lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCN(C[C@@]12CC[C@@H](c3cc(-c4c(F)cccc4F)nnc31)C2(C)C)S(=O)(=O)c1ccc(OC(F)F)cc1
InChIKey: MYITZYRPJKUUHU-PPHZAIPVSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro RORc Ligand Binding Assay: Consumable Supplier and product code GFB Unifilter plates Perkin Elmer 6005177 3-[(3- Sigma C5070 Cholamidopropyl)dimethylammonio]- 1-propanesulfonate (CHAPS) 96-well polypropylene U-bottom Nunc 267245 assay plate HEPES buffer, 1M Sigma H3375 Magnesium chloride (MgCl2) Sigma M8266 D,L-Dithiothreitol (DTT) Sigma D0632 Sodium chloride (NaCl) Sigma 71382 Bovine serum albumin (BSA) Sigma A7030 [lyophilized powder, ≥98% (agarose gel electrophoresis), Essentially fatty acid free, essentially globulin free] 25-hydroxycholesterol Sigma H1015 25-[26,27-3H]hydroxycholesterol Perkin Elmer NET674250UC American Radiolabeled Chemicals ART0766 RORc ligand binding domain Genentech (e.g., PUR 28048), expressed in E. coli Plate seals Perkin Elmer 6005185 Microscint 0 Perkin Elmer 6013611. Final concentrations were as follows: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2, 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 uM 25-hydroxycholesterol was also present.
Measurement: IC50 = 10.9 nM (pChEMBL 7.960000038146973)
Descriptors: MW 549.59, ALogP 5.89, HBA 5, HBD 0, PSA 72.39, QED 0.33
Compound (SMILES): COc1ccc(C)c(CNC(=O)c2nc(N)nc3c(F)cccc23)n1
InChIKey: RMFOGOQJELCEON-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: A2a Ki assay: Binding affinities of compounds of the invention for the human A2a receptor were determined in a competition binding assay using Scintillation Proximity technology. Thus, 1* μg, or preferably 0.25 μg of membranes from HEK293 cells expressing the human A2a receptor were incubated with a compound of the invention at concentrations ranging from 3000 nM to 0.15 nM in a reaction mixture containing also 0.5 nM of a tritiated form of 5-amino-7-[2-phenethyl]-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (the tritiated compound) and 25 μg of wheat germ agglutin-coated yttrium silicate SPA beads for one hour at room temperature with agitation. The beads were then allowed to settle to the bottom of the wells for 1 hr, after which the membrane-associated radioactivity was determined by scintillation counting in a TopCount microplate reader. Ki values were determined using the Cheng-Prusoff equation.
Measurement: Ki = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 341.35, ALogP 1.99, HBA 6, HBD 2, PSA 103.02, QED 0.75
Compound (SMILES): C[C@H]1CC2(CCN(C[C@H](O)c3ccc4c(ccc5nnnn54)c3)CC2)C(=O)N1C1=CC(=O)OC1
InChIKey: SUFBPVQFOFQPFD-YWZLYKJASA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: A Thallium Flux Assay was performed on the compounds of the Examples. This assay has been described previously; see, e.g., PCT Published Application WO 2013/062900.Data collected for compounds in the Examples of the present invention using the Thallium Flux Assay are shown in Table 3 below. All of the tested final product compounds in the Examples (diastereomeric mixtures and individual diastereomers) had IC50 potencies less than 1 μM in the Thallium Flux Assay.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 462.51, ALogP 1.45, HBA 9, HBD 1, PSA 113.16, QED 0.58
