lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC1(S(=O)(=O)NC(=O)c2ccccc2-c2ccc3c(c2)OC[C@@H](Cc2coc(-c4ccccc4)n2)[C@@H]3O)CC1
InChIKey: MOTSXHJJBPRMGF-ZBLYBZFDSA-N
Target: Leukotriene B4 receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15722
Assay: BLT-1 cAMP Assay: The ability of compounds to antagonize the human BLT1 receptor was determined using a kit to measure changes in intracellular cyclic AMP levels (cAMP dynamic assay kit, Cisbio Cat. No. 62AM4PEC). HEK293 cells recombinantly expressing human BLT1 receptor, previously frozen in Recovery Medium (Life Technologies, Cat. No. 12648-010) were thawed and diluted into assay medium (HBSS (Hyclone SH 30268.01), 20 mM HEPES (Gibco 15630-106), 800 μM IBMX (Sigma I5879), 0.1% DTPA BSA (Perkin Elmer CR84-100)). The cell suspension was centrifuged at 200×g for 10 min and then resuspended in fresh assay medium to a density of 2.5×105 cells/mL. A Labcyte Echo 550 acoustic dispenser was used to transfer 25 nL of test compound dissolved in DMSO into the wells of a dry 384-well plate (Greiner 784075). All subsequent liquid additions were performed using a BIORAPTR (FRD; Beckman Coulter). Next, 5 μL of cell suspension was added and incubated for 20 min. at 37° C. and 5% CO2 in a humidified plastic tray. To test for agonist activity 51 μL of assay buffer containing forskolin (Sigma F-6886. 4 μM for BLT1) was added and incubated for 30 minutes at 37° C. and 5% CO2 in a humidified plastic tray. To test for antagonist activity 5 μL of assay buffer containing forskolin (Sigma F-6886. 4 μM for BLT1) and either LTB4 (Sigma Aldrich L0517; 2 nM BLT1) was added and incubated for 30 minutes at 37° C. and 5% CO2 in a humidified plastic tray.The levels of cAMP were detected using the CisBio kit following the manufacturer's instructions: cAMP-d2 vial was reconstituted with distilled water and then diluted accordingly with conjugate & lysis buffer. 5 μL of cAMP-d2 working solution was added to all of the wells of the assay plate. cAMP-Cryptate vial was reconstituted with distilled water and then diluted accordingly with conjugate & lysis buffer. 5 μL of cAMP-Cryptate working solution were added to the assay plate. The assay plate was shaken for 3 minutes and incubated at room temperature for 45 minutes, then read on Perkin Elmer Envision. cAMP standard curve and fit data were plotted using a 4 parameter dose response curve fitting algorithm to fit curve. A 4-parameter curve fit (Max, min, log EC50 and slope) was used to transform fluorescent 665 nm/615 nm ratio signal to cAMP concentration. After normalization to treated and untreated controls, the percent effect of signal at each compound concentration was calculated. The plot of percent effect versus the log of compound concentration was fit with a 4-parameter concentration response equation to calculate EC50 values. Compound concentrations tested were 10 000, 3 333, 1 111, 370.4, 123.4, 41.2, 13.7, 4.6, 1.5 and 0.5 nM with 0.25% residual DMSO.
