombinant cells harbor luciferase reporter gene under the control of cAMP response element.The above cells were grown in 96 well clear bottom white plates in Hams F12 medium containing 10% fetal bovine serum (FBS). Prior to the addition of compounds or standard agonist, cells were serum starved overnight. Increasing concentrations of test compounds were added along with EC20 of acetylcholine in OptiMEM medium to the cells. The incubation was continued at 37° C. in CO2 incubator for 4 hours. Medium was removed and cells were washed with phosphate buffered saline. The cells were lysed and luciferase activity was measured in a Luminometer. Luminescence units were plotted against the compound concentrations using Graphpad software. EC50 values of the compounds were defined as the concentration required in stimulating the luciferase activity by 50% in presence of EC20 of acetylcholine.
Measurement: EC50 = 534 nM (pChEMBL 6.269999980926514)
Descriptors: MW 432.44, ALogP 4.37, HBA 4, HBD 2, PSA 61.8, QED 0.75
Compound (SMILES): CCOC(=O)COc1ccc(/N=c2\[nH]c(=O)n(CC)c(=O)n2Cc2ccc(Cl)cc2)cc1
InChIKey: QYNVCOLNGPPXTQ-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 619 nM (pChEMBL 6.210000038146973)
Descriptors: MW 458.9, ALogP 2.23, HBA 8, HBD 1, PSA 107.68, QED 0.52
Compound (SMILES): O=C(Nc1ccc(N2CCOCC2=O)cc1O)OCc1ccccc1
InChIKey: FFBHFFJDDLITSX-UHFFFAOYSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
Assay: Human Coagulation Factor Xa Assay: The inhibitory activity on coagulation factor Xa activity in human was measured by using Tris-HCl buffer (50 mM, pH 8.3, 150 mM NaCl). A buffer of 50 mL human coagulation factor Xa (Enzyme Research Laboratories, Inc; final concentration 8.36 nM) or a buffer of 50 μL rat coagulation factor Xa (Enzyme Research Laboratories, Inc; final concentration 57.5 nM) was added dropwise to the appropriate wells of the Greiner 384 microtiter plate to determine IC50. The buffer containing 2 μL 2% (V/V) DMSO (control group which was free of inhibition) or various concentrations of the compounds to be tested were diluted in the buffer containing 2% (V/V) DMSO, and 48 μL buffer of the supporting base S-2222 (Chromogenix; chemical formula: Bz-IIe-Glu(γ-OR)-Gly-Arg-pNA.HCl R═H (50%), wherein R═CH3 (50%)) was added, the final concentration was 0.172 mM. In this experiment, the compounds to be tested and the enzyme were incubated for 10 minutes, and then the substrate S-2222 was added to give a final volume of 100 μL to start the assay.The compounds to be tested were regarded as being active when Ki<10 μM. The compounds whose Ki<1 μM were preferred in the present invention, more preferably Ki<0.1 μM, further more preferably Ki<0.01 μM, and further preferably Ki<0.001 μM. Determined by the above method, some compounds of the present invention were of K1<0.1 μM, thus, the compounds of the present invention can be used as effective factor Xa inhibitors.
Measurement: Ki = 1.01 nM (pChEMBL 9)
Descriptors: MW 342.35, ALogP 2.5, HBA 5, HBD 2, PSA 88.1, QED 0.83
Compound (SMILES): CCn1cc(Nc2ncc(C(N)=O)c(NCc3cccc(F)c3Cl)n2)cn1
InChIKey: TXDKYSNRVAVETF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Kinase Activity Assay: JAK3 (the final concentration was 0.36 nM); ATP (the final concentration was 10 μM); ULight-labeled JAK-1 (Tyr1023) Peptide (the final concentration was 50 nM); the enzymatic reaction time was 1 hour. The maximum final concentration of compounds was 2.5 μM. After a 3-fold gradient dilution, 11 concentrations were obtained, with the minimum final concentration of 0.042 nM. The final DMSO concentration was 1%.
