lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCC(C)NC(=O)c1n[nH]c2ccc(-c3cccnc3)cc12
InChIKey: HDURSVVEJSZELF-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: DYRK1A activity assay: SH-SY5Y cells are cultured in DMEM/F-12 medium supplemented with 15% FBS, Non-essential Amino Acid and Penicillin/Streptamycin. Two days before treatment, cells are seeded onto 96 well plates at 20e5 cells/well.DMSO-resuspended compounds are dispensed to 8 wells as a serial titration from 10 μM to 4.6 nM final and cells are exposed overnight (16-18 h) before harvest. Wells are visualized checked for cell death or change in morphology and supernatants are tested for cytotoxicity by measurement of lactate dehydrogenase release (LDH, CytoToxOne kit, Progema) if necessary. As controls, commercially available DYRK1A inhibitors, Harmine and Indy were shown to have good DYRK1A inhibition in the kinase assay with no CDK1 activity (EC50 18 and 53n M respectively, 6 μM for CDK1) but weak EC50 in the Tau assay: about 10 μM for Harmine and 30 μM for Indy.Cells are lyzed with RIPA buffer complemented with phosphatase and protease inhibitors (Thermo Scientific) then lysates are sonicated and spun down at 12,000 g for 10 min to remove any cellular debris. Lysates are then either directly tested for pSer396 by ELISA (Life Technology, Kit KHB7031) or loaded on NuPage Bis-Tris gels for western blot analysis. Colorimetric detection of ELISA signal is performed by Cytation3 plate reader (Biotek) and the chemoluminecence signal for HRP-linked antibodies used in western blotting is detected using a Carestream Image Station. The same pSer396 antibody is used for detection of pTau in both assays.Blot densitometry for pSer396 and beta-actin are analyzed using ImageJ (NIH) and pSer396/Total Tau ELISA signal was used to plot, draw the curve fitting, and determine each compounds EC50 in Prism (GraphPad).
Measurement: EC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 294.36, ALogP 3.15, HBA 3, HBD 2, PSA 70.67, QED 0.78
Compound (SMILES): O=C1NC(=O)[C@@H](C2CCC(COCCC(F)(F)F)CC2)C2c3cc[nH]c3N=CC12
InChIKey: UHMAIQLCRXRLIU-PGTPHSSJSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzyme Assay: The JAK inhibitory activities of compounds of the present invention were measured.The enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As the substrate for the enzymes (hereinafter referred to as the substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Co., Ltd.) was used.As the antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Co., Ltd.) was used.The other reagents are purchased from the following suppliers.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIESMagnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for the assays after diluted with the assay buffer.The composition of the assay buffer is given below.HEPES (pH7.5): 50 mMEGTA: 1 mMMgCl2: 10 mMDTT: 2 mMTween 20: 0.01% (wt/wt)Dilutions were made at such concentrations and dispensed on a well plate, which will be described later, in such volumes that the following final concentrations would be achieved on the well plate. The enzyme concentrations and the ATP concentrations in the respective enzyme (JAK1, JAK2, JAK3 and Tyk2) assays were as follows.JAK1 enzyme assay; the enzyme concentration was 0.5 μg/mL, and the ATP concentration was 70 μM.JAK2 enzyme assay; the enzyme concentration was 0.013 μg/mL, and the ATP concentration was 10 μM.JAK3 enzyme assay; the enzyme concentration was 0.020 μg/mL, and the ATP concentration was 3 μM.Tyk2 enzyme assay; the enzyme concentration was 0.25 μg/mL and the ATP concentration was 20 μM.The concentration of the substrate for the enzymes was 25 nM.The concentration of EDTA was 15 mM.The concentration of PT66 was 2 nM.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 411.42, ALogP 3.48, HBA 4, HBD 2, PSA 83.55, QED 0.57
Compound (SMILES): C=CC(=O)N1CCN(C(CC)c2ccc([C@H](C)Nc3ncc4c(n3)N(CC)C(=O)OC4)cc2)CC1
