lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): NC(=O)c1ccc2c(c1)C1(CC2)CC1N
InChIKey: WLGGBVLPQJAUKU-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biochemical Assay for LSD1 Activity (15 minutes): LSD1 demethylase reactions were carried out in 50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. All enzymatic reactions were performed for either 15 minutes at room temperature in a 10-μL volume. Five microliters of 800 nM biotinylated H3K4me1 peptide solution was added to each well of a black 384 well Proxiplate containing 80 nL compound (final concentration of 0.8% DMSO). Reactions were initiated by the addition of a mixture containing 20 nM LSD1 and 20 nM FAD (5 μL). Enzyme activity was stopped by the addition of 5 μL of high salt buffer consisting of 50 mM HEPES pH 7.4, 1.5 M NaCl, 1 mM DTT, 0.01% Tween-20, and 0.1 mg/mL BSA. Capture of the product peptide by the anti-H3K4me0 antibody and Streptavidin APC was allowed to proceed for 60 mM at room temperature before measuring the TR-FRET signal. Europium-labeled antibody and Streptavidin APC were used at final concentrations of 0.003 nM and 100 nM, respectively (total assay volume of 20 μL). Plates were read on a Perkin Elmer EnVision. Percent inhibition was calculated using Max (no inhibitor) and Min (quenched with stop buffer) controls and inhibition curves plotted to determine IC50 values.
Measurement: IC50 = 2400 nM (pChEMBL 5.619999885559082)
Descriptors: MW 202.26, ALogP 0.7, HBA 2, HBD 2, PSA 69.11, QED 0.7
Compound (SMILES): Nc1ncnn2c(-c3ccc(CF)c(C(=O)N[C@@H]4CN(C(=O)CC(F)(F)F)C[C@@H]4F)c3)cc(C(F)(F)F)c12
InChIKey: FLXMYLFXHWSITJ-LSDHHAIUSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 13000 nM (pChEMBL 4.889999866485596)
Descriptors: MW 550.41, ALogP 3.7, HBA 6, HBD 2, PSA 105.62, QED 0.47
Compound (SMILES): CCOC(=O)c1ccc(NC(=O)NCc2ccnc(N)c2)cc1
InChIKey: LBLMGMSJBQAQKL-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Enzyme Assay and NMN Detection: NAMPT reaction was performed in 50 mM HEPES, pH 7.5, containing 5 mM MgCl2, 0.1% Prionex, 0.005% Tween 20, and 1 mM TCEP at room temperature in Greiner 384-well black polypropylene plates. The concentration of substrates and enzymes in the final assay were as follows: ATP (120 uM), nicotinamide (5 uM), phosphoribosylpyrophosphate (6.25 uM), NAMPT enzyme (16 nM), pyrophosphatase (0.04 U/ml) in the final reaction volume 6 uL. Following 2 h incubation the reaction was stopped by addition 2.5 ul 20% acetophenone in dimethyl sulfoxide (DMSO) and 2.5 ul 2 MKOH. Plates were centrifuges and added with 10.5 ul 88% formic acid. The fluorescence (ex380/em460) was measured following another short incubation and 30 min incubation. The increased concentration of NMN was determined from a calibration curve obtained at the time of experiment.
Measurement: EC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 314.35, ALogP 2.16, HBA 5, HBD 3, PSA 106.34, QED 0.73
Compound (SMILES): Cc1cc(-c2cc(NCCCC3CCCNC3)c3ccccc3n2)cnc1C#N
InChIKey: MOIYHWSQCZRFFF-UHFFFAOYSA-N
Target: Low molecular weight phosphotyrosine protein phosphatase (SINGLE PROTEIN), Homo sapiens
UniProt: P24666
Assay: LMPTP Primary Screening Protocol: A listing of materials is provided:    Item, source, catalog no.    LMPTP-A Enzyme Stock Solution (4.22 mg/ml or 206.8 μM), SBMRI Protein Facility,    N/A    OMFP, Sigma, M2629-100 MG    Bis-Tris pH 6.0, Fisher Sci, BP301-100    Triton-X 100, Sigma, T9284    DTT, Sigma, D9779    Mol. Grade Water, Mediatech, Inc., 46-000-CM    1536-well black High base opaque bottom plate, Nexus Biosystems, 00019120Final assay conditions are:    0.625 nM LMPTP-A Enzyme    40 μM OMFP    50 mM Bis-Tris pH 6.0    1 mM DTT    0.01% Triton-X 100    20 μM test compound    3% DMSO (2% from substrate and 1% from compounds)    6 μL reaction volume    50 minutes incubation at room tempAssay Procedure    1. Prepare Reagents as described in section F. Recipe.    2. Using LabCyte Echo, transfer 60 nL from 2 mM test compound source plate into assay plate Col. 5-48 (final concentration of test compounds is 20 μM). 60 nL of DMSO should be transferred to col. 1-4 for positive and negative control wells.    3. Spin plates at 1000 rpm for 1 minute in centrifuge.    4. Using the Beckman Coulter Bioraptr, add 3 μL/well of control buffer to columns 1 and 2.    5. Using the Bioraptr, add 3 μL/well of enzyme solution to col. 3-48.    6. Using the Bioraptr, add 3 μL/well of substrate solution to col. 1-48.    7. Spin plates at 1000 rpm for 1 minute in centrifuge.    8. Incubate plates in the dark at room temperature for 50 minutes.    9. Read plates on PerkinElmer Viewlux using a FI protocol.