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5ccc(C(C)O)cc5)nc4n3CC3CC3)n(C)c12
InChIKey: YOOIOHGUTVMABZ-YXDIFBQUSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 149 nM (pChEMBL 6.829999923706055)
Descriptors: MW 590.73, ALogP 5.29, HBA 8, HBD 2, PSA 111.43, QED 0.27
Compound (SMILES): COc1ccc(NC(=O)c2ccc(-c3ccc(F)cc3)s2)cc1NC(=O)CN1CCOCC1
InChIKey: NBQLKDMGEXFHCR-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: HCT116 TOPFlash Assay: In preparation of the assay, the two cell lines were plated 24 hours before at 10000 cells per well of a 384 micro titre plate (MTP) in 30 μL growth medium. Selective inhibitory activity for small molecules on the mutated Wnt pathway was determined after parallel incubation of both (TOP and FOP) HCT116 reporter cell lines with a compound dilution series from 50 μM to 15 nM in steps of 3.16-fold dilutions in CAFTY buffer (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 5 mM NaHCO3, pH 7.4) containing 2 mM Ca2+ and 0.01% BSA. The compounds were thereby serially prediluted in 100% DMSO and thereafter in addition 50 fold into the CAFTY compound dilution buffer (described above). From this dilution 10 μL were added to the cells in 30 μL growth medium and incubated for 36 hours at 37° C. and 5% CO2. Thereafter luciferase assay buffer (1:1 mixture of luciferase substrate buffer (20 mM Tricine, 2.67 mM MgSO4, 0.1 mM EDTA, 4 mM DTT, 270 μM Coenzyme A, 470 μM Luciferin, 530 μM ATP, ph adjusted to pH 7.8 with a sufficient volume of 5M NaOH) and Triton buffer (30 mL Triton X-100, 115 mL glycerol, 308 mg Dithiothreitol, 4.45 g Na2HPO4.2H2O, 3.03 g TRIS HCl, ad 1I H2O, pH 7.8) was added as equal volume to the compound solution on the cells to determine luciferase expression as a measure of Wnt signaling activity in a luminometer.n order to determine the inhibitory activity of compounds for the WT Wnt signaling pathway, the Super TopFlash vector were cotransfected with pcDNA3 into HEK293 cells.
Measurement: IC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 469.54, ALogP 4.09, HBA 6, HBD 2, PSA 79.9, QED 0.55
Compound (SMILES): Cc1nscc1Nc1nccc(-c2cc(C#N)c3c(c2)[C@@](C)(CO)CN3)n1
InChIKey: FNRUFLMAYSGBJS-LJQANCHMSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition of auto-phosphorylation of recombinant human NF-kappaB-inducing kinase (NIK/MAP3K14) activity: NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen® (αscreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. Final DMSO concentration was 1% (v/v) in assays. Assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% Tween® 20. Assays were carried out in 384 well Alphaplates (Perkin Elmer). Incubations consisted of compound, 25 microM Adenosine-5′-triphosphate (ATP), and 0.2 nM NIK/MAP3K14. Incubations were initiated by addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 1 h at 25° C. and terminated by addition of stop buffer containing anti-phospho-IKK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision® Multilabel Plate Reader (Perkin Elmer). Signal obtained in the wells containing blank samples was subtracted from all other wells and IC50's were determined by fitting a sigmoidal curve to % inhibition of control versus Log10 compound concentration.