InChIKey: BASWGFWYCDZRSM-HXBUSHRASA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Biochemical Assay: IDH1-R132H, IDH1-R132C, IDH2-R172K and IDH2-R140Q mutant enzymes catalyze the conversion of αKG to 2HG. 2HG is analyzed using in-line solid phase extraction and mass spectrometry. This analysis is carried out in a RapidFire® instrument coupled to a 6460 triple quadrupole mass spectrometer (G6460A Agilent).IDH1 mutant (R132H and R132C) and IDH2 mutant (R140Q and R172K) proteins containing N-terminal His-tag are expressed in E.coli and purified using nickel affinity chromatography. The enzyme assays are carried out in V-bottom 96 well polypropylene plates containing 100 mM Tris-HCl buffer, 1 mM DTT, 0.005% TRITON X-100, 120 mM NaCl. For IDH1 R132H, α-ketoglutarate, NADPH and MnCl2 are included at final concentrations of 300 μM, 2.5 μM and 300 μM respectively. For IDH1 R132C, α-ketoglutarate, NADPH and MnCl2 are included at final concentrations of 100 μM, 10 μM and 100 μM respectively. For IDH2 R172K, α-ketoglutarate, NADPH and MnCl2 are included at final concentrations of 150 μM, 10 μM and 150 μM respectively. For IDH2 R140Q, α-ketoglutarate, NADPH and MnCl2 are included at final concentrations of 3000 μM, 10 μM and 100 μM respectively. Final pH=7.0. Test compound dissolved in DMSO stock is diluted in the reaction mix at a final DMSO concentration of 4%. Compounds are tested in dose-response format. The assay is started by addition of enzyme. Enzymes are used at the following final concentrations: IDH1 R132H, 2 nM; IDH1 R132C, 0.5 nM; IDH2 R172K, 1.2 nM; IDH2 R140Q, 1.2 nM. After 90 minutes the reaction is quenched by adding ACN (50:50) containing 3-hydroxy-1,5-pentanedioic-2,2,3,4,4-d5 acid (5d5-3HG) as an internal standard for mass spectrometry analysis and quantitation of reaction product. 2-Hydroxyglutarate (2HG) in quenched samples is separated using strong anionic exchange column chromatography (Phenomenex Strata-X-A SecurityGuard) and analyzed by mass spectrometry in a 6460 triple quadrupole mass spectrometer (G6460A Agilent). The 2HG signal detected is transformed into an analyte concentration using a calibration curve generated using known 2HG concentrations. For each compound tested, the % inhibition is calculated using a DMSO control sample as 0% inhibition and a no enzyme control as 100% inhibition.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 478.6, ALogP 3.91, HBA 7, HBD 1, PSA 90.9, QED 0.58
Compound (SMILES): O=C1CN(CCOc2ccc(Nc3nc(N4CCCCCC4)nc4cn[nH]c(=O)c34)cc2)CCN1
InChIKey: ZLKXZPXDOZIMOT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzymatic JAK2 Kinase Activity: Two μL of 10% DMSO in blank, substrate control and positive control wells and 2 μL of test compound in test wells was added. Thirteen μL of assay buffer in blank and substrate control wells and 13 μL of Enzyme buffer mix in positive and test wells was added. The reaction mixture was incubated for 30 minutes at RT on a plate shaker. Ultra Light-pGT substrate (5 μL) [poly Glu-Tyr (4:1) labeled with U Light dye, a tyrosine kinase substrate] and ATP mix was added to all wells. The reaction plate was incubated for 60 minutes at RT on a plate shaker. The reaction was stopped by adding 40 mM EDTA (10 μL) in buffer. Ten μL of antibody was added to all the wells. The plate was read in a Wallac® 1420 Multilabel Counter Victor 3 instrument (Ex: 340 nm Em: 615 & 665 nm)
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 478.56, ALogP 1.65, HBA 9, HBD 3, PSA 128.37, QED 0.46
Compound (SMILES): CC(C)c1ncnc2[nH]cc(C(=O)c3c(F)ccc(NS(=O)(=O)c4ccc(C(F)(F)F)cc4)c3F)c12
InChIKey: CDCCJJDIKJZUGO-UHFFFAOYSA-N
Target: Cytochrome P450 2C19 (SINGLE PROTEIN), Homo sapiens
UniProt: P33261
Assay: Inhibition Assay: The inhibition of CYP enzyme activity (IC50) for each of CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4(BFC) and CYP3A4(BQ) is determined for compounds, where inhibition of metabolism of a known substrate leads to a decrease in the fluorescence of the metabolized product. The fluorescence of the product is monitored as a function of compound concentration. Compounds are dissolved in dimethyl sulfoxide to a concentration of 100 mM. These are diluted 1 μL into 82 μL of acetonitrile. An 11 μL aliquot of this solution is then added to 204 μL of cofactor mix (1.3% NADPH Regeneration system Solution A, 1.04% NADPH Regeneration system Solution B from BD Biosciences, 5% acetonitrile and 0.05% dimethyl sulfoxide). These are then serially diluted 1:1 (160 μL to 160 μL co-factor mix) for a total of 10 points. A 10 μL aliquot of this final mixture is dispensed into 384 well assay plates and incubated for 10 minutes at 37° C. Enzyme and substrate mix (10 μL; 0.5 pmol CYP1A2/5 μM CEC; 1.0 pmol CYP2C9/75 μM MFC; 0.5 pmol CYP2C19/25 μM CEC; 1.5 pmol CYP2D6/1.5 μM AMMC; 1.0 pmol CYP3A4/50 μM BFC; or 1.0 pmol CYP3A4/40 μM BQ) is added to these assay plates. Assay plates are incubated at 37° C. (CYP1A2-15 min; CYP2C9-45 min; CYP2C19, 2D6 and 3A4-30 min) and read in a Tecan Safire 2 plate reader (CYP1A2, 2C19 and 3A4 409 ex/460 em; CYP2C9 and 2D6 409 ex/530 em).