Measurement: IC50 = 2900 nM (pChEMBL 5.539999961853027)
Descriptors: MW 385.52, ALogP 4.67, HBA 5, HBD 2, PSA 73.63, QED 0.6
Compound (SMILES): Cc1nc(N2CCC3(CCC[C@H]3N)CC2)n2ccnc2c1-c1ccc(Cl)c(Cl)c1
InChIKey: RDMACMFSOMURQF-GOSISDBHSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Biochemical Assay: Inhibition of SHP-2 activity by small molecule inhibitors was monitored by a biochemical DiFMUP pseudosubstrate-fluorogenic assay. SHP-2 enzyme at final concentration of 0.2 nM was incubated with an IRS-1 activator peptide at a final concentration of 1 μM in SHP-2 assay buffer (60 mM HEPES, pH 7.2-7.5, 0.05% Tween-20, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, and 5 mM DTT) for 75 min at room temperature. The reaction mix was added to the 384-well non-binding plate which had been pre-spotted with 1:3 serial diluted compounds from 10,000-0.005 nM, and incubated at room temperature for 90 min. DiFMUP substrate at final concentration of 50 μM was then added to the wells and incubated for additional 30 min at room temperature. The final reaction volume was 20 μl/well. The fluorescence intensity of the reaction product was measured by Envision at excitation and emission wavelengths of 340 nm and 450 nm, respectively. The inhibitor dose response curves were generated using normalized IC50 regression curve fitting by in house dose response analysis tools. The wells with DMSO vehicle were used as controls (100% signal). The percent (%) control of SHP-2 compound was calculated as: (Compound well fluorescence counts/DMSO fluorescence counts)×100%.
Measurement: IC50 = 95 nM (pChEMBL 7.019999980926514)
Descriptors: MW 430.38, ALogP 5.11, HBA 5, HBD 1, PSA 59.45, QED 0.61
Compound (SMILES): Cn1c(Cc2c(Cl)ncc(C(=O)N3CCC(CC(=O)O)CC3)c2Cl)cc2c(F)cc(C3CC3)cc21
InChIKey: DXHUJHAGTASXAO-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: transient transactivation assay: The primary screen was performed by transient transactivation assays. These cell-based assays were carried out using Cos-7 cells transfected with a chimeric human RORγ-Gal4 receptor expression plasmid and a 5Gal4 pGL3 TK Luc reporter plasmid. Transfections were performed by a chemical agent (Jet PEI). Transfected cells were distributed in 384-wells plates and were allowed to recover for 24 h. The culture medium was then removed and fresh medium containing the compounds to be tested was added (final concentration ranging from 10−4 M to 3 10−10 M). After an overnight incubation, luciferase expression was measured by adding SteadyGlo according to the manufacturer's instructions (Promega). T0901317 at 10−5 M was used as reference. Results were expressed as fold induction compared to basal level or as percentage activity compared to references taken as 100% (basal) or 0% (T0901317). Dose-effect curves and IC50 were calculated using the software Assay Explorer (MDL).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 518.42, ALogP 5.81, HBA 4, HBD 1, PSA 75.43, QED 0.42
Compound (SMILES): C[C@@H]1CN(c2ncc(-c3cc(NC(=O)c4ccc(F)cc4C(F)(F)F)c(N4C[C@@H](C)N(C)[C@@H](C)C4)cc3F)cn2)C[C@H](C)N1C
InChIKey: DYHKUOKFQBAXGE-FXELSEDVSA-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 = 0.31 nM (pChEMBL 9.510000228881836)
Descriptors: MW 631.69, ALogP 5.75, HBA 7, HBD 1, PSA 67.84, QED 0.36
Compound (SMILES): N#Cc1ccc(-c2cnn3ccc(-c4ccc(C(=O)N5CCOCC5)cc4)nc23)cc1
InChIKey: IHBSDYYVQMKNMJ-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: In Vitro Kinase Assay: MNK1 and MNK2 inhibitor activity was determined using recombinant kinase domains expressed in E. coli. MNK1 and MNK2 were expressed as GST fusion proteins and the GST tag was removed using PreScission protease. After concentration to 10-15 mg/ml the proteins were flash frozen in liquid nitrogen and stored at −80° C. MNK1 and MNK2 were activated using recombinant ERK2 which was activated using a constitutively active mutant of MEK1, both ERK2 and MEK1 were expressed in E. coli as N-terminally his tagged proteins. Recombinant ERK2 was activated by incubating 11.3 μM of the kinase with 1 μM MEK1 and 100 μM ATP. This reaction mixture was then used immediately for the activation of the MNKs. The activation of the MNK1 was performed by incubating 5.0 μM of MNK1 with 0.3 μM of activated ERK2 and 500 μM ATP at 30° C. for 6 hours. The activation of MNK2 was performed by incubating 50 μM of MNK2 with 3.0 μM of activated ERK2 and 500 μM ATP at 30° C. for 2 hours. The activated MNKs were stored at −20° C. until required for assay.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 409.45, ALogP 3.41, HBA 6, HBD 0, PSA 83.52, QED 0.52
Compound (SMILES): Nc1ccc2c(c1)NC(=O)CCCCC(c1ccc(-c3c(F)ccc(Cl)c3F)c[n+]1[O-])c1cccc-2c1
InChIKey: GREDTIZAEYKOPI-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Factor XIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIa can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at rt or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 40 pM (Sekisui Diagnostics) and he synthetic substrate, Z-Gly-Pro-Arg-AFC, TFA salt (Sigma #C0980) at a concentration of 100 μM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ≤0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (Vo) or presence of inhibitor (Vi) were measured. Assuming competitive inhibition, and that unity is negligible compared Km/[S], [I]/e, and [I]/e (where [S], [I], and e respectively represent the total concentrations, of substrate, inhibitor and enzyme), the equilibrium constant (Ki) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of Vo/Vi on [I] shown in the following equation.