Measurement: IC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 378.46, ALogP 3.2, HBA 8, HBD 3, PSA 106.75, QED 0.64
Compound (SMILES): CC(C)Oc1c(-c2cnn(C)c2)cnc2ccc(Sc3nnc4ccc(-c5cnn(C)c5)cn34)cc12
InChIKey: QZRSPASIAMAJGP-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Inhibition Assay: Experimental Purpose: The purpose is to study the effect of the compounds on c-MET tyrosine kinase activity at the molecular level. IC50 values were calculated from the data of the inhibition rates of the compounds on c-MET tyrosine kinase phosphorylation at various concentrations, thereby evaluating the compounds.Experimental Method: 1. The positive control and the test compounds were first dissolved in 100% dimethyl sulfoxide to prepare a 20 mM stock solution, which was stored at â¿¿20° C. in a freezer. 2. Just before the experiment, the above stock solution was diluted to a final concentration of 4% in dimethyl sulfoxide. 3. 5x kinase buffer was diluted with water to 1.33x kinase buffer. 4. A mixture of 4 umol/L polypeptide substrate and 2x kinase was prepared in 1.33x kinase buffer. 5. 4 umol/L phosphorylated peptide substrates were prepared in 1.33x kinase buffer. 6. Appropriate concentration of ATP was prepared in 1.33x kinase buffer. 7. According to the need of dilution ratio, endonuclease was diluted in display buffer B. 8. All of the compounds were diluted within a 96-well plate. 9. The mixture of 4 umol/L polypeptide substrate and 2x kinase, and the 4x ATP solution were added to a 96-well plate, and the reagents were then transferred to a 384-well plate by a 12 channel pipettor. 10. The plate was incubated at room temperature for 1 hour. 11. 5 uL display buffer were added, and the plate was incubated at room temperature for another 1 hour. 12. Fluorescence was read on a NovoStar microplate reader, with excitation wavelength: 400 nm, and emission wavelength: 445 nm and 520 nm. 13. Using GraphPad software, a non-linear regression curve was generated based on the inhibition ratio as a function of the concentration of the compounds. The IC50 was calculated using a S-shaped dose-effect curve fitting. The inhibition rates of the compounds were calculated according to the following formula. 14. Emission light wave ratio=(Coumarin emission wave 445 nm)/(Fluorescein emission wave 520 nm). 15. Inhibition percent=1-phosphorylation (absorption)%/phosphorylation (divergence)%.
Measurement: IC50 = 8.6 nM (pChEMBL 8.069999694824219)
Descriptors: MW 496.6, ALogP 5.02, HBA 10, HBD 0, PSA 87.95, QED 0.32
Compound (SMILES): NC1(CN2CCC(c3ccc(-c4ccc(C(F)(F)F)c(S(N)(=O)=O)c4-c4nnn[nH]4)cc3)CC2)CC1
InChIKey: VIWDEKVIIKABHL-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 3.66 nM (pChEMBL 8.4399995803833)
Descriptors: MW 521.57, ALogP 2.87, HBA 7, HBD 3, PSA 143.88, QED 0.45
Compound (SMILES): Cc1c(-c2ccccc2)nc2ccc(Br)cc2c1C(=O)NC[C@@H](CC(=O)O)c1ccccc1Cl
InChIKey: RTPIJAVXJNAJOS-GOSISDBHSA-N
Target: Prostaglandin F2-alpha receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43088
Assay: In Vitro Test of Inhibition of Human FP Receptor Activity: For the characterization of test substances in respect of FP antagonism, PGF2α-induced calcium flux in FP-expressing CHEM1 cells (Millipore, HTS093C) was used.3000 cells in 30 μl of full medium [DMEM F12, 10% FCS, 1.35 mM sodium pyruvate, 20 mM HEPES, 4 mM GlutaMAX, 2% sodium bicarbonate, 1% Pen/Strep, 1% 100× non-essential amino acids] are sown per well of a 384 multititre plate (from Greiner, TC plate, black with clear base) and incubated at 33° C., 5% CO2 for 24 hours. Prior to the measurement, the medium is replaced by 30 μl of Fluo-8 AM loading buffer [calcium-free Tyrode (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 4.8 mM NaHCO3, pH 7.4), 2 mM CaCl2, 6.3 mM Probenecid, 5 μM Fluo-8 AM, 0.0112% Pluronic®] and incubated at 37° C., 5% CO2 for 30 minutes. The test substance is prepared in DMSO in various concentrations as a dose-response curve (starting concentration 10 mM, dilution factor 3.16) and prediluted 1:50 with calcium-free Tyrode, 2 mM CaCl2, 0.002% SmartBlock (from CANDOR Bioscience GmbH). 10 μl of the prediluted substance solution are added to the Fluo-8-laden cells and incubated at 37° C., 5% CO2 for 10 minutes. The FP receptor is activated by adding 40 μl of 2 nM (final concentration) PGF2α in calcium-free Tyrode, 2 mM CaCl2, 0.002% SmartBlock, and the calcium flux is determined by measuring the fluorescence at ex. 470 nm/em. 525 nm in a fluorescence measuring instrument (FLIPR Tetra®, Molecular Devices) for 120 seconds.