Measurement: IC50 = 7500 nM (pChEMBL 5.119999885559082)
Descriptors: MW 524.47, ALogP 5.41, HBA 5, HBD 2, PSA 104.81, QED 0.26
Compound (SMILES): COC(=O)N1c2ccc3c(nc(C(O)c4cccc5c4COC5)n3C3CCCC(C(=O)O)C3)c2CC[C@@H]1C
InChIKey: WBRLENDDMAMVNQ-SXWHCCASSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: HTRF Biochemical Assay for CBP and BRD4 Activity: The assay was performed in a final volume of 6 μL in assay buffer containing 50 mM Hepes (pH 7.5, (0.5M Hepes, pH 7.5 solution; Teknova H1575)), 0.5 mM GSH, 0.01% BGG (0.22 μM filtered, Sigma, G7516-25G), 0.005% BSA (0.22 μM filtered, EMD Millipore Corporation, 126575) and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 33 μM to 1.7 nM, top to lowest dose, respectively. 3 of 2× Protein and 3 μL of 2× Peptide Ligand were added to assay plates (pre-stamped with compound). Plates were incubated for varying times at room temperature prior to measuring the signal. TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) was measured on the PHERAstar (BMG, equipped with HTRF optic module [337/520/490]) or on the Envision (PerkinElmer, equipped with the TRF Laser unit, TRF dual mirror D400/D505 and emission filters M520 and M495). Data were reported as percent inhibition compared with control wells based on the following equation: % inh=1−((TR-FRET ratio−AveLow)/(AveHigh−AveLow)) where TR-FRET ratio=(Fluorescence at 520 nm/Fluorescence at 490 nm)*10000), AveLow=average TR-FRET ratio of no enzyme control (n=32), and AveHigh=average TR-FRET ratio of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm. For all assay formats data were reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured Fluorescence, AveLow=average Fluorescence of no enzyme control (n=32), and AveHigh=average Fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 519.6, ALogP 4.87, HBA 7, HBD 2, PSA 114.12, QED 0.51
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(-c4cn(C)nc4C)cc3N(CC3CCOCC3)C2=O)c1F
InChIKey: PAECKJXBNAKEJP-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 513.55, ALogP 4.46, HBA 7, HBD 0, PSA 81.95, QED 0.48
Compound (SMILES): C=CC(=O)Nc1cc(Nc2ncc(C(=O)OCC3CC3)c(-c3cn(C)c4ccccc34)n2)c(OC)cc1N(C)CCN(C)C
InChIKey: PFWGSFHALNIOLP-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: HER2 Exon 20 YVMA Insertion Mutation: A compound's ability in selectively inhibiting Her2 exon 20 YVMA insertion mutations can be assessed using Ba/F3 cells, a murine pro-B cell line, which have been transduced with Her2 Exon20 YVMA insertions. An expression vector, pLVX-IRES puro (Clontech) coding for human EGFR exon 20 insertions YVMA (A775_G776ins YVMA), was transfected into HEK293 cells by the Trans-Lentiviral ORF Packaging System (Thermo Scientific), to produce virus encoding EGFR exon 20 insertions. Ba/F3 (DSMZ) cells maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 200 μM L-glutamine/200 μg/mL penicillin/200 μg/mL streptomycin (Life Technology) and 10 ng/mL IL-3 (R&D system), were infected by EGFR Exon20 virus and subsequently selected by puromycin (Life Technology) selection and IL-3 depletion. Ba/F3 cells expressing Her2 Exon20 YVMA insertions (named Ba/F3-Her2 Exon20 YVMA) can proliferate in the absence of IL-3. The anti-proliferative activity of compounds was determined as follows: BaF3-Her2 Exon20 YVMA cells seeded in 96 well plates (2500 cells/well) were treated with test compound (dissolved in DMSO) at a series of concentrations (4-fold dilution, top concentration: 10,000 nM). The plates were incubated for 72 h in a 37° C. incubator with 5% CO2, and the number of viable cells in each well were measured indirectly by CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega; this assay is a colorimetric method for determining the number of viable cells through measurement of their metabolic activity by detection of enzymatic conversion of tetrazolium salts into blue formazan derivatives). Reagent (20 μL) was added into each well, and the plates were returned to the incubator for 2 h. The absorbance in each well was then measured at 490 nm using an Envision plate reader (Perkin Elmer). IC50 values were calculated by determining the concentration of compound required to decrease the MTS signal by 50% comparing to the DMSO control in best-fit curves using Microsoft XLfit software or Accelrys Pipeline Pilot.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 597.72, ALogP 5.08, HBA 10, HBD 2, PSA 113.85, QED 0.16