Measurement: EC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 544.63, ALogP 4.91, HBA 7, HBD 2, PSA 118.73, QED 0.34
Compound (SMILES): C[C@@H]1CN(Cc2cnc3c(c2)c2ncnn2c(=O)n3Cc2ccc(OC(F)(F)F)cc2)C[C@H](C)O1
InChIKey: GRHRWDJPZQUGNO-GASCZTMLSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: PDE1B Inhibitory Assay: PDE1B inhibition was determined by an IMAP TR-FRET assay. The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, Calmodulin, cAMP or cGMP substrate, DMSO tolerance, and incubation time. Into each well of a solid white 1536 well plate (Corning) was dispensed 250 pg full-length recombinant NH-terminal GST tagged human PDE1B enzyme (BPS Bioscience Cat #60011, San Diego, Calif.) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 10 U/ml Calmodulin and 2.5 mM CaCl2 (Sigma Aldrich.) After a brief centrifugation, 30 nl compound was added by transfer from 1 mM stock in DMSO using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 533 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 200 nM. After a brief centrifugation, the plates were incubated for 30 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer). The instrument was set to excite using the DUG11 filter and measure using 490/10 nm and 520/10 nm filters. Ratios of acceptor and donor were then calculated.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 488.47, ALogP 3, HBA 9, HBD 0, PSA 86.78, QED 0.43
Compound (SMILES): C[C@@H]1CN(S(=O)(=O)CCCN)CC[C@@H]1NC(=O)c1cc(C2CC2)on1
InChIKey: LRMHFTKQCYRREO-YPMHNXCESA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 370.48, ALogP 0.67, HBA 6, HBD 2, PSA 118.53, QED 0.73
Compound (SMILES): CC(C)(C)c1ccc(NC(=O)N[C@@H]2CCCN(c3nnc(C(N)=O)c(Nc4ccc(C(=O)N5CCOCC5)cc4)n3)C2)cc1
InChIKey: GIBGWAMSRVOAKH-XMMPIXPASA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk In Vitro Inhibitory Assay: Human Btk kinase (Genbank accession # NP_000052) was purified from insect cells as a full-length construct containing a N-terminal 6×-His tag. Btk kinase activity was determined using a radiometric filter binding assay. Measurements are performed in a low μL reaction volume 384-well assay plates. BTK enzyme (8 nM final in reaction), inhibitor (at requested doses), and 0.2 mg/mL peptide substrate (Poly-Glu-Tyr, 4:1 ratio) are incubated in a reaction buffer composed of 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO for 15 min. followed by addition of 1 μM ATP to start the assay. Kinase reactions are carried out for 120 min. at room temperature. The reaction was stopped by spotting of reaction sample onto P81 cationic exchange paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% Phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme (via addition of saturating EDTA), kinase activity data for each dose of compound tested was expressed as the percent of remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 601.71, ALogP 3.27, HBA 9, HBD 4, PSA 167.7, QED 0.32
Compound (SMILES): OCC1CN(c2cnc3ccc(N4CCC[C@@H]4c4cc(F)ccc4F)nn23)C1
InChIKey: XGMUKTARXHHSOG-QGZVFWFLSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Omnia Kinase Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (either TrkA or TrkB from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog #267462). Omnia Tyr Peptide #4 (for TrkA) or #5 (for TrkB), as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP for TrkA assay or 1 mM ATP for TrkB assay, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were then calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 385.42, ALogP 2.78, HBA 6, HBD 1, PSA 56.9, QED 0.75
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn(C[C@H](O)[C@H](C)O)c3)cn3nccc23)cn1
InChIKey: RLBJLTMGMDYGHH-XOBRGWDASA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Tyk2 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit Tyk2 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 8 μM Omnia Y12 peptide (Catalog # IVGN KPZ3121C; Invitrogen Corporation, Carlsbad, Calif.) and 1 nM Tyk2 in a total volume of 25 μL. Human Tyk2 kinase domain, comprising amino acids 886 to 1187 with 10 additional histidine residues (histidine tag) on the carboxy terminus, was expressed and purified from bacculovirus in-house at Array BioPharma Inc. (Boulder, Colo.). The histidine tag was cleaved after purification using standard conditions.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 409.49, ALogP 2.56, HBA 9, HBD 2, PSA 106.29, QED 0.46