Measurement: Ki = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 519.98, ALogP 6.81, HBA 3, HBD 2, PSA 82.06, QED 0.13
Compound (SMILES): Fc1cccc(-c2cc(NCCCC3CCCNC3)c3ccccc3n2)c1
InChIKey: QMFUOEWWYXSTQO-UHFFFAOYSA-N
Target: Low molecular weight phosphotyrosine protein phosphatase (SINGLE PROTEIN), Homo sapiens
UniProt: P24666
Assay: LMPTP Primary Screening Protocol: A listing of materials is provided:    Item, source, catalog no.    LMPTP-A Enzyme Stock Solution (4.22 mg/ml or 206.8 μM), SBMRI Protein Facility,    N/A    OMFP, Sigma, M2629-100 MG    Bis-Tris pH 6.0, Fisher Sci, BP301-100    Triton-X 100, Sigma, T9284    DTT, Sigma, D9779    Mol. Grade Water, Mediatech, Inc., 46-000-CM    1536-well black High base opaque bottom plate, Nexus Biosystems, 00019120Final assay conditions are:    0.625 nM LMPTP-A Enzyme    40 μM OMFP    50 mM Bis-Tris pH 6.0    1 mM DTT    0.01% Triton-X 100    20 μM test compound    3% DMSO (2% from substrate and 1% from compounds)    6 μL reaction volume    50 minutes incubation at room tempAssay Procedure    1. Prepare Reagents as described in section F. Recipe.    2. Using LabCyte Echo, transfer 60 nL from 2 mM test compound source plate into assay plate Col. 5-48 (final concentration of test compounds is 20 μM). 60 nL of DMSO should be transferred to col. 1-4 for positive and negative control wells.    3. Spin plates at 1000 rpm for 1 minute in centrifuge.    4. Using the Beckman Coulter Bioraptr, add 3 μL/well of control buffer to columns 1 and 2.    5. Using the Bioraptr, add 3 μL/well of enzyme solution to col. 3-48.    6. Using the Bioraptr, add 3 μL/well of substrate solution to col. 1-48.    7. Spin plates at 1000 rpm for 1 minute in centrifuge.    8. Incubate plates in the dark at room temperature for 50 minutes.    9. Read plates on PerkinElmer Viewlux using a FI protocol.
Measurement: IC50 = 2900 nM (pChEMBL 5.539999961853027)
Descriptors: MW 363.48, ALogP 5.23, HBA 3, HBD 2, PSA 36.95, QED 0.59
Compound (SMILES): CC1(C)CC(CN2CCc3c(-c4ccc(Cl)cc4F)cc(C(N)=O)nc3C2)CCO1
InChIKey: CSOAHWZMIVAVCY-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assays: The utility of the compounds of the invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr-cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) were treated with various concentrations of antagonists of compounds and the Ca2+ response was monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity. Then 2,500 nM glutamate was added and the antagonist response was monitored. The maximum calcium response at each concentration of compound for agonist or antagonist were plotted as dose responses and the curves were fitted with a four parameter logistic equation giving IC50 and Hill coefficient using the iterative non linear curve fitting software ADA (Merck & Co).