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(-c4cc(N)c(F)cc4C)cc3C3(CC3)C2=O)c1F
InChIKey: LWWCEHXQPQZJKB-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 469.46, ALogP 4.65, HBA 5, HBD 1, PSA 77.68, QED 0.56
Compound (SMILES): C[C@]1(O)CC[C@H](NC(=O)c2cc(-c3ccc([C@]45C[C@H]4CN(C4CCC(F)(F)CC4)C5)cc3)cnc2N)CC1
InChIKey: NZDLTZYLNOKWBF-VHRRBUMHSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: ADPGlo Assay: The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 6 ul. Dose-response curves were generated by incubating 10 nM of each kinase in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 μm Na3VO4, 10 mM β-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 and 15 μm ATP for 60 min at 32° C. in the presence or absence of compound diluted in DMSO. The amount of generated ADP is a measure of kinase activity and is quantified using the ADP-Glo™ Kinase Assay (Promega) according to manufacturer's instructions. ADP is converted to ATP by adding 3 ul of ADP-Glo™ Reagent and incubation at 32° C. for 60 min. ATP is subsequently converted into a bioluminescent signal by adding 6 ul luciferase assay reagents (Kinase detection buffer+Kinase Detection Substrate (Promega)) and further incubation at 32° C. for 60 min. For the measurement of luminescence a PHERAstar™ Multilabel Reader was used at a measurement interval time of 0.1 second (optical module for luminescence measurements in the 230 nm to 750 nm wavelength range).
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 524.66, ALogP 4.91, HBA 5, HBD 3, PSA 91.48, QED 0.52
Compound (SMILES): CCc1cc(O)c(F)cc1-c1cc2[nH]nc(C(=O)NC)c2c(NCC(C)C)n1
InChIKey: SLDNTHWIALNKOG-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 385.44, ALogP 3.46, HBA 5, HBD 4, PSA 102.93, QED 0.52
Compound (SMILES): CC(C(=O)Nc1cc(C(=O)Nc2ccc(-c3ccccc3F)nc2)ccc1OC(F)(F)F)N1CCOCC1
InChIKey: HFPJXXWOFQHZSR-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: HEK FOP Assay: HEK293 cells were cotransfected with the FOP control vector and pcDNA3. The FOP vector is identical to the TOP construct, but it contains instead of functional TCF elements a randomized, non-functional sequence
Measurement: IC50 = 7400 nM (pChEMBL 5.130000114440918)
Descriptors: MW 532.49, ALogP 4.7, HBA 6, HBD 2, PSA 92.79, QED 0.43
Compound (SMILES): COc1cc2nc(N)n3nc([C@@H]4CCCN(c5ncn(C(C)C(C)(C)O)n5)C4)nc3c2cc1F
InChIKey: XXTWQFUSENGCGM-ZGTCLIOFSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Measurement of A2b Binding Affinity: The reported affinity of the compounds of the invention for the human A2b adenosine receptor was determined experimentally using a radioligand filtration binding assay. This assay measures the amount of binding of a tritiated proprietary A2b receptor antagonist, in the presence and absence of a compound of the invention, to membranes made from HEK293 cells recombinantly expressing the human A2b adenosine receptor (Perkin Elmer, Cat. No. ES-013-C).To perform the assay, compounds of the invention to be tested were first solubilized in 100% DMSO and further diluted in 100% DMSO to generate, typically, a 10-point titration at half-log intervals such that the final assay concentrations did not exceed 10 μM of compound or 1% DMSO. 148 μL (135 μg/mL) membranes and 2 μL test compounds were transferred to individual wells of a 96-well polypropylene assay plate and incubated for 15 to 30 minutes at room temperature with agitation. Tritiated radioligand was diluted to a concentration of 14 nM in assay buffer (phosphate buffered saline without Magnesium and Calcium, pH 7.4; GE Healthcare Life Sciences, Cat. No. SH30256.01) and then 50 μL of the solution were