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 537.84, ALogP 6.61, HBA 3, HBD 2, PSA 79.29, QED 0.28
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCC2(CCCN2Cc2cc(N3CCCCC3)cc(S(F)(F)(F)(F)F)c2)CC1
InChIKey: BYLBBZCPCLPKDB-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Competitive Activity-Based Protein Profiling Assay: Human: Proteomes (human prefrontal cortex or cell membrane fractions) (50 μL, 1.0-2.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP-Rh or JW912 (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at room temperature. Reactions were quenched with SDS loading buffer (15 μL-4X) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL using ImageJ 1.49 k software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 633.57, ALogP 8.39, HBA 4, HBD 0, PSA 36.02, QED 0.31
Compound (SMILES): COC1CCN(c2cccc3c2CCN(C(=O)c2cn(-c4cccc(Cl)c4F)nn2)C3C(=O)Nc2ccc(C(N)=O)cc2)CC1
InChIKey: QYVCEYOGBUMRBR-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Enzyme Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 632.1, ALogP 4.15, HBA 8, HBD 2, PSA 135.68, QED 0.31
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@H]3C[C@@]4(Cc5nc(no5)/C=C\COCc5ccc(Br)nc5NC3=O)C[C@@H]24)c2c(C)cc(-c3cnc(C)nc3)cc12
InChIKey: KBPSLKREMFZJNC-FWZNKYJXSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 M each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 minutes using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of Complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 738.6, ALogP 4.64, HBA 12, HBD 1, PSA 171.12, QED 0.2
Compound (SMILES): CCC(=O)Nc1cc(Nc2nc(-c3cccc(C(F)(F)F)n3)nn3cccc23)ccn1
InChIKey: TZTCSORERJEKKZ-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 0.89 nM (pChEMBL 9.050000190734863)
Descriptors: MW 427.39, ALogP 4.3, HBA 7, HBD 2, PSA 97.1, QED 0.49
Compound (SMILES): COC(=O)N1CCC(n2ncc3c(N4CCOCC4)nc(-c4ccc5oc(N)nc5c4)nc32)CC1
InChIKey: LMIIHDYGRLQNPC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: LanthaScreen Kinase Assay: The kinase reaction is performed in a white 384 well microtitre plate. The total reaction volume is 20 ul per well and the reaction buffer composition is 50 mM HEPES pH7.5, 0.01% Polysorbate 20, 1 mM EGTA, 10 mM MnCl2, and 2 mM DTT. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, 8 ul of mTOR diluted in reaction buffer is added per well for a 60 ng/ml final concentration. To start the reaction, 10 ul of an ATP/GFP-4EBP1 mixture (diluted in reaction buffer) is added per well for a final concentration of 10 uM ATP and 0.5 uM GFP-4EBP1. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 10 ul per well of a Tb-anti-pT46 4EBP1 antibody/EDTA mixture (diluted in TR-FRET buffer) for a final concentration of 1.3 nM antibody and 6.7 mM EDTA. The plate is sealed, incubated for 1 hour at room temperature, and then read on a plate reader set up for LanthaScreen TR-FRET.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 478.51, ALogP 2.46, HBA 11, HBD 1, PSA 137.66, QED 0.47
Compound (SMILES): Cc1ncc(-c2ccc3cnc(NC(=O)C4CCN(C)CC4)cc3c2)cn1
InChIKey: JHMDORVAPDFOPC-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Kinase Assay: The DYRK1A kinase assay was run using the Ser/Thr 18 peptide Z-lyte assay kit according to manufacturer's instructions (Life Technologiesa Division of Thermo-Fisher). This is a non-radioactive assay using fluorescence resonance energy transfer (FRET) between coumarin and fluorescein to detect kinase activity which is represented as a ratio of coumarin emission/fluorescein emission.Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated ("0% Control") and phosphorylated ("100% control") forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (luM top) was run to serve as a positive compound control.