Measurement: IC50 = 3.14 nM (pChEMBL 8.5)
Descriptors: MW 389.82, ALogP 2.94, HBA 7, HBD 3, PSA 110.75, QED 0.57
Compound (SMILES): CC(C)(C)OC(=O)N1CCC[C@H]1C(=O)N1CCc2c(cccc2-c2ccccc2)C1
InChIKey: GFWQTQCAFKEBJY-QFIPXVFZSA-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 hr 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 406.53, ALogP 4.64, HBA 3, HBD 0, PSA 49.85, QED 0.73
Compound (SMILES): Cc1cccc(C#N)c1Oc1nc(NS(=O)(=O)CCC(F)(F)F)cc(-c2ccc(=O)n(C)c2)n1
InChIKey: BELJQQRHBGKATE-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro Enzyme Inhibition Assay-CREBBP Inhibition: Determination of the IC50 for the CREBBP inhibitors disclosed herein was performed as follows: CREBBP was cloned and expressed in E. Coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CREBBP binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CREBBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20-minute incubation at room temperature, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After two hours of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.The ability of the compounds disclosed herein to inhibit CBP activity was quantified and the respective IC50 value was determined.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 493.47, ALogP 3.51, HBA 8, HBD 1, PSA 126.97, QED 0.53
Compound (SMILES): Fc1ccccc1CN1CCN(c2ccnc(-c3cnc4ccc(C(F)(F)F)cn34)n2)CC1c1cn[nH]c1
InChIKey: HMCGYWNCAMBVHY-UHFFFAOYSA-N
Target: eIF-2-alpha kinase GCN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P2K8
Assay: Enzyme Inhibition Assay: An assay buffer containing GCN2 kinase and (RS)7 was prepared. 4.7 μL of this stock solution was placed per well of a black, low volume, 384-well microtitre plate (e.g. catalogue number 3676, Corning Inc., NY). To this was added 0.65 μM of DMSO containing serial dilutions of the test compound (typical final concentrations of test compound were 0 to 8 μM). The plate was incubated for 10 minutes at 25° C. prior to the addition of 4.7 μL of ATP stock buffer to initiate the enzyme reaction. The reaction was allowed to proceed for 1 hour at 25° C., prior to the addition of 10 μL detection buffer (consisting of appropriate concentrations of ADP2 antibody and ADP Alexa633 tracer in 1× stop and detect buffer as supplied by BellBrook Labs). The reaction was left to incubate for 1 hour at 25° C., prior to measuring the fluorescence polarisation signal (mP) in each well using a PHERAstar FS reader (BMG Labtech, Germany).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 522.51, ALogP 4.74, HBA 7, HBD 1, PSA 78.24, QED 0.34
Compound (SMILES): CNCCC(Oc1cccc(N2CCCCCC2=O)c1)c1ccccc1
InChIKey: RZFDJGLCCAEWMU-UHFFFAOYSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay : Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. The binding of the test compound was measured in five different concentrations. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 352.48, ALogP 4.32, HBA 3, HBD 1, PSA 41.57, QED 0.81
Compound (SMILES): COCCn1cccc(-c2cc(NCc3ccc(Cl)s3)n(C(=O)C(C)(C)CO)n2)c1=O
InChIKey: GNYAUENMEQGNTM-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 = 55000 nM (pChEMBL 4.260000228881836)
Descriptors: MW 464.98, ALogP 3.34, HBA 9, HBD 2, PSA 98.38, QED 0.5
Compound (SMILES): COCCc1nc(-c2ccc(NS(=O)(=O)c3ccccc3F)cc2)nc2[nH]nc(N)c12
InChIKey: YXWZSUIQESWPIU-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Sgk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00141