transferred to each well of the assay plate. To define total and non-specific binding, wells containing 1% DMSO and 20 μM N-ethylcarboxamidoadenosine (Tocris Bioscience, Cat. No. 1691) respectively, were also included. The wells of the assay plate were incubated at room temperature for 60 minutes with agitation, then filtered using a FilterMate Harvester® (Perkin Elmer) or similar equipment through a UniFilter-96® PEI coated plate (Perkin Elmer Cat. No. 6005274 or 6005277). Filtering was achieved by aspirating the contents of the assay plate for 5 seconds, then washing and aspirating the contents three times with ice-cooled wash buffer (assay buffer supplemented with 0.0025% Brij58) and allowing the vacuum manifold to dry the plate for 30 seconds. The filter plate was incubated for at least 1 hour at 55° C. and allowed to dry. The bottom of the filter plate was then sealed with backing tape. 40 μL Ultima Gold (Perkin Elmer, Cat. No. 6013329) was added to each well of the filter plate and the top of the plate was sealed with TopSeal-A PLUS® clear plate seal (Perkin Elmer, Cat. No. 6050185). The plates were then incubated for at least 20 minutes, and then the amount of radioactivity remaining in each well was determined using a TopCount® (Perkin Elmer) scintillation counter. After normalization to total and non-specific binding, the percent effect at each compound concentration was calculated. The plot of percent effect versus the log of compound concentration was analyzed electronically using a 4-parameter logistic fit based on the Levenberg-Marquardt algorithm to generate IC50 values.
Measurement: IC50 = 228 nM (pChEMBL 6.639999866485596)
Descriptors: MW 469.53, ALogP 2.31, HBA 11, HBD 2, PSA 132.51, QED 0.45
Compound (SMILES): CCOc1cc(C)ccc1S(=O)(=O)Nc1noc2cc(Cn3cccn3)cc(OC)c12
InChIKey: JZRRUENUBDVCFY-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92993
Assay: Kat5 Assay: A. Compound preparation    1. Prepare 10 mM stock solutions in 100% DMSO from solid material    2. Serial dilute 10 mM, 1 mM or 0.1 mM compound stocks 3-fold in 100% DMSO for 11-point dose responseB. Reagent preparation    1. Prepare 1× assay buffer containing 10 mM Tris HCL pH 8.0, 2.5 mM NaCl, 0.5 mM EDTA, 0.005% BSG and 0.02% Tween-20    2. Dilute Histone peptide (CPC Scientific) and AcCoA (Sigma) together in assay buffer to 2×.    3. Dilute KAT enzyme in assay buffer to 2×.C. Enzyme reaction    1. Final reaction conditions for each KAT assay in a 20 ul assay reaction volume:  KAT5 25 nM, 1 uM AcCoA, 2 uM H4 1-21 peptide, 30-minute reaction     2. Add 0.5 ul of diluted compound to the assay plate (384-well V-bottom polypropylene plates) or 0.5 ul of DMSO for control wells.    3. Add 10 ul of 2× Histone peptide/2×AcCoA mix to the assay plate.    4. Add 10 ul of 2× enzyme to the assay plate.    5. Stop the reaction after the indicated time with the addition of 2 ul of 5% formic acid    6. Each reaction was analyzed using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, Milan (2008) Mass Spectrometry of Self-Assembled Monolayers: A New Tool for Molecular Surface Science ACS Nano 2008 2 (1), 7-18; SAMDI Tech, Inc. (Chicago, Ill.)).    7. Area under the curve (AUC) for both substrate and product peaks was determined for KAT5 at M.W. 2561 [Substrate+H]+ and 2603 [Product+H]+ with a +/−1 Da tolerance, respectively    8. Area under the curve (AUC) for both substrate and product peaks was determined for KAT6a, KAT6B, KAT7 and KAT8 at M.W. 2723 [Substrate+H]+ and 2765 [Product+H]+ with a +/−1 Da tolerance, respectively. 9. Percent conversion to product was calculated by: AUCProduct/(AUCsubstrate+AUCProduct).D. Data analysis    1. IC50 values were determined by fitting the % conversion at each inhibitor concentration to the 4-parameter IC50 equation using Pfizer proprietary curve fitting software.    2. Ki values were determined by fitting the % conversion at each inhibitor concentration to the Morrison equation for tightbinding competitive inhibitors using Pfizer proprietary curve fitting software.