Measurement: IC50 = 5600 nM (pChEMBL 5.25)
Descriptors: MW 431.94, ALogP 4.2, HBA 4, HBD 1, PSA 68.45, QED 0.79
Compound (SMILES): CC(C)(O)[C@@H](N)C(=O)N[C@@H]1CC[C@@]2(S(=O)(=O)c3ccc(F)cc3)c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3CC[C@@H]12
InChIKey: UQDGAZQNJRFPBL-SEDAZVQISA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: General RORgamma Gal4 Reporter Assay: Inverse agonist activity of potential ligands to RORγ was measured by inhibition of luminescence in a Gal4-luciferase reporter assay in Jurkat cells.Jurkat cells stably over-expressing the RORγ receptor, Jurkat pEx/Gal/hRORγ CLBD/HYG pG5luc/blast, were plated at a concentration of 10,000 cells/well in a 384-well solid white cell culture plate (Perkin Elmer #6007899) in assay buffer RPMI 1640 (Gibco 11875-085 1L) containing 0.1% BSA, 100×HEPES (Gibco 15360-080), 100 mM sodium pyruvate (Gibco 11360-040), 50 mg/mL Hygromycin B (Invitrogen 10687-010) and 10 mg/mL blasticidin (Invitrogen R210-01). 100 nL of test compound in a 3-fold serial dilution, with final concentrations ranging from 40 μM to 0.67 nM, were added to the cells which were then incubated overnight.The following day, cells were lysed with 10 μL of Steady-Glo Luciferase Assay System (Promega Cat. No. EZ550), and analyzed immediately. IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce luciferase activity by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 628.58, ALogP 4.72, HBA 5, HBD 3, PSA 109.49, QED 0.32
Compound (SMILES): C=CC(=O)N1CCC[C@H](c2cn(-c3ccc(Oc4c(F)cccc4F)cc3)c3c(N)n[nH]c(=O)c23)C1
InChIKey: NYMXGINEAUXDEN-HNNXBMFYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Cell Proliferation Assays: MTS testing kit was purchased from Promega. The DMEM, Fetal bovine serum and Penicillin-Streptomycin were purchased from Gibco. Dimethyl sulfoxide (DMSO) was purchased from Sigma.To investigate whether a compound is able to inhibit the activity of BTK in cells, a mechanism-based assay using DOHH2 (DSMZ catalog#: ACC47) and Mino (ATCC® Number CRL-3000) cells was developed. In this assay, inhibition of BTK was detected by the inhibition of DOHH2 and Mino cell proliferations. DOHH2 or Mino cells were cultured in culture flasks to 40-80% confluence in RPMI-1640 plus 10% fetal bovine serum. C ells were collected and plated onto 96-well plates at desired cell density (DOHH2: 5000 cells/well; Mino: 10000 cells/well). Plates were incubated overnight at 37° C., with 5% CO2 to adhere. Compounds were added to the plates, and the final compound concentrations were 10000, 3333, 1111, 270, 124, 41, 14, 4.6 and 1.5 nM. Plates were placed at 37° C., with 5% CO2 for 120 h (DOHH2) or 72 h (Mino). 20 μl MTS/100 μl medium mixture solution were added to each well and the plates were incubated for exactly 2 hours. The reaction was stopped by adding 25 μl 10% SDS per well. Absorbance at 490 nm and 650 nm (reference wavelength) were measured and IC50 was calculated using GraphPad Prism 5.0.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 491.5, ALogP 4.26, HBA 6, HBD 2, PSA 106.24, QED 0.41
Compound (SMILES): CC[C@@H](CC(F)(F)F)Nc1cc(OC(F)F)c(-c2c(Cl)c(C(=O)NC[C@]3(O)CC[C@@H](S(C)(=O)=O)CC3)nn2CC)cn1
InChIKey: DDBCZGXUUVTEKV-ZEUIUJOJSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: For the RORγt construct used in the ThermoFluor® assay, numbering for the nucleotide sequences was based on the reference sequence for human RORγt, transcript variant 2, NCBI Accession: NM 001001523.1 (SEQ ID NO:1). Nucleotides 850-1635 (SEQ ID NO:2) coding for the wild type human RORγt ligand binding domain (RORγt LBD) were cloned into the pHIS1 vector, a modified pET E. coli expression vector (Accelagen, San Diego), containing an in-frame N-terminal His-tag and a TurboTEV protease cleavage site (ENLYFQG, SEQ ID NO:3) upstream of the cloned insert sequence. The amino acid sequence for the RORγt construct used in the Thermofluor® assay is shown as SEQ ID NO:4.ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Development, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.
Measurement: Kd = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 646.08, ALogP 4.81, HBA 9, HBD 3, PSA 135.44, QED 0.28
Compound (SMILES): COc1ncc(-c2cc(CN3CCC(F)(F)CC3)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)c2ncccc2F)C[C@@H]1F
InChIKey: KIHNRVHUSLNNSV-LEWJYISDSA-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 = 2100 nM (pChEMBL 5.679999828338623)
Descriptors: MW 627.6, ALogP 2.74, HBA 10, HBD 2, PSA 143.87, QED 0.3
Compound (SMILES): CC(C)CNc1cc(N2CC[C@@H](N)C2)ccn1
InChIKey: IAPFMZATXNRZAV-LLVKDONJSA-N
Target: Histamine H4 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3N8
Assay: Binding Assay: Cell pellets from SK-N-MC cells stably or transiently transfected with human H4 receptor (NCBI accession No. AF312230) were used for the binding assays. Cell pellets were homogenized in 50 mM Tris/5 mM EDTA buffer and supernatants from an 800 g spin were collected and recentrifuged at 30,000 g for 30 min. Pellets were rehomogenized in 50 mM Tris/5 mM EDTA buffer. For competition binding studies, cell membranes were incubated with 2x KD (10 nM),[3H] histamine (Specific activity: 14.2 to 23 Ci/mmol), with or without test compounds for 45 to 60 min at 4 to 25° C. Ki values were calculated based on an experimentally determined appropriate KD values according to Cheng and Prusoff (Biochem. Pharmacol. 1973, 22(23):3099-3108).