Assay: Enzymatic Activity Assay: The compounds were tested for serum and glucocorticoid-regulated kinase 1 (SGK-1) inhibitory activity in a substrate phosphorylation assay designed to measure the ability of the isolated enzyme to catalyze the transfer of phosphate from ATP to serine/threonine residues in a fluorescein-labeled substrate peptide, using recombinant human SGK-1 enzyme produced in a baculovirus system (Biomol, Hamburg, Germany, Cat. No. 4-331). The synthesized fluorescent labeled peptide substrate contained (5(6)-Carboxyfluorescein)-RPRAATF-NH2. The phosphorylated substrate peptide and non-phosphorylated substrate peptide were separated with caliper life science's lab-chip technology based on a micro fluidics method. All fluid flow was established on the chip by applying a vacuum of a few psi to the waste well transporting fluid from various sources through interconnecting channels. Because the phosphoryl group is doubly negatively charged, under the pressure-driven hydrodynamic flow and the voltage-driven flow within the electric field, the fluorescent labeled peptide substrate and its phosphorylation product appear at different times in the detection window to the detection point. Substrate turnover can thus be determined as the ratio of the product peak area and the sum of substrate peak area and product peak area.The enzyme reaction was carried out in a buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM MnCl2, 2 mM DTT, and 0.03% bovine serum albumine. The enzyme was pre-incubated with the test compound for 30 min at 24° C. The kinase reaction was initiated by addition of the substrate mixture containing the peptide substrate (final concentration 1 μM) and ATP (final concentration 10 μM). After 60 min incubation at 37° C., the enzyme reaction was terminated by adding a buffer containing 100 mM Hepes (pH 7.4) and 35 mM EDTA.
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 442.48, ALogP 2.73, HBA 7, HBD 3, PSA 135.88, QED 0.4
Compound (SMILES): COc1ccc(-c2ccc3c(N4C5CCC4COC5)nc(N4C[C@@H](C)O[C@@H](C)C4)nc3n2)cc1CO
InChIKey: KVMLDDLEDRJZCM-AGXSTUJCSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Kinase Assay: The test on activity against mTOR Kinase was performed using a 1.7 nM mTOR (Millipore, 14-770M), a 50 nM ULight-4EBP1 (Perkin-Elmer, TRF0128M) and a 100 μM ATP on a detection system LANCE® Ultra (PerkinElmer). The compound was first prepared into a 20 mM stock solution, and then gradientle diluted so as to be added into the mTOR enzyme reaction system on a 384 well plates. The test concentrations were 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, 0.000256 μM (n=3). After the mTOR enzyme reaction was performed for 1.5 hours, it was terminated, and LANCE® Ultra systeme (PerkinElmer) was used to perform the test for 1 hour. The test results were recorded on a multimode microplate reader Synergy II (BioTek). Relative activity against mTOR %=(Lance light value of the well with drug−light value of the blank group (without mTOR)/(light value of the DMSO group−light value of the blank group)×100%.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 491.59, ALogP 3.17, HBA 9, HBD 1, PSA 93.07, QED 0.58
Compound (SMILES): Cc1cnn2c(C(=O)Nc3cc4c(cc3N3CCOCC3)C(=O)N(C[C@@H](F)C(C)(C)O)C4)cnc2c1
InChIKey: VSPPTQKSBGOGPS-OAQYLSRUSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Transcreener-Fluorecescence Polarization Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 μM ATP; 10 μM MgCl2; 125 μM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 1.5 nM IRAK1; 62.5 μM ATP; 5 μM MgCl2, and 62.5 μM IRAK1 peptide in reaction buffer for 60 min.
Measurement: Ki = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 496.54, ALogP 2.19, HBA 8, HBD 2, PSA 112.3, QED 0.54
Compound (SMILES): C[C@@H]1CC[C@@H](n2ncc(C(C)(C)O)n2)CN1C(=O)c1nccnc1-c1ccccn1
InChIKey: BYXIWMURUZACQQ-HUUCEWRRSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: FLIPR Ca2+ Flux Assay: For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist.