Measurement: EC50 = 221 nM (pChEMBL 6.659999847412109)
Descriptors: MW 361.45, ALogP 3.28, HBA 5, HBD 1, PSA 71.01, QED 0.78
Compound (SMILES): CCOC1CCN(C[C@H]2C[C@@H](c3ccc4nc(-c5cccc(Cl)c5)n(CC(=O)NC(C)C)c(=O)c4c3)C2)CC1
InChIKey: UJTMNUBXALNSOP-PEPAQOBHSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: Binding Assay: The binding assay was carried out by the method based on that of Tahara et al. (Tahara A et al., Brit. J. Pharmacol. 125, 1463-1470 (1998)).The incubation buffer was: 50 mM Tris, 10 mM MgCl2, 0.1% BSA, pH 7.4.In the assay mixture (200 μl), membranes (40 μg protein in incubation buffer) from CHO-K1 cells with stably expressed human V1a receptors (cell line hV1a_5_CHO) were incubated with 0.04 nM 125I-AVP (8-Arg-vasopressin, PerkinElmer NEX 128) in incubation buffer (50 mM Tris, 10 mM MgCl2, 0.1% BSA, pH 7.4) (total binding) or additionally with increasing concentrations of test substance (displacement experiment). The nonspecific binding was determined with 1 μM AVP (Fluka 94836). Duplicate determinations were carried out.After incubation (60 minutes at room temperature), the samples were processed as described for V1b.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 551.13, ALogP 5.24, HBA 6, HBD 1, PSA 76.46, QED 0.39
Compound (SMILES): CC(C)(O)COc1ccc2c(C(=O)N[C@H]3CC4(C3)C[C@H](Oc3nn5cc(C(F)(F)F)ccc5c3C(N)=O)C4)nn(C(F)F)c2c1
InChIKey: SQSRAMFLLZQUPB-MVLNGOCHSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH) (SEQ ID NO. 1). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 611 nM (pChEMBL 6.210000038146973)
Descriptors: MW 636.58, ALogP 4.47, HBA 9, HBD 3, PSA 146, QED 0.23
Compound (SMILES): CCNC(=O)CCCOc1ccc(Cc2cc([C@@H]3O[C@H](SC)[C@@H](O)[C@H](O)[C@H]3O)ccc2C)cc1
InChIKey: MLMUZJXNMQRMIX-PUHDZGQXSA-N
Target: Sodium/glucose cotransporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P13866
Assay: In Vitro Human SGLT1 Inhibition Assay: Human sodium/glucose co-transporter type 1 (SGLT1; accession number NP_000334; GI: 4507031) was cloned into pIRESpuro2 vector for mammalian expression (construct: HA-SGLT1-pIRESpuro2). HEK293 cells were transfected with the human HA-SGLT1-pIRESpuro2 vector and the bulk stable cell line was selected in presence of 0.5 μg/mL of puromycin. Human HA-SGLT1 cells were maintained in DMEM media containing 10% FBS, 1% GPS and 0.5 μg/mL of puromycin. The HEK293 cells expressing the human HA-SGLT1 were seeded in 384 well plates (30,000 cells/well) in DMEM media containing 10% FBS, 1% GPS and 0.5 μg/mL of puromycin, then incubated overnight at 37 C, 5% CO2. Cells were then washed with uptake buffer (140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM Tris, 1 mg/mL bovine serum albumin (BSA), pH 7.3). Twenty microliters of uptake buffer with or without testing compounds were added to the cells. Then, 20 microliters of uptake buffer containing 14C-AMG (100 nCi) were also added to cells. The cell plates were incubated at 37° C., 5% CO2 for 1-2 hours. After washing the cells with uptake buffer, scintillation fluid was added (40 microliters/well) and 14C-AMG uptake was measured by counting radioactivity using a scintillation coulter (TopCoulter NXT; Packard Instruments).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 489.63, ALogP 2.72, HBA 7, HBD 4, PSA 108.25, QED 0.38
Compound (SMILES): CNC(=O)Cn1cc(-c2ccc(NC(=O)[C@H](NC[C@H](C)c3ccc(C#N)cc3)c3ccccc3)nc2)cn1
InChIKey: IZDVOEWRWQYODV-WTYVLRPYSA-N
Target: Histone acetyltransferase p300 (SINGLE PROTEIN), Homo sapiens
UniProt: Q09472