Measurement: Ki = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 234.35, ALogP 1.69, HBA 4, HBD 2, PSA 54.18, QED 0.83
Compound (SMILES): O=C(NC1CCC(=Cc2cccc(Oc3ccc(C(F)(F)F)cn3)c2)CC1)c1ccc(-n2cnnc2)nc1
InChIKey: BOCWHKDIBVAVFW-PDGQHHTCSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: FAAH Assay: All reagents were purchased from Sigma-Aldrich unless specified. Human and Rat Fatty Acid Amide Hydrolase (FAAH) genes used in assay have been described by Patricelli et al. (Biochemistry. 1998, 37(43), 15177-87). The transmembrane domain-deleted Fatty Acid Amide Hydrolase (FAAH) genes were cloned into pET15b (Novagen, #69661) (human FAAH)/pET 28a (Novagen, #69864-3) (rat FAAH gene) plasmids and expressed in E coli BL21 DE3. Chaperone protein groEL-groES in pGRO7 plasmid (Takara Bio Inc, Japan) was co-expressed with Fatty Acid Amide Hydrolase (FAAH) to improve solubility of the protein expressed in E coli. The protein was expressed and enriched as described in Mileni et al. (Proc Natl Acad Sci USA. 2008, 105(35),12820-4). Briefly, the bacterial cultures in Luria Broth (2 L) were induced with arabinose (2 mM) and Isopropyl (β-D-1-thiogalactopyranoside, IPTG (1 mM), for 20 h at room temperature. The cultures were centrifuged at 1200×g for 10 min and the cell pellet was resuspended in 100 mL buffer containing of 20 mM NaPi (pH 7.4), 100 mM NaCl, Benzonase (500u), Aprotinin (1 μg/mL) and Leupeptin (1 μg/mL). Cells were lyzed by sonication (Amp 20%, Pulse 15 s×15, on ice) and the cell debris removed by centrifugation at 5000×g for 20 min. The supernatant was enriched via ultracentrifugation at 100,000×g for 1 h and the cell pellet was resuspended in 16 mL buffer containing 20 mM NaPi (pH 7.8), 500 mM NaCl, 1% Triton X-100. The resuspended cell extract was subjected to ultra-centrifugation at 100,000×g for 1 h and the enriched supernatant was used in the in vitro assay. All protein extraction steps were carried out on ice or at 4° C.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 520.52, ALogP 5.62, HBA 7, HBD 1, PSA 94.82, QED 0.35
Compound (SMILES): CCO[C@H](C(=O)Nc1nnc(N[C@@H]2CCN(c3cccnn3)C2)s1)c1cccc(N2CC(OC)C2)c1
InChIKey: WAPXAOQFTGRVPA-UTKZUKDTSA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: GLS Enzyme Potency Assay: A Glutamate Oxidase/AmplexRed coupled assay was used to measure the ability of compounds to bind to and inhibit the activity of GLS1 in vitro. 6His tagged GLS protein (amino acids 63-669) expressed in E. Coli was purified and stored at −80° C. in aliquots. GLS1 was diluted to 2× working concentration and incubated at room temperature to allow the tetrameric/dimeric forms to reach steady state. Assay measurements were performed in buffer comprising 50 mM TRIS pH 7.8, 100 mM NaPO4, pH 7.8, 0.001% v/v Tween20. Purified recombinant GLS1 protein was diluted in assay buffer to 12 nM and pre-incubated at room temperature for 30 minutes. Test compounds were prepared by dilution in 100% DMSO to give the correct dose range for 12 point concentration response and an appropriate volume (2.5-60 nl) dispensed into 384 well micro assay plates (Greiner product code 784900) using a Labcyte Echo 555 acoustic dispenser. DMSO concentration was maintained at 2% by back filling with DMSO solution. 3 μL of diluted GLS1 protein (12 nM) was then dispensed into each well using a BioRaptr automated dispenser (Beckman-Coulter) and incubated for 15 minutes at room temperature. 3 μL of 100 mM glutamine diluted in assay buffer was then added and the reaction incubated at room temperature for 60 minutes. The reaction was then stopped by addition of 45 μM 6-(2-bromoethynyl)-2,3-dimethyl-quinazolin-4-one, 75 μM Amplex Red, 0.375 units/mL Horseradish Peroxidase, 0.12 units/mL Glutamate Oxidase in 100 mM TRIS pH7.5. After 30 minutes at room temp in the dark, plates were read on a Perkin Elmer EnVision using 535/590 nm optic filters and raw data analysed using Genedata to generate IC50 values. An artefact version of the assay where the 6His tagged GLS protein and glutamine were replaced with assay buffer was also used to rule out non specific effects on the assay components.
Measurement: IC50 = 68.5 nM (pChEMBL 7.159999847412109)
Descriptors: MW 510.62, ALogP 2.57, HBA 11, HBD 2, PSA 117.63, QED 0.42
Compound (SMILES): Oc1ccc(C(O)CN2CC3(C2)CC(O)(Cc2ccccc2)C3)cc1
InChIKey: QRPVJEWWXDDUJD-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: FDSS Assay: To each well of the plate, 10 μL test compound, control (MK801) or HHnoCa buffer was added to a final concentration of 10 μM with a final concentration of DMSO of 0.1%. Following 10 minutes pre-incubation in the dark, plates are loaded onto the Hamamatsu FDSS 6000. After collecting baseline fluorescence images, 3 μM glutamate, 3 glycine, and 1 mM Ca2+ in HHnoCa buffer is added to each well, and Ca2+ is recorded for 3 minutes. Data were processed by computing ratio of fluorescence at the end of data collection to baseline fluorescence to assess degree of Ca′ influx inhibition relative to that observed in MK801.