Measurement: IC50 = 622 nM (pChEMBL 6.210000038146973)
Descriptors: MW 407.48, ALogP 2.22, HBA 8, HBD 1, PSA 109.92, QED 0.71
Compound (SMILES): O=C(Nc1ccc(Oc2ncnc3cc(OCCN4CCCC4)c4c(c23)OCCO4)cc1)Nc1cc(C(F)(F)F)ccc1F
InChIKey: VJFVAVLVIQQUHJ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Test of Small Molecule Compounds for Inhibiting the Activity of VEGFR-2 Kinase: 1. Dilution of the compound: a total of 12 concentrations were obtained using a 4-fold gradient dilution from the highest concentration of 10000 nM (the maximum final concentration of the drug used in this experiment is 10000 nM, and the minimum final concentration is 0.002384 nM),2. 2.5 μl of the gradient-diluted compounds was taken with a transfer pipette to a 384-well plate,3. Addition of enzyme: 5 μl of 2×VEGFR-2 kinase was taken with a transfer pipette to the corresponding reaction well of the 384-well plate, which was mixed and pre-reacted at room temperature for 30 min,4. 2.5 μl of 4× substrate/ATP Mix was taken with a transfer pipette to the corresponding reaction well of the 384-well plate,5. Negative control: 2.5 μl/well 4× substrate/ATP Mix and 7.5 μl 1× Kinase Assay Buffer were added to the wells of the 384-well plate,Positive control: 2.5 μl/well 4× substrate/ATP Mix, 2.5 μl/well 1× Kinase Assay Buffer containing 4% DMSO, and 5 μl/well 2×VEGFR-2 solution were added to the 384-well plate. The final concentration of DMSO in the reaction system is 4%,6. The mixture was mixed well and then centrifuged and reacted at room temperature in dark for 60 min,7. Termination of the enzymatic reaction: 5 μl of 4× Stop solution was taken with a transfer pipette to the wells of the 384-well plate, mixed and then centrifuged, and reacted at room temperature for 5 min,8. Development of the reaction: 5 μl of 4× Detection Mix was taken with a transfer pipette to the wells of the 384-well plate for color development, and the mixture was mixed and then centrifuged and reacted at room temperature for 60 min,9. The 384-well plate was placed into the Envision plate reader and the signal was detected using the appropriate program,10. Analysis and processing of the raw data:The drug concentrations and the corresponding inhibition rates were input into GraphPad Prism5 for calculation, and the inhibition rate of the compound was calculated as follows: inhibition rate (%)=[1−(experimental well reading value−negative control well reading value)/(positive control well reading value−negative control well reading value)]×100%. Processing with GraphPad Prism5 software yielded the corresponding IC50 value (the concentration of the compound at which 50% of the highest inhibition of the enzyme is achieved).
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 613.57, ALogP 6.47, HBA 8, HBD 2, PSA 107.07, QED 0.22
Compound (SMILES): N#Cc1cncc(Cn2ncc3ncc(-c4cccc(C(F)(F)F)c4)cc32)c1
InChIKey: FXMUNFGJHTYIMS-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: Effects of Test Articles on Cloned Human NR1/GuN2B Ion Channels Expressed in Mammalian Cells: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty-four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed, and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2), 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 483 nM (pChEMBL 6.320000171661377)
Descriptors: MW 379.35, ALogP 4.43, HBA 5, HBD 0, PSA 67.39, QED 0.53
Compound (SMILES): Cc1nc(CN2CCN(c3cccc4c3cnn4-c3ccc(F)cc3F)C2=O)cs1
InChIKey: SDLYKPOQSGRZQW-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 μg/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 μL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 μM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 μL assay buffer containing 2.5 μM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR®TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics.
Measurement: IC50 = 1610 nM (pChEMBL 5.789999961853027)
Descriptors: MW 425.46, ALogP 4.51, HBA 5, HBD 0, PSA 54.26, QED 0.48
Compound (SMILES): CN(C)[C@]1(c2cccc(F)c2)CC[C@]2(CC1)CN(c1cnc(C(F)(F)F)nc1)C(=O)N2
InChIKey: KSYKXUUKQMFQQB-BGYRXZFFSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Human Nociceptin/Orphanin FQ Peptide (hNOP) Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl. 10 mM MgCl2. 1 mM EDTA (pH 7.4). The final assay volume (250 μl/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 1 μM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux β-counter (PerkinElmer Life Sciences/Wallac. Turku. Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]nociceptin-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation. (Cheng and Prusoff. 1973).