Compound (SMILES): COCCN1CC2(CCN(c3cnc(C#N)c(-c4cnn(C)c4)n3)CC2)N(CC2CC2)C1=O
InChIKey: HNAWXNUUWXTXGE-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Biochemical Assay: The PI3-Kinase biochemical assay was optimized using the HTRF kit provided by Upstate (Millipore). The assay kit contains six reagents: 1) 4×Reaction Buffer; 2) native PIP2 (substrate); 3) Stop (EDTA); 4) Detection Mix A (Streptavidin-APC); 5) Detection Mix B (Eu-labeled Anti-GST plus GST-tagged PH-domain): 6) Detection Mix C. In addition, the following items were obtained or purchased; PI3Kinase (catalog #'s alpha 14-602, beta 14-603, gamma 14-558 and delta 14-604 from Upstate Biotechnology now EMD Millipore, Mahopac, N.Y., USA), dithiothreitol (Sigma-Aldrich Corp. (St. Louis, Mo., USA) Catalog #D-5545), Adenosine-5′ triphosphate (InVitrogen (Waltham, Mass., USA), Cat#AS001 Å), native PIP3 (PI(3,4,5)P3, diC8, H+, CELLSIGNALS, INC., (Columbus, Ohio, USA) Cat #907) DMSO ((Sigma-Aldrich Corp. (St. Louis. Mo., USA) Catalog #472301).PI3Kinase Reaction Buffer is prepared by dilution the stock 1:4 with de-ionized water. DTT (DL-Dithiothreitol), phosphatidylinositol 4,5-bisphosphate (PIP2) and Biotin-PIP3 were added to 1536 assay plate at a final concentration of 5 mM, 5 mM and 25 nM on the day of use. Enzyme addition and compound pre-incubation are initiated by the addition of 1.25 ul of PI3K (at twice its final concentration) in the 1× reaction buffer to all wells using a BioRaptor. Plates are incubated at room temperature for 15 minutes. Reactions are initiated by addition of 1.25 ul of 2× substrate solution (PIP2 and ATP in 1× reaction buffer) using BioRaptor. Plates are incubated in humidified chamber at room temperature for one hour. Reactions are quenched by addition of 0.625 uL of stop solution to all wells using the BioRaptor. The quenched reactions are then processed to detect product formation by adding 0.625 uL of Detection Solution to all wells using the BioRaptor (Detection mix C, Detection Mix A and Detection Mix B combined together in an 18:1:1 ratio prepared 2 hours prior to use). Following a one hour incubation in the dark, the HTRF signal is measured on the Envision plate reader set for 330 nm excitation and dual emission detection at 620 nM (Eu) and 665 nM (APC).
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 450.55, ALogP 1.88, HBA 8, HBD 0, PSA 103.41, QED 0.63
Compound (SMILES): CC(C)(C)[C@@H](N)C(=O)N1CC[C@H]1C(=O)NCc1ccc(C(=N)N)cc1
InChIKey: WAFJWVBMFWWNCB-KBPBESRZSA-N
Target: Mannan-binding lectin serine protease 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00187
Assay: Enzymatic Assay for MASP-2: The assay was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50 values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed., John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki = 6250 nM (pChEMBL 5.199999809265137)
Descriptors: MW 345.45, ALogP 0.56, HBA 4, HBD 4, PSA 125.3, QED 0.46
Compound (SMILES): O=c1cc(C2CCNCC2)n2ncc(-c3ccccn3)c2[nH]1
InChIKey: QEHPDHUFOPTPFK-UHFFFAOYSA-N
Target: Tissue-type plasminogen activator (SINGLE PROTEIN), Homo sapiens
UniProt: P00750
Assay: Plasma-based Clot Lysis Assay: The clot-lysis test system configures the kinetics of clot formation and degradation in vitro and allows quantifying modulation of the process by selected test compounds.The test compounds were dissolved in 1% acetic acid and further complemented with an equal volume of DMSO. The resulting stock solutions were serially diluted in 0.5% acetic acid/50% DMSO. 1 μL aliquots of these solutions were placed into 384 well microplates (Greiner, black, transparent bottom), followed by 30 μL of diluted human citrated plasma (platelet-poor, final concentration: 5%; supplemented with fibrinogen, final concentration: 3 μM; dilution buffer: 20 mM HEPES, 150 mM NaCl, 0.01% Brij (pH 7)). The reactions were started by addition of 20 μL of CaCl2 (final concentration: 10 mM), and tPA (tissue plasminogen activator, final concentration: 0.2 nM) in dilution buffer, followed by an additional volume of 20 μL dilution buffer for improved mixing. The reactions were incubated at 37° C. Clot formation and degradation was monitored spectrophotometrically by kinetic optical density measurements at 405 nm. IC50 values were determined by comparing the resulting time courses with the time course of a blank control reaction.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 295.35, ALogP 1.55, HBA 5, HBD 2, PSA 75.08, QED 0.75