Assay: Biochemical Assay: The full length p300 SPA assay was run following the same protocol as p300 HAT SPA assay, but used 6 nM purified full length p300 (purchased from Active Motif) in place of the purified p300-HAT domain.Select compounds were also evaluated in a H3K18Ac MSD cellular assay that measures the ability of compounds to inhibit the the acetylation of chromatin at H3K18, a process catalyzed by p300 and CBP. In a typical experiment the p300 HAT inhibitory activity inside cells of the compounds described herein was determined in accordance with the following experimental method. 20 k HCT-116 cells per well are plated in 75 μL RPMI+10% FBS media the night before treatment. Compounds plated in DMSO at 4× final concentration are resuspended in 30 μL RPMI+10% FBS, then 25 μL is combined with corresponding wells containing cells. Treated cells are incubated for 2 hr at 37° C., then lysed in 500 μL final volume and frozen at −80° C. MSD plates (Meso Scale Discovery) are coated overnight at 4° C. with 60 μL 1:500 α-total histone antibody (Millipore MAB3422) in PBS. Plates are then blocked with 5% BSA in TBST shaking at RT for 1 hr, washed, and 30 μL lysate added to each well for 2 hr shaking at RT. Plates are washed and 25 μL 1:216 α-H3K18ac antibody (CST 9675) in PBS added, then incubated for 1 hr shaking at RT. Plates are washed again, then 25 μL 1:1000 Sulfo-Tag goat α-rabbit antibody (Meso Scale Discovery R32Ab-1) in PBS is added for 1 hr shaking at RT. Plates are washed once more, then 150 μL 1× Read Buffer (MSD #R92TD-3) is added to all wells and read on MSD SECTOR Imager 2400 using the conventional read setup.
Measurement: IC50 = 993 nM (pChEMBL 6)
Descriptors: MW 507.6, ALogP 3.64, HBA 7, HBD 3, PSA 124.73, QED 0.3
Compound (SMILES): CN1C(N)=N[C@](C)(c2cc(/C=C(\F)c3cnc(Cl)cn3)ccc2F)CS1(=O)=O
InChIKey: XKGVHXNXXZBFGM-LBWZGELYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assays: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05 M acetate, pH 4.5, 8% DMSO final, 100 μM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The β-secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2), which was pre-incubated for one hour with the test compound, typically in about 1 uL of DMSO according to a serial dilution, was added thereto. The assay was effectively started by the addition of FRET substrate (50 nM) and the combination was incubated for one hour. The FRET assay was terminated by the addition of tris buffer, which raised the pH to neutrality, and the fluorescence was determined. The FRET substrate was a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. The specific FRET substrate used in this assay was made by Amgen in-house. Commercially available FRET substrates, for example, the FRET substrate offered with the BACE1 FRET Assay Kit sold by ThermoFisher Scientific (Catalog Number P2985), may be used in this assay with the appropriate modifications, which are within the purview of the ability of a person with ordinary skill in the art. Proteolytic cleavage of the FRET substrate released quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 427.86, ALogP 2.54, HBA 6, HBD 1, PSA 101.54, QED 0.81
Compound (SMILES): COC(=O)N1CC(c2ccc3cc(-c4nc5cc(C(=O)N6C[C@H](N)C[C@@H](F)C6)cc(OC)c5n4C)n(CC4CC4)c3n2)C1
InChIKey: BRSGVFMESYIADP-FGZHOGPDSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 589.67, ALogP 3.69, HBA 9, HBD 1, PSA 120.74, QED 0.36
Compound (SMILES): CC1C[C@@H]2C[C@H]1OC1Cn3cc(C(=O)NCc4ccc(F)cc4F)c(=O)c(O)c3C(=O)N12
InChIKey: DOTHVMMWDNQOGS-KHVRFWMWSA-N