Measurement: IC50 = 506 nM (pChEMBL 6.300000190734863)
Descriptors: MW 339.44, ALogP 2.5, HBA 4, HBD 3, PSA 63.93, QED 0.78
Compound (SMILES): CN(C)CCn1cc(-c2cc3c(cc2F)OCC/C=C\Cn2cnnc2-c2cccc(n2)NC3=O)cn1
InChIKey: LBOSPCLNMAZISL-ARJAWSKDSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: TR-FRET Assay: Table 2: Alternatively, the protein kinase activity of the compounds described herein were tested using the ASK1/MAP3K5 assay by Reaction Biology Corp. The assay procedure can be found on Reaction Biology Corp.website at http://www.reactionbiology.com/webapps/site/KinasePDFs/ASK1_MAP3K5.pdf. ASK1/MAP3K5 was used as the protein kinase and 20 μM of myelin basic protein (MBP) and 10 μM ATP were used as the substrate.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 502.55, ALogP 3.5, HBA 9, HBD 1, PSA 102.99, QED 0.43
Compound (SMILES): CCS(=O)(=O)Nc1cccc(-c2nnc(Nc3ccc4[nH]ncc4c3)s2)c1
InChIKey: JQTWKEQQCOFNEM-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: Biological Assays for Inhibition of Rho-Associated Protein Kinase: Assays for ROCK inhibition were performed using the following protein constructs: glutathione S-transferase (GST)-tagged human ROCK1 catalytic domain 1-477 from Carna Biosciences (cat #01-109; apparent Km value for ATP is 10 μM) and GST-tagged human ROCK2 catalytic domain 1-553 from Carna Biosciences (Cat#01-110; apparent Km value for ATP is 15 μM). Protein constructs were purified from a baculovirus expression system. The peptide substrate was fluorescent LANCE® Ultra ULight-CREBtide: CKRREILSRRPSYRK (PerkinElmer, # TRF0107-D). Kinase reactions were carried out in in a 10 μL volume in 384-well plates: 50 nM ULight-CREBtide substrate, 2 nM constitutively active ROCK1 or ROCK2 kinase, and test compound in DMSO (or DMSO only for controls) were diluted into assay buffer containing 50 mM Tris-HCl (pH=7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, and 2 mM DTT such that the final concentration of DMSO was 0.5%. After a 90 minute incubation at room temperature on a shaker table, the kinase reaction was stopped by addition of 10 mM EDTA, and phosphorylation of the substrate was detected by adding 1 nM LANCE® Ultra Europium-anti-phospho-CREB (ser133) antibody (PerkinElmer, # TRF0200-D) and incubating for 60 minutes on a shaker at room temperature. The flurorescence resonance energy transfer (FRET) signals were read and analyzed on an Envision¿ 2103 Multilabel Reader (Perkin Elmer). The concentration of test compound required to inhibit substrate phosphorylation by 50% (the IC50) was calculated by non-linear regression using GraphPad PRIZM.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 400.49, ALogP 3.59, HBA 7, HBD 3, PSA 112.66, QED 0.46
Compound (SMILES): CC1(c2ccc(CC(=O)Nc3cnn(Cc4ccc(C#N)c(F)c4)n3)cc2)COC1
InChIKey: IVNKNTVCPREBJZ-UHFFFAOYSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Measurement of Calcium Channel Flux by Means of FLIPR Assay: Stock solutions of test compounds are prepared to a concentration of 10 mM in DMSO. For the Cav3.2 assay, serial dilutions of the compounds are prepared in TEAC buffer (100 mM tetraethylammonium chloride; 20 mM Hepes; 2.5 mM CaCl2; 5 mM KCl; 1 mM MgCl2; 1% FBS; pH 7.2), for the Cav1.2 assay serial dilutions are prepared in assay buffer. Test compounds are added to the cells to give a 3-fold dilution range from 10 μM to 0.05 nM. The compounds are incubated with the cells for 3 minutes and Ca2+ entry is stimulated by adding either CaCl2 to a final concentration of 10 mM (Cav3.2 assay) or by adding KCl to a final concentration of 20 mM (Cav1.2 assay). The kinetics of fluorescence increase are recorded for every well and the area under the fluorescence trace for every compound concentration is used to generate inhibition curves using non-linear regression sigmoidal concentration-response curve analysis with in-house software. IC50 values are calculated and represent the compound concentration required to inhibit 50% of the signal that is obtained in the presence of vehicle instead of test compound. In analogy, antagonistic activities (IC50 values) of all exemplified compounds have been measured for the for the Cav3.1- and the Cav3.3-channel. Antagonistic activities (IC50 values) of all exemplified compounds are in the range of 1.7 to 970 nM with respect to Cav3.1; and in the range of 1.1 to 620 nM with respect to Cav3.3.
Measurement: IC50 = 74 nM (pChEMBL 7.130000114440918)
Descriptors: MW 405.43, ALogP 2.81, HBA 6, HBD 1, PSA 92.83, QED 0.68
Compound (SMILES): Cc1cc(C(F)(F)F)cc(O)c1-c1ccc(N[C@@H]2CCCN(C)C2=O)nn1
InChIKey: VMQWHVSIOQFVJQ-CYBMUJFWSA-N
Target: Interleukin-1 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P01584
Assay: IL-1 Secretion Assa: Monocytic THP-1 cells (ATCC: TIB-202) were maintained according to providers' instructions in RPMI media (RPMI/Hepes+10% fetal bovine serum+Sodium Pyruvate+0.05 mM Beta-mercaptoethanol (1000× stock)+Pen-Strep). Cells were differentiated in bulk with 0.5 μM phorbol 12-myristate 13-acetate (PMA; Sigma #P8139) for 3 hours, media was exchanged, and cells were plated at 50,000 cells per well in a 384-well flat-bottom cell culture plates (Greiner, #781986), and allowed to differentiate overnight. Compound in a 1:3.16 serial dilution series in DMSO was added 1:100 to the cells and incubated for 1 hour. The NLRP3 inflammasome was activated with the addition of 15 μM (final concentration) Nigericin (Enzo Life Sciences, #BML-CA421-0005), and cells were incubated for 3 hours. 10 μL supernatant was removed, and IL-1p levels were monitored using an HTRF assay (CisBio, #621L1PEC) according to manufacturers' instructions. Viability and pyroptosis was monitored with the addition of PrestoBlue cell viability reagent (Life Technologies, #A13261) directly to the cell culture plate.