Compound (SMILES): CS(=O)(=O)n1ccc2c(-c3cccc(CN)c3)cc(COc3ccccc3CC(=O)O)cc21
InChIKey: MFRHJNYEZCPWOE-UHFFFAOYSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Esterolytic Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399.) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 464.54, ALogP 3.78, HBA 6, HBD 2, PSA 111.62, QED 0.41
Compound (SMILES): Cn1nc(C(C)(C)NC(=O)c2cccc3c2OCCNC3)c2cccc(F)c21
InChIKey: ABDNZFSCDFEQDP-UHFFFAOYSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: cAMP ASSAY: The activation of the SSTR4 receptor (Gi coupled) causes an inhibition of intracellular cAMP after stimulation with Forskolin, which can be quantifiable by use of a suitable assay Kit and an adequate plate reader. This technique is used to characterize pharmacological effects of the SSTR4 receptor agonists by use of hSSTR4 expressing H4 cells.Compounds are dissolved and diluted in DMSO. The final test solution contains 1 DMSO. The cAMP standard (Lance cAMP 384 Kit; PerkinElmer, Cat #AD0264) is prepared in assay buffer (HBSS with 0.1% BSA, 5 mM HEPES, 0.5 M IBMX, pH 7.4) containing 1% DMSO and the cAMP standard curve is included at least on one plate. Cells are centrifuged and suspended in assay buffer (incl. 1:100 diluted Alexa antibody).For the assay 5 μl of a cell suspension (approximately 5000 cells/well) incl. Alexa antibody (diluted 1:100) are added into a 384 well MTP microtitre plate excepting one row or column (depending on the plate layout), which is reserved for the standard curve. Then 2 μl of compound sample is added as concentration response curve (e.g. 1e-5 M to 6e-10 M), usually in triplicates. Each assay contains incubations with vehicle controls instead of compound as controls for non-inhibited cAMP generation (100% CTL; high values) and incubations with 1 μM Somatosatin as controls for full inhibition and background (0% CTL; low values). After approximately 10-15 min incubation time 3 μl Forskolin (dissolved in DMSO, final conc. 15 μM) is added. Then the plates are shaken briefly and incubated for 60 min at room temperature. After 60 min 10 μl of the detection mix is added into all wells followed by an additional incubation period of 1 h. The plates are read in a suitable plate reader.The analysis of the data is based on the ratio of the time-resolved fluorescence measurements of donor and acceptor fluorophore (Ex: 320 nm; Em1: 665 nm; Em2: 615 nm; ratio 665/615). From this ratio, cAMP concentrations are calculated from standard curve and the EC50 is estimated by least square curve fit program.
Measurement: EC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 382.44, ALogP 2.86, HBA 5, HBD 2, PSA 68.18, QED 0.73
Compound (SMILES): C[C@@H]1C[C@H](NC(=O)c2cc(C3CC3)on2)CCN1S(=O)(=O)c1c[nH]cn1
InChIKey: GHIBULXPNZLKHG-ZYHUDNBSSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 = 732 nM (pChEMBL 6.130000114440918)
Descriptors: MW 379.44, ALogP 1.25, HBA 6, HBD 2, PSA 121.19, QED 0.81
Compound (SMILES): NCCN(Cc1cccc(Cl)c1)C(=S)Nc1ccc2cnccc2c1