Measurement: Ki = 260 nM (pChEMBL 6.579999923706055)
Descriptors: MW 442.5, ALogP 3.59, HBA 8, HBD 1, PSA 108.48, QED 0.44
Compound (SMILES): COC[C@@](C)(CCO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4ccnn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: GMPHJJCBRBCQTF-GDLZYMKVSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 591.58, ALogP 4.03, HBA 10, HBD 4, PSA 156.28, QED 0.18
Compound (SMILES): C=Cc1cncnc1N(C)[C@H]1C[C@@H](NS(=O)(=O)[C@@H]2CC[C@@H](C#N)C2)C1
InChIKey: YRFJLQAZMZVENW-DMRZNYOFSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 1024 nM (pChEMBL 5.989999771118164)
Descriptors: MW 361.47, ALogP 1.7, HBA 6, HBD 1, PSA 98.98, QED 0.83
Compound (SMILES): CCC(Nc1cc(F)c(S(=O)(=O)Nc2nccs2)cc1Cl)c1ccccc1F
InChIKey: HBBSMJFEJAGCAU-UHFFFAOYSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 2545 nM (pChEMBL 5.590000152587891)
Descriptors: MW 443.93, ALogP 5.44, HBA 5, HBD 2, PSA 71.09, QED 0.51
Compound (SMILES): CN1CCN(c2ccnc3ccc(-c4ccc5oc(N)nc5c4)cc23)CC1
InChIKey: FFKJOVARIRXUNG-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 359.43, ALogP 3.38, HBA 6, HBD 1, PSA 71.42, QED 0.59
Compound (SMILES): C=CC(=O)Nc1cc(Nc2ncc(C(=O)OC(C)C)c(-c3cn(C)c4ncccc34)n2)c(OC)cc1N(C)CCNC
InChIKey: FABVPXAVMGBCGS-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: EGFR Exon 21 L858R and Exon 20 T790M Concurrent Mutations: A compound's ability in selectively inhibiting EGFR L858R and T790M concurrent mutations can be assessed using Ba/F3 cells, a murine pro-B cell line, which have been transduced with EGFR L858R and T790M double mutations. An expression vector, pLVX-IRES puro (Clontech) coding for human EGFR L858R and T790M double mutation, was transfected into HEK293 cells by the Trans-Lentiviral ORF Packaging System (Thermo Scientific), to produce virus encoding EGFR L858R and T790M double mutations. Ba/F3 (DSMZ) cells maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 200 μM L-glutamine/200 μg/mL penicillin/200 μg/mL streptomycin (Life Technology) and 10 ng/mL IL-3 (R&D system), were infected by EGFR L858R and T790M double mutation virus and subsequently selected by puromycin (Life Technology) selection and IL-3 depletion. Ba/F3 cells expressing EGFR L858R and T790M double mutation (named Ba/F3-EGFR L858R/T790M) can proliferate in the absence of IL-3. The anti-proliferative activity of compounds was determined as follows: BaF3-EGFR L858R/T790M cells seeded in 96 well plates (2500 cells/well) were treated with test compound (dissolved in DMSO) at a series of concentrations (4-fold dilution, top concentration: 10,000 nM). The plates were incubated for 72 h in a 37° C. incubator with 5% CO2, and the number of viable cells in each well were measured indirectly by CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega; this assay is a colorimetric method for determining the number of viable cells through measurement of their metabolic activity by detection of enzymatic conversion of tetrazolium salts into blue formazan derivatives). Reagent (20 μL) was added into each well, and the plates were returned to the incubator for 2 h. The absorbance in each well was then measured at 490 nm using an Envision plate reader (Perkin Elmer). IC50 values were calculated by determining the concentration of compound required to decrease the MTS signal by 50% comparing to the DMSO control in best-fit curves using Microsoft XLfit software or Accelrys Pipeline Pilot.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 572.67, ALogP 4.13, HBA 11, HBD 3, PSA 135.53, QED 0.17
Compound (SMILES): C=CC(=O)N1CC2(COCCN2C(=O)N(C)[C@H](C(=O)N[C@H]2Cc3nc(cs3)-c3ccc4c(c3)c(c(-c3cccnc3[C@H](C)OC)n4CC)CC(C)(C)COC(=O)[C@@H]3CCCN(N3)C2=O)C(C)C)C1
InChIKey: GWRKWZIWOUBESZ-DHUQGQIFSA-N
Target: GTPase NRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01111