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 437.44, ALogP 3.93, HBA 4, HBD 1, PSA 61.36, QED 0.74
Compound (SMILES): CN[C@H]1CCCCC[C@@H]1N(C)c1ccc2c(c1)C(=O)N(C1CCC(=O)NC1=O)C2=O
InChIKey: PBWMTPNJEUCDEA-UBFHEZILSA-N
Target: Zinc finger protein Helios (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UKS7
Assay: Prolabel Quantification of IKZF1, IKZF2, or GSPT1 Protein Levels in 293GT Cells: The Prolabel system from DiscoverX was used to develop high-throughput and quantitative assays to measure changes in IKZF1, IKZF2, and GSPT1 protein levels in response to compounds. The prolabel tag is derived from the alpha fragment of beta galactosidase and has the following protein sequence: mssnslavvlqrrdwenpgvtqlnrlaahppfaswrnseeartdrpsqqlrslnge. The complementary fragment of beta-galactosidase (from DiscoverX), is added to the prolabel tag to form an active beta galactosidase enzyme whose activity can be precisely measured. In this way, the levels of a fusion protein with the prolabel tag can be quantified in cell lysates.Lentiviral vectors, based on the Invitrogen pLenti6.2/V5 DEST backbone, were constructed that placed the prolabel tag upstream of IKZF1, IKZF2, or GSPT1 and expressed the fusion protein from a CMV promoter.To ensure moderate and consistent expression of the prolabel fusion proteins across all cells in the population, stable cell lines were constructed from cells expressing a single copy of the construct. Lentivirus packaged with the constructs was made using the Virapower kit from Invitrogen. Strongly adherent 293GT cell, GripTite 293 MSR cells from Thermo Fisher Scientific (Catalog number: R79507), were infected with the virus at low multiplicity of infection and selected by 5 μg/mL blasticidin for 2 weeks.
Measurement: EC50 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 412.49, ALogP 1.44, HBA 6, HBD 2, PSA 98.82, QED 0.57
Compound (SMILES): Cc1cccc(S(=O)(=O)N2CC(N)COc3ncc(NC(=O)c4c(Cl)cccc4Cl)cc32)c1
InChIKey: WJEOKQCHXLIESG-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: TR-FRET Assay Protocol II: HIS-tagged RORγ-LBD protein was expressed in SF9 cells using a baculovirus expression system. The RORγ-LBD protein was purified by glutathione sepharose chromatography. Separately, SF9 cells not expressing recombinant protein were lysed in TBS buffer (25 mM Tris, pH 8.0, 150 mM NaCl) under sonication. The lysate was added to the purified RORγ-LBD in a volume equivalent of 0.75 μL lysate (from 30,000 SF9 cells) per 75 femtomol of RORγ-LBD protein. The resulting mixture was diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 0.01% BSA) to obtain RORγ-LBD protein at a final concentration of 3 nM.Compounds to be tested were injected to the assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, Calif.).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-CPSSHSSLTERHKILHRLLQEGSPS) was prepared in assay buffer and added to each well (100 nM final concentration). A solution of Europium tagged anti-HIS antibody (1.25 nM final concentration) and APC-conjugated streptavidin (8 nM final concentration) were also added to each well.The final assay mixture was incubated overnight at 4° C., and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 μs, integration time=200 μs). The EC50 value for test compounds was calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm
Measurement: EC50 = 174 nM (pChEMBL 6.760000228881836)
Descriptors: MW 507.4, ALogP 3.86, HBA 6, HBD 2, PSA 114.62, QED 0.55
Compound (SMILES): Cc1ncc(-c2cc(NC(=O)c3c[nH]c(=O)cc3C(F)(F)F)c(N3C[C@@H](C)N(C)[C@@H](C)C3)cc2F)cn1
InChIKey: NSDXRTZOEXSSPU-OKILXGFUSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin labelled protein in vivo. Purification of the protein was performed using Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000 RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40 μl/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds respectively when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 518.52, ALogP 4.08, HBA 6, HBD 2, PSA 94.22, QED 0.51