InChIKey: BEBPKEULDKOBRT-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: ELISA Assay: ROCK Substrate Coated Plate (Part No 241601): One strip well 96-well plate pre-coated with recombinant MYPT1.Buffer: 25 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM Glycerol-2-Phosphate, 0.1 mM Na3VO4; 1% DMSO; 2.5 mM DTT; (Enzyme: ROCK active-II) (Cell Biolabs, Catalog #STA-406, Part No. 241505) 0.1 ng/μl.ATP Solution (Part No. 241604): 100 mM ATP. Final concentration of ATP in reaction mixture: 250 μM.Anti-phospho-MYPT1 (Thr696) (Part No. 241603).Secondary Antibody, HRP Conjugate (Part No. 231003).Biotinylated substrate, diluted to 0.25 μM with buffer described above (without ATP).Steps:1. Purified kinase or cell lysate sample can be used directly in the kinase assay or further diluted with 1× Kinase Buffer. Each sample should be assayed in duplicate.2. Add 90 μL of the diluted active ROCK-II positive control or unknown ROCK samples to the wells of the substrate plate.3. Initiate the kinase reaction by adding 10 μL of the 10× Kinase Reaction Buffer containing DTT and ATP. Mix well.4. Cover with a plate cover and incubate the wells at 30° C. for 30-60 minutes with gentle agitation.5. Stop kinase reaction by flicking out the content or by adding 50 μL of 0.5M EDTA, pH 8.0, to each well.6. Remove the plate cover and empty wells. Wash microwell strips 3 times with 250 μL 1× Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1× Wash Buffer.7. Add 100 μL of the diluted anti-phospho-MYPT1 (Thr696) antibody to each well.8. Cover with the plate cover and incubate at room temperature for 1 hour on an orbital shaker.9. Remove the plate cover and empty wells. Wash the strip wells 3 times according to step 6 above.10. Add 100 μL of the diluted HRP-conjugated secondary antibody to each well.11. Cover with the plate cover and incubate at room temperature for 1 hour on an orbital shaker.12. Remove the plate cover and empty wells. Wash microwell strips 3 times according to step 6 above. Proceed immediately to the next step.13. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature for 5-20 minutes on an orbital shaker.14. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).15. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 370.91, ALogP 4.05, HBA 3, HBD 2, PSA 54.18, QED 0.66
Compound (SMILES): O=C(Cn1cc(-c2cnc(NC3Cc4ccccc4C3)nc2)c(O)n1)N1CCc2[nH]nnc2C1
InChIKey: MGGVALXERJRIRO-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Inhibitory Activity Assay: Two-fold dilution of each test compound solution (10 μM, 100% dimethyl sulfoxide) is carried out on 96-well V bottom plate (Costar 3363). After ten-fold dilution of each the test compound solution (100% dimethyl sulfoxide) with deionized distilled water, 10 μL of the diluted each mpound solution (10% dimethyl sulfoxide) was aliquoted to a black flat bottom 96-well plate (Costar 3915). 50 μL of 1.6× Assay solution (224 mM NaCl, 80 mM Tris-HCl (pH 8.0), 8 mM KCl, 1.6 mM CaCl2, 1.6 mM MgCl2, and 1.6 mg/mL fatty acid free BSA) was added thereto, and subsequently 20 μL of 20 nM human ENPP2 solution (buffer solution: 140 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mg/mL fatty acid free BSA) and 20 μL of 5 μM FS-3 solution (buffer solution:deionized distilled water) were added thereto, respectively, followed by mixing. Under reaction for 30 minutes at 37° C., fluorescence intensity measurement (Ex: 485 mm, Em: 528 mm) was carried out at every 5 minutes by Envision Xcite Multilabel Reader. ΔCFU30 min value (CFU value measured at 30 minutes−CFU value measured at 0 minute) is obtained for each test solution and based on the equation of 100−(ΔCFU30 min of test solution/Mean value of ΔCFU30 min of control group)×100, the inhibitory activity percentage ratio (i.e., % inhibition) is obtained
Measurement: IC50 < 1.56 nM (pChEMBL 8.807000160217285)
Descriptors: MW 457.5, ALogP 1.33, HBA 9, HBD 3, PSA 137.74, QED 0.41
Compound (SMILES): CC(=O)CCc1oc2ccc(Br)cc2c1CCc1ccc(C)cc1
InChIKey: SZICHZCHTJLSOO-UHFFFAOYSA-N
Target: Microtubule-associated protein tau (SINGLE PROTEIN), Homo sapiens
UniProt: P10636
Assay: AlphaLisa Assay: Tau target (equal mixture of each construct at 300 nM) was prepared in buffer (Tris-HCl pH 7.4) and was incubated in 96-well plates at room temperature for 4 hours with vehicle control (DMSO) and a dose range of a compound of the disclosure (0.098 uM-50 uM). AlphaLisa acceptor beads & donor beads with ligands to the epitope tags (Perkin Elmer) were diluted (final 20 ug/mL per bead) in bead buffer (25 mM HEPES pH 7.4, 100 mM NaCl, 0.1% Tween-20), added to the wells and incubated 1 hour at room temperature. The positive control (non-incubated target) & blank (no tau) were prepared and mixed with beads in bead buffer and the plate was read immediately using the EnVision plate reader (Perkin Elmer).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 385.3, ALogP 5.81, HBA 2, HBD 0, PSA 30.21, QED 0.54