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 952.19, ALogP 5.38, HBA 13, HBD 2, PSA 180.77, QED 0.16
Compound (SMILES): COC1C(=O)C(C)CC/C=C\C=C/C=C(/C)[C@H](NS(=O)(=O)CC2CCOCC2)C[C@H]2CC[C@H](C)[C@](O)(O2)C(=O)C(=O)N(C)C(C)C(=O)OC([C@H](C)C[C@@H]2CC[C@@H](O)[C@H](OC)C2)CC(=O)C(C)/C=C(/C)C1O
InChIKey: SBZKSSDEBCUAKP-YMNQKHCBSA-N
Target: Peptidyl-prolyl cis-trans isomerase FKBP5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13451
Assay: SPR Assay to Characterize Ternary Complex Formation with FKBP51: Biotinylated avi-FKBP51 was immobilized on a streptavidin chip (Cytiva Series S SA) using a Biacore 8K or 8 k+ (Cytiva). To achieve an immobilization level of 200 RU, 0.6 ��g/ml biotinylated avi-FKBP51 were injected for 150 sec at a flow rate of 10 ��l/min. Serial dilution of FRB was prepared (12 concentrations, 3-fold dilutions, 0.00011-20 ��M) and supplemented with 100 nM of rapalog. A-B-A injection mode was used to ensure saturation immobilized FKBP12 with respective rapalog. 100 nM solution of the respective rapalog was injected before FRB injection for 120 sec and during dissociation for 420 sec. The FRB dilutions were then injected 120 seconds contact time with increasing concentrations. Rapamycin was used as reference sample. 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20, 1% DMSO was used as running buffer at a flow rate of 30 ��l/min. The multi-cycle kinetics data were fit to a 1:1 binding model to measure the association rate ka (1/Ms), the dissociation rate kd (1/s) and the affinity Kd (M). In case of fast association and dissociation, steady state affinity analysis following the law of mass action was used to determine the affinity Kd (M).
Measurement: Kd > 800 nM (pChEMBL 6.0970001220703125)
Descriptors: MW 1021.32
Compound (SMILES): CC(N)C1CCN(S(=O)(=O)c2cccc3cnccc23)CC1
InChIKey: MGLIYKZFLDTAMW-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Competition Binding Assay (ATP 10 uM) : Compounds were tested using an 11-point curve with 3-fold serial dilutions. IC50 determinations were made using an ATP concentration of 10 uM. The highest concentration tested was 30 μM. Test compounds were prepared in 100% DMSO at 100x final test concentration and were diluted to 1x in the assay with a final DMSO concentration of 1%. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 min at RT to generate affinity resins for kinase assays. The ligand-bound beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinases, ligand-bound affinity beads, and test compounds in 1x binding buffer (20% SeaBlock, 0.17xPBS, 0.05% Tween 20, 6 mM DTT). All reactions were performed in polystyrene 96-well plates in a final volume of 0.135 mL . The assay plates were incubated at RT with shaking for 1 hr. The affinity bead s were washed with wash buffer (1?PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1?PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at RT with shaking for 30 min. The kinase concentration in the eluates was measured by qPCR.
Measurement: Kd = 66 nM (pChEMBL 7.179999828338623)
Descriptors: MW 319.43, ALogP 1.98, HBA 4, HBD 1, PSA 76.29, QED 0.94
Compound (SMILES): COc1cccc(-c2nc(NC(=O)c3ccc(N4CCC(C(=O)O)CC4)nc3)sc2C#N)c1
InChIKey: WDOWFPMPTLZQSK-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Competition Radioligand Binding Assay: Adenosine A1 Receptor: Competition assays were carried out by incubation of human recombinant membranes of adenosine receptors from hA1 receptors transfected to CHO cells, [3H] DPCPX as radioligand, buffer (HEPES 20 mM (pH=7.4), 10 mM MgCl2, 100 mM NaCl, 2 U/mL of deaminase adenosine and non-labelled ligand in a total volume of 0.2 mL for 90 min at 25° C. R-PIA was used to determinate non-specific binding. Filter over Schleicher & Schuell GF/52 (pre-soaked 0.5% polyethylenimine) in a Brandel cell harvester. Unbound radioligand was removed with HEPES 30 mM (3×250 μl), NaCl (100 mM) and MgCl2 (10 mM).