Target: Solute carrier family 22 member 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15244
Assay: Inhibition Assay: The dose dependent inhibition of OCT2 mediated uptake of a model substrate 14C-Tetraethylammonium (TEA) by test compounds was studied in wild-type and OCT2-transfected MDCKII cells at 7 concentrations from 0.014 μM to 10 μM.MDCKII cells were maintained in minimal essential medium (MEM) with 1% Pen/Strep, 10% fetal bovine serum, and 0.25 mg/mL hygromycin B in an incubator set at 37° C., 90% humidity and 5% CO2. 24 hours prior to assay, media containing 5 mM sodium butyrate were added to MDCKII cells in flasks, and cells were grown to 80-90% confluence. On assay day, cells were trypsinized and resuspended in Krebs-Henseleit Buffer (KHB), pH 7.4 at 5×106 million cells/mL. Cells were preincubated for 15 min in assay plate before addition of test compound or substrate.
Measurement: IC50 = 358 nM (pChEMBL 6.449999809265137)
Descriptors: MW 445.42, ALogP 1.74, HBA 6, HBD 2, PSA 100.87, QED 0.75
Compound (SMILES): COc1cc2ncnc(Oc3ccc(NC(=O)Nc4cccc(CN(C)CCO)c4)c(Cl)c3)c2cc1OC
InChIKey: OOZJOGKFFWVVDR-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Binding Assay: A competition binding assay (DISCOVERX KINOMESCAN) was used to measure the ability of a compound to compete for binding of an immobilized adenosine triphosphosphate (ATP) site directed ligand using a DNA-tagged vascular endothelial growth receptor 2 (VEGFR2) as the target. The ability of the test compound to compete with the immobilized ligand was measured using quantitative polymerase chain reaction (qPCR) of the DNA tag (Fabian, M. A. et al., 23 Nature Biotechnology 329-336 (2005); Karaman, M. W. et al., 26 Nature Biotechnology 127-132 (2008)).A VEGFR2 tagged T7 phage strain was prepared in an Escherichia coli (E. coli) derived from the BL21 strain. The E. coli were grown to log-phase, infected with VEGFR2 tagged T7 phage and then incubated with shaking at 32° C. until lysis. The lysate containing the kinase was then centrifuged and filtered to remove cell debris. Affinity resin for the VEGFR2 assay was prepared by treating Streptavidin-coated magnetic beads with a biotinylated small molecule ligand for 30 minutes at room temperature. The beads were blocked with excess biotin and then washed with blocking buffer (SEABLOCK (PIERCE), 1% bovine serum albumin, 0.17% phosphate buffered saline, 0.05% TWEEN 20, 6 mM dithiothreitol). The binding reaction was initiated by combining in a well of a polystyrene 96-well plate, DNA tagged VEGFR2, liganded affinity beads and the serial diluted test compound in 1× binding buffer (20% SEABLOCK, 0.17× phosphate buffered saline, 0.05% TWEEN 20, 6 mM dithiothreitol) in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and then the beads were washed with wash buffer (1× phosphate buffered saline, 0.05% TWEEN 20). The beads were re-suspended in elution buffer (1× phosphate buffered saline, 0.05% TWEEN 20, 0.05 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The VEGFR2 concentration in the eluate was measured using qPCR.An 11-point dose response curve of 3-fold serial diluted test compound starting at 1 μM was used to determine the VEGFR2 binding constant (Kd). The compounds were prepared in 100% DMSO at 100× the final test concentration and the diluted to 1× in the assay for final DMSO concentration of 1%. Binding constants were calculated with standard dose-response curve using the Hill equation with Hill slope set to −1. Curves were fit using a non-linear least square fit with the Levenberg-Marquardt algorithm.