Measurement: IC50 = 35.9 nM (pChEMBL 7.449999809265137)
Descriptors: MW 380.37, ALogP 3.21, HBA 5, HBD 2, PSA 78.35, QED 0.86
Compound (SMILES): Cc1nnn(Cc2cc(Cl)ccc2/C=C/C(=O)N2CCC(n3ncnc3C)CC2)n1
InChIKey: JWYDCYLTFCDGBZ-GQCTYLIASA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: ATX Assay: Reagents LPC (oleoyl (18:1)) was purchased from Avanti Polar Lipids (Alabaster, Ala.) and solubilized in methanol to 20 mM. Amplex Red was obtained from Invitrogen Life Technologies (Paisley, UK) and dissolved in DMSO to 10 mM. Choline oxidase and horseradish peroxidase (HRP) were obtained from Sigma Aldrich (Dorset, UK) and dissolved in HBSS to 20 U/ml and 200 U/ml respectively. All reagents were stored at −20° C. in single use aliquots. All experimental measurements were performed in assay buffer made up immediately prior to use (HBSS, 0.01% BSA essentially fatty acid free).Protein Recombinant human ATX was prepared at Novartis (Basel, CH) in a human embryonic kidney (HEK) cell preparation, and stored in single use aliquots of 26 mg/ml (26 μM) stocks stored at −80° C.Method All experimental measurements were performed in black 384 well polystyrene (low volume, round bottom, Corning (3676)) plates. PerkinElmer EnVision (Fluorescence Intensity/Absorbance Monochromator) or Tecan Infinite 200 PRO series plate reader was used to detect change in fluorescent intensity.Assessing ATX inhibition ATX activity was determined by measurement of released choline in reactions containing ATX (10 nM), choline oxidase (0.1 U/ml), HRP (100 U/ml), amplex red (50 μM) and LPC 18:1 (10 μM). Compounds of the invention were prepared as 10 point serial dilutions from 1 μM in duplicate and pre-incubated with ATX at 37° C. for 20 minutes prior to the addition of remaining reagents. The liberated choline was measured from changes in fluorescence intensity (λex 530 nm, λem 590 nm) of the product resurofin at 37° C. every 2 minutes over a 40-minute period. ATX activity was measured as a slope of the linear portion of the progress curve, typically between 14 to 24 minutes.Data analysis Slope data was exported to Graphpad prism (Graphpad software, San Diego, Calif.)
Measurement: IC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 426.91, ALogP 2.46, HBA 8, HBD 0, PSA 94.62, QED 0.58
Compound (SMILES): CN1C(=O)CCc2cc(-n3cnc4c3C(=O)CC3(CCOCC3)C4)ccc21
InChIKey: SBPVMADMMNMHGX-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibition of Aldosterone Synthase: Assays are performed in 96-well format in a final volume of 60 microL/well, containing 100 mM potassium phosphate, pH 7.4, 1% (v/v) DMSO, and additionally, 2 μM of corticosterone and 50 units of CYP11B2 activity. Reactions are started by the addition of NADPH to 1 mM and allowed to proceed for 90 minutes at 37° C. Reactions are terminated by the addition of 60 μL of MeCN containing an internal standard for mass spectrometry. One hundred microliters are then transferred to a glass filter plate and centrifuged at 570×g for 5 minutes and the filtrate is collected. Reaction product aldosterone is quantified by mass spectrometry. To determine the assay blank value (0% activity), NADPH is omitted from some reactions.Dose dependent inhibition is quantified by the inclusion of compound at various concentrations. Maximum activity (100%) is defined by reactions containing NADPH, but without compound. Activities at each concentration are expressed as a percentage of the maximum activity (y-axis) and plotted against concentration of compound (x-axis) and the concentration corresponding to 50% activity (IC50) determined using the XLFit curve-fitting program using a 4-parameter logistic model.
Measurement: IC50 = 460 nM (pChEMBL 6.340000152587891)
Descriptors: MW 365.43, ALogP 2.71, HBA 5, HBD 0, PSA 64.43, QED 0.78
Compound (SMILES): CNC(=O)c1ccc2c(c1)nc(C1CC1)n2-c1ccc2c(c1)CCC(=O)N2
InChIKey: JXGSPMMTSNVXPO-UHFFFAOYSA-N
Target: Protein cereblon (SINGLE PROTEIN), Homo sapiens
UniProt: Q96SW2
Assay: Â Fluorescence Direct Binding Protocol: Process 1) Optimization of measurement parameters to minimize protein consumption and to minimize the dilution effect and the DMSO content 2) Titration measurements of the protein against ligand by at least 12 titration steps to obtain a good s-curve fit 3) Repeat the same titration measurements with the ligand alone to enable correction 4) Check the stability of the protein once by titration against DMSO alone 5) Determination of the molar extinction coefficients of the ligand at 280 and 340 nm with help of a UV-spectrophotometer 6) Use Excel template for the correction of the measured raw data 7) Use GraphPad Prism software for the quadratic binding fit and the Kd evaluation.