Compound (SMILES): Cc1nc2c(-c3nnc(N)[nH]3)cc(N3CCOCC3)cc2n1-c1ccnc2c(F)ccc(F)c12
InChIKey: BWCYDCFMOJSYCC-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET PI3K Beta Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 μM, and PI3Kγ at 2 nM. The compounds were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).The results were normalized based on positive (1 μM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ, and γ were calculated from the fit of the dose-response curves to a four-parameter equation. These assays generally produced results within 3-fold of the reported mean. As shown in Table 1, the compounds provided herein are inhibitors of PI3Kβ.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 462.46, ALogP 3.36, HBA 8, HBD 2, PSA 110.77, QED 0.42
Compound (SMILES): CCNC(=O)c1noc(-c2cc(C(C)C)c(O)cc2O)c1-c1ccc(CNCCN(CC)CC)cc1
InChIKey: RUTBSCTYOKEKBU-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Malachite Green ATPase assay: Chemicals are of the highest purity commercially available and all aqueous solutions are made up in AR water. Because of the need to minimise contamination with inorganic phosphate, precautions should be taken with solutions and apparatus used in the assays. Glassware and pH meters are rinsed with double distilled or deionised water before use and, wherever possible, plastic ware should be used.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 494.64, ALogP 4.72, HBA 7, HBD 4, PSA 110.86, QED 0.27
Compound (SMILES): C=CC(=O)N1CC2(CC(n3nc(-c4ccc(N)nc4)c(-c4c(Cl)c(C)cc5[nH]ncc45)c3C)C2)C1
InChIKey: NJSYGOZOJMGADH-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Scintillation Proximity Assay (SPA): Assays were run using 384-well plates (781207/Greiner) in which one column was designated as the high signal (no inhibition) control, and contained DMSO with no test compound, and another column was designated as the low signal control (maximal inhibition), and contained no protein. Serial dilutions of compounds to be tested were added to the assay plate (resulting in duplicate 11-point dose response with semi-log compound dilutions from 50 μM to 0.5 nM or from 5 μM to 0.05 nM for the most potent compounds). A 1/20 isotopic dilution of labeled (Compound B) and non-labeled covalent (Compound A) probe was prepared and added to all wells on the plate. The reaction was started by addition of KRasG12C (M1-K169, biotinylated on the N-terminus) to the compounds and incubated for 2 hours with continuous agitation allowing for full modification of KRasG12C with probe or test compounds. Final concentrations in an assay volume of 40 μL were 10 nM KRasG12C, 25 nM radio-ligand and 475 nM unlabelled ligand. The assay buffer contained 20 mM Tris-HCl pH 7.5 (Invitrogen), 150 mM NaCl (Sigma Aldrich), 0.1 mM MgCl2 (Sigma Aldrich), and 0.01% Tween-20 (Sigma Aldrich). Following addition of 50 μL of a 400 μg/ml. suspension of streptavidin-coated YSi beads (Perkin Elmer), plates were incubated for a further 30 min with continuous agitation before reading the plates on a scintillation counter (Topcount NXT 384 (Packard).
Measurement: IC50 = 92 nM (pChEMBL 7.039999961853027)
Descriptors: MW 488, ALogP 4.69, HBA 6, HBD 2, PSA 105.72, QED 0.41
Compound (SMILES): CC[C@@H]1[C@H](C(=O)Nc2cc3cc(-c4cncc(N)c4C)c(F)c(N)c3cn2)[C@H]1c1cncn1C
InChIKey: QBOGUDWLMQAKJK-CUJSYCCXSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1-FL HTRF Enzymatic Assay: To a 384 well Proxiplate with 80 nL compound or DMSO spotted on was added 4 μl/well kinase mix. The mixture was preincubated for 30 minutes and then 4 μl/well substrate mix was added. The solution was incubated for 60 min and then 4 μl/well detection mix was added. The solution was incubated for another 60 min. The plates were then loaded onto a Perkin Elmer Envision and the TR-FRET signal was measured at 615 and 665 nm. A ratio of 665/620 was used to calculate the % activity at each concentration of compound.