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 463.52, ALogP 3.64, HBA 8, HBD 2, PSA 128.44, QED 0.57
Compound (SMILES): COc1cccc(OC)c1S(=O)(=O)Nc1noc2cc(Cn3cccn3)c(F)c(OC)c12
InChIKey: NWQPURPZPWUJPB-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT7 (SINGLE PROTEIN), Homo sapiens
UniProt: O95251
Assay: Kat7 Assay: A. Compound preparation    1. Prepare 10 mM stock solutions in 100% DMSO from solid material    2. Serial dilute 10 mM, 1 mM or 0.1 mM compound stocks 3-fold in 100% DMSO for 11-point dose responseB. Reagent preparation    1. Prepare 1× assay buffer containing 10 mM Tris HCL pH 8.0, 2.5 mM NaCl, 0.5 mM EDTA, 0.005% BSG and 0.02% Tween-20    2. Dilute Histone peptide (CPC Scientific) and AcCoA (Sigma) together in assay buffer to 2×.    3. Dilute KAT enzyme in assay buffer to 2×.C. Enzyme reaction    1. Final reaction conditions for each KAT assay in a 20 ul assay reaction volume:  KAT7 12.5 nM, 1 uM AcCoA, 2 uM H3 1-21 peptide, 45-minute reaction    2. Add 0.5 ul of diluted compound to the assay plate (384-well V-bottom polypropylene plates) or 0.5 ul of DMSO for control wells.    3. Add 10 ul of 2× Histone peptide/2×AcCoA mix to the assay plate.    4. Add 10 ul of 2× enzyme to the assay plate.    5. Stop the reaction after the indicated time with the addition of 2 ul of 5% formic acid    6. Each reaction was analyzed using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, Milan (2008) Mass Spectrometry of Self-Assembled Monolayers: A New Tool for Molecular Surface Science ACS Nano 2008 2 (1), 7-18; SAMDI Tech, Inc. (Chicago, Ill.)).    7. Area under the curve (AUC) for both substrate and product peaks was determined for KAT5 at M.W. 2561 [Substrate+H]+ and 2603 [Product+H]+ with a +/−1 Da tolerance, respectively    8. Area under the curve (AUC) for both substrate and product peaks was determined for KAT6a, KAT6B, KAT7 and KAT8 at M.W. 2723 [Substrate+H]+ and 2765 [Product+H]+ with a +/−1 Da tolerance, respectively. 9. Percent conversion to product was calculated by: AUCProduct/(AUCsubstrate+AUCProduct).D. Data analysis    1. IC50 values were determined by fitting the % conversion at each inhibitor concentration to the 4-parameter IC50 equation using Pfizer proprietary curve fitting software.    2. Ki values were determined by fitting the % conversion at each inhibitor concentration to the Morrison equation for tightbinding competitive inhibitors using Pfizer proprietary curve fitting software.
Measurement: Ki = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 462.46, ALogP 3.04, HBA 9, HBD 1, PSA 117.71, QED 0.42
Compound (SMILES): C[C@H]1CN(C(=O)CCC2(c3cnccn3)NC(=O)NC2=O)CCN1c1cc(F)cc(Cl)c1
InChIKey: KARIJUOWBYDHHJ-JRTLGTJJSA-N
Target: Matrix metalloproteinase-14 (SINGLE PROTEIN), Homo sapiens
UniProt: P50281
Assay: In Vitro Assay: MMP-14:The basis for the assay is the cleavage of the substrate 390 MMP FRET Substrate I (Anaspec Cat # AS-27076) by human MMP14 (Biomol, Cat #SE-259).For the dose response (10 point), 4 μL of a dilution series of compound 2 mM highest concentration, 1/5 dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM MOPS pH7, 5 mM CaCl2, 1 μM ZnCl2, 0.1% Brij-35) containing MMP14 (0.05 ng/μL) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate 390 MMP FRET Substrate I (10 μL, 2.5 μM) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 60 min at room temperature (Excitation 485 nm, Emission 530).
Measurement: IC50 > 4000 nM (pChEMBL 5.3979997634887695)
Descriptors: MW 460.9, ALogP 1.82, HBA 6, HBD 2, PSA 107.53, QED 0.66
Compound (SMILES): CN1CCCN(c2cnc3ccc(-c4ccc5oc(N)nc5c4)cc3n2)CC1
InChIKey: NWHOAJOKVRQOOA-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 374.45, ALogP 3.16, HBA 7, HBD 1, PSA 84.31, QED 0.58