Measurement: Kd = 456 nM (pChEMBL 6.340000152587891)
Descriptors: MW 360.42, ALogP 3.15, HBA 4, HBD 2, PSA 76.02, QED 0.75
Compound (SMILES): Cn1cc(-c2cnc(NCc3c(F)ccc4c3CCO4)n3cnnc23)c(C(F)(F)F)n1
InChIKey: XAFJTQTWRKCBFK-UHFFFAOYSA-N
Target: Polycomb protein EED (SINGLE PROTEIN), Homo sapiens
UniProt: O75530
Assay: EED-H3K27Me3 Peptide Competition Binding Assay by AlphaScreen: To assess the compounds potency in the EED-H3K27Me3 competition binding assay, compounds were serially diluted 3-fold in DMSO to obtain a total of twelve concentrations. Then compounds at each concentration (75 nL of each) were transferred by Mosquito into a 384-well Perkin Elmer ProxiPlate 384 plus plates. 8 uL of solutions containing 30 nM EED (1-441)-His protein and 15 nM biotin-H3K27Me3 (19-33) peptide in the buffer (25 mM HEPES, pH 8, 0.02% Tween-20, 0.5% BSA) were added to the wells and then incubated with compound for 20 min. AlphaScreen detection beads mix was prepared immediately before use by mixing nickel chelate acceptor beads and streptavidin donor beads in a 1:1 ratio (Perkin Elmer, Product No. 6760619C/M/R) into the buffer described above. Then 4 μL of detection beads mix was added to the plate and incubate in the dark at the rt for 1 h. The final concentration of donor and acceptor beads was 10 μg/mL for each. Plates were read on EnVision (PerkinElmer) using the AlphaScreen setting adapted for optimal signal detection with a 615 nm filter, after sample excitation at 680 nm. The emission signal at 615 nm was used to quantify compounds inhibition. AlphaScreen signals were normalized based on the reading coming from the positive (maximum signal control) and negative controls (minimum signal control) to give percentage of activities left. The data were then fit to a dose response equation using the program Helios (Novartis) to get the IC50 values. Helios is a Novartis in-house assay data analysis software using the methods described by Normolle, D. P., Statistics in Medicine, 12:2025-2042 (1993); Formenko, I. et al, Computer Methods and Programs in Biomedicine, 82, 31-37 (2006); Sebaugh, J. L., Pharmaceutical Statistics, 10:128-134 (2011); Kelly, C. et al., Biometrics, 46(4):1071-1085 (1990); and Kahm, M. et al., Journal of Statistical Software, 33(7): (2010) (grofit: Fitting Biological Growth Curves with R, pages 1-21, available at http://www.jstatsoft.org/).Each compound was counterscreened to determine if it interfered with the AlphaScreen beads. Compounds were diluted as described in the preceding section, and the assay was performed by adding 12 μL of 10 nM biotin-miniPEG-His6 peptide in the above buffer and incubating for 20 min at rt prior to addition of the beads to 10 μg/mL each. The plates were then incubated for 1 h at rt in dark before being read on EnVison.
Measurement: IC50 = 10.7 nM (pChEMBL 7.96999979019165)
Descriptors: MW 433.37, ALogP 3.23, HBA 8, HBD 1, PSA 82.16, QED 0.5
Compound (SMILES): CC(C)(C)OC(=O)N1CCCC(n2nc(-c3ccc(Oc4cccc(OCc5cncs5)c4)cc3)c3c(N)ncnc32)C1
InChIKey: MFUFNTDKMOXQEA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Fluorescence Polarization-Based Kinase Assay: Fluorescence polarization-based kinase assays were performed in 384 well-plate format using histidine tagged recombinant human full-length Bruton Agammaglobulinemia Tyrosine Kinase (Btk) and a modified protocol of the KinEASE FP Fluorescein Green Assay supplied from Millipore. Kinase reaction were performed at room temperature for 60 minutes in presence of 250 μM substrate, 10 μM ATP and variable test article concentrations. The reaction was stopped with EDTA/kinase detection reagents and the polarization measured on a Tecan 500 instrument. From the dose-response curve obtained, the IC50 was calculated using Graph Pad Prisms® using a non linear fit curve.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 599.72, ALogP 6.48, HBA 11, HBD 1, PSA 130.51, QED 0.22
Compound (SMILES): CN1C(=N)N[C@](C)(c2cc(NC(=O)c3ccn(C(F)F)n3)ccc2F)C2(CC2)S1(=O)=O
InChIKey: UIDKDNIVBINBTH-QGZVFWFLSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 456.45, ALogP 2.22, HBA 6, HBD 3, PSA 120.18, QED 0.65
Compound (SMILES): C[C@H](Nc1nccc([S@+](C)[O-])n1)c1cc2cc(Cl)ccc2[nH]c1=O
InChIKey: AXXSHNHVVCXWHF-VHMAXNEBSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: IDH1-R132H and IDH1-R132C Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 M Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 362.84, ALogP 2.88, HBA 5, HBD 2, PSA 93.73, QED 0.55