Measurement: Ki = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 459.53, ALogP 4.02, HBA 7, HBD 3, PSA 124.74, QED 0.39
Compound (SMILES): Cn1ccc(NC(=O)c2cc3ccc(-c4nn(C(C)(C)C)c5ncnc(N)c45)cc3[nH]2)n1
InChIKey: SIPNZNLJPIXBEH-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET and KDR Enzyme Assay: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer was used for the KDR biochemical assay with the addition of 2 mM MnCl2.Inhibitors were pre-incubated in the plate for 15 mins with 5 μL kinase and assay buffer at the following concentrations; 13 pM RET (Carna Biosciences; 08-159) and 150 pM KDR (Millipore; 14-630). The reaction was initiated by the addition of 5 μL ATP and substrate at 2× final reaction concentrations. For RET, this was 18 μM and 2 μM; for KDR, this was 16 μM and 1 μM, respectively. Reactions were performed at ATP Km for each target. The assay was allowed to proceed at room temperature for 20 mins before terminating with the addition of 10 μL HTRF detection buffer containing EDTA supplemented with TK-antibody labelled with Eu3+-Cryptate (1:100 dilution) and streptavidin-XL665 (128 nM). Following incubation at room temperature for 1 hour, FRET signal was measured using the Pherastar FS Microplate Reader.
Measurement: IC50 = 17.6 nM (pChEMBL 7.75)
Descriptors: MW 429.49, ALogP 3.3, HBA 8, HBD 3, PSA 132.33, QED 0.4
Compound (SMILES): O=c1cc(C2CCNCC2)n2ncc(-c3cccnc3)c2[nH]1
InChIKey: QWGJWIMGFBFDNZ-UHFFFAOYSA-N
Target: Tissue-type plasminogen activator (SINGLE PROTEIN), Homo sapiens
UniProt: P00750
Assay: Plasma-based Clot Lysis Assay: The clot-lysis test system configures the kinetics of clot formation and degradation in vitro and allows quantifying modulation of the process by selected test compounds.The test compounds were dissolved in 1% acetic acid and further complemented with an equal volume of DMSO. The resulting stock solutions were serially diluted in 0.5% acetic acid/50% DMSO. 1 μL aliquots of these solutions were placed into 384 well microplates (Greiner, black, transparent bottom), followed by 30 μL of diluted human citrated plasma (platelet-poor, final concentration: 5%; supplemented with fibrinogen, final concentration: 3 μM; dilution buffer: 20 mM HEPES, 150 mM NaCl, 0.01% Brij (pH 7)). The reactions were started by addition of 20 μL of CaCl2 (final concentration: 10 mM), and tPA (tissue plasminogen activator, final concentration: 0.2 nM) in dilution buffer, followed by an additional volume of 20 μL dilution buffer for improved mixing. The reactions were incubated at 37° C. Clot formation and degradation was monitored spectrophotometrically by kinetic optical density measurements at 405 nm. IC50 values were determined by comparing the resulting time courses with the time course of a blank control reaction.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 295.35, ALogP 1.55, HBA 5, HBD 2, PSA 75.08, QED 0.75
Compound (SMILES): Cc1nc(C(=O)N2CCC[C@@H](C)[C@H]2COc2nc3ccccc3o2)c(-c2ccc(F)cc2)s1
InChIKey: GWXZQMCHFMLSDI-FOIQADDNSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 465.55, ALogP 5.72, HBA 6, HBD 0, PSA 68.46, QED 0.38
Compound (SMILES): Cc1sc2c(-c3cc(Cl)ccc3OCCn3c(C)nc4cc(C(F)(F)F)c(N5CCN(C)CC5=O)c(C#N)c4c3=O)ccnc2c1C(=O)O
InChIKey: WJOMFZNHQGHNHK-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 = 28.1 nM (pChEMBL 7.550000190734863)
Descriptors: MW 711.12, ALogP 5.89, HBA 10, HBD 1, PSA 141.65, QED 0.22
Compound (SMILES): CS(=O)(=O)c1ccc(-c2cccc(Oc3cnn[nH]3)c2)cc1
InChIKey: WAGIHJSOVWCEPR-UHFFFAOYSA-N
Target: 2-Hydroxyacid oxidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UJM8
Assay: In-Vitro Human Glycolate Oxidase (hGOX) Assay: The in-vitro glycolate oxidase assay was performed using recombinant full-length human hydroxyacid oxidase 1 (HAO1), the equivalent of hGOX. The enzyme was obtained from AbCam (Catalog #ab113144) and was purified using conventional chromatography to >95% purity. Purified HAO1 was dissolved in assay buffer consisting of 10 mM NaCl, 110 mM KCl, 2 mM MgCl2, 50 mM HEPES (pH 7.4) and 0.01% Triton X-100. The assay used Corning 3575 384-well flat bottom, low flange, non-binding surface, black polystyrene plates.To determine HAO1 inhibition, 2 μL of compound concentrations in 100% DMSO were added to 28 μL of 36 nM HAO1 and incubated at room temperature for 10 min. Subsequently 40 μL of 100 μM Amplex red/0.2 U/mL horseradish peroxidase (HRP) was added followed by 10 μL of 680 μM glycolate (pH 7.4). Thus, final concentrations in the 80 μL reaction well were 12.5 nM HAO1, 50 μM Amplex Red, 0.1 U/mL HRP, and 85 μM glycolate in a final concentration of 2.5% DMSO. The fluorescence signal was measured every 53 seconds using a GENios microplate reader (Tecan) with Ex=530±10 nm and Em=585±10 nm. Linear data were plotted to calculate the reaction velocity values.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 315.35, ALogP 2.67, HBA 5, HBD 1, PSA 84.94, QED 0.8
Compound (SMILES): CN1CCC(N(C)C(=O)c2ccc(Nc3nc4c(N5CCN(C(=O)N6CC(C)(C)C6)CC5)cccn4n3)cc2)CC1
InChIKey: BSSLLFIVRHOGBI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 18.7 nM (pChEMBL 7.730000019073486)
Descriptors: MW 559.72, ALogP 3.22, HBA 8, HBD 1, PSA 92.56, QED 0.51
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(C#CC3CC3)cc1C21N=C(SCC2CCN(C)C2)N(CC2CCN(C)C2)C1=O
InChIKey: GNDPHSGXXUSCGI-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Assay: For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 μl OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 μl water containing the desired concentration of test compound and 1% DMSO, 1 μM substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 μl in a 384 well plate. In general, 25 μl of compound dilution were given to the plate followed by the addition of 10 μl of BACE containing OptiMEM diluted 1:2 in water with 0.2% Triton X-100. The reaction was started with the addition of 15 μl substrate in NaOAc buffer. The reaction was incubated at 30° C. in a fluorimeter and the cleavage of the substrate was recorded as kinetic for 60 min. at ex: 530 nm, em: 590 nm. Blank wells containing either no inhibitor or no enzyme were included on each plate.The intensity of fluorescence was regressed against time in order to derive velocities of reaction in all 384 wells. These velocities were used for calculating percent inhibition using an uninhibited control containing 1% DMSO and a fully inhibited control incubations performed in the absence of enzyme. IC50 values were calculated by fitting percent inhibition vs. inhibitor concentration using standard software like GraphPadPrism.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 574.84, ALogP 4.61, HBA 6, HBD 0, PSA 48.38, QED 0.48
Compound (SMILES): N#CCC1(N2CC=C(c3cc(NC(=O)C4CC4)nc4[nH]ccc34)CC2)CN(C2CCNCC2)C1
InChIKey: XCSLPZCFYOUZSD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 > 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 459.6, ALogP 2.72, HBA 6, HBD 3, PSA 100.08, QED 0.61
Compound (SMILES): COc1c(Nc2cc(NC(=O)C3CC3)nc3[nH]cnc23)cccc1-c1ncn(C)n1
InChIKey: GACNNAYACADTTF-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Binding Assay: Binding constants for compounds of the present invention against the JH2 domain were determined by the following protocol for a KINOMEscan® assay (DiscoveRx). A fusion protein of a partial length construct of human TYK2 (JH2domain-pseudokinase) (amino acids G556 to D888 based on reference sequence NP_003322.3) and the DNA binding domain of NFkB was expressed in transiently transfected HEK293 cells. From these HEK 293 cells, extracts were prepared in M-PER extraction buffer (Pierce) in the presence of Protease Inhibitor Cocktail Complete (Roche) and Phosphatase Inhibitor Cocktail Set II (Merck) per manufacturers' instructions. The TYK2 (JH2domain-pseudokinase) fusion protein was labeled with a chimeric double-stranded DNA tag containing the NFkB binding site (5′-GGGAATTCCC-3′) fused to an amplicon for qPCR readout, which was added directly to the expression extract (the final concentration of DNA-tag in the binding reaction is 0.1 nM).
Measurement: Kd = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 404.43, ALogP 2.85, HBA 8, HBD 3, PSA 122.64, QED 0.45