Measurement: Kd = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 538, ALogP 5.16, HBA 8, HBD 3, PSA 118.07, QED 0.25
Compound (SMILES): O=C(NO)C(Cc1ccccc1)n1ncc2cc(-c3ccccc3)ncc21
InChIKey: ABSYYVBMIOTHSN-UHFFFAOYSA-N
Target: Histone deacetylase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BY41
Assay: Enzymatic Assay: HDAC-Glo I/II Assay Kit was purchased from Promega Corporation (Madison, Wis.), including the following components: HDAC-Glo I/II Buffer (25 mM Tris buffer, pH 8.0, supplemented with 137 mM: NaCl, 2.7 mM KCl, 1% v/v Triton X-100 and 1 mM: MgCl2), luminogenic substrates Boc-GAK(Ac)-aminoluciferin (HDAC-Glo I/II substrate), proprietary developer reagent (containing trypsin). Assay:The HDAC8 reaction was performed at room temperature in white 384-well polystyrene, flat-bottom microtiter plate (Greiner Bio-one, Monroe, N.C.) in a final volume of 20 μL. Test compounds were first diluted serially in DMSO and 100 nL were transferred to the plate wells by Echo 550 (Labcyte, Sunnyvale, Calif.) before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The HDAC-Glo I/II assay reagent was prepared by rehydration of lyophilized HDAC-Glo I/II substrate in 10 mL. HDAC-Glo I/II assay buffer followed with the addition of 10 μL of developer reagent. 10 uL of 3 nM HDAC8 in the HDAC-Glo I/II assay buffer were first dispensed into the microliter plate containing the compounds and incubated at room temperature for 30 min. The reactions were initiated with the addition of 10 uL HDAC-Glo I/II assay reagent with Multidrop.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 358.4, ALogP 3.39, HBA 5, HBD 2, PSA 80.04, QED 0.42
Compound (SMILES): CC(C)(NC(=O)Cc1csc(-c2ccc(Cl)cc2-c2cccc(Cl)c2)n1)C(=O)O
InChIKey: IEOBPNNUZFRSKC-UHFFFAOYSA-N
Target: Serine hydroxymethyltransferase, cytosolic (SINGLE PROTEIN), Homo sapiens
UniProt: P34896
Assay: SHMT1 and SHMT2 Activity Assays: Full length human cytosolic Serine Hydroxymethyl transferase 1 (SHMT1, residues 1-483 in Uniport ID P34896) was expressed as an N-terminal His6 tagged protein and purified in E. coli using nickel capture followed by size-exclusion chromatography. Human mitochondrial SHMT2 (residues 30-504 in Uniprot ID P34897) with mitochondrial leader sequence deleted was expressed as an N-terminal His6 tagged protein and purified in E. coli using nickel capture followed by size-exclusion chromatography.Serine hydroxymethyltransferases catalyze the reversible hydroxymethylation of glycine to serine, with methylene tetrahydrofolate (CH2-THF) providing the additional carbon. SHMT1 and SHMT2 activity was determined by measuring serine production from glycine and CH2-THF using mass spectroscopy. Briefly, 15 ul of 0.5 mM glycine and 0.2 mM CH2-THF in 20 mM TEA, pH8.0, 0.2 mM NADPH were added to 384 well plate containing 0, 0.05 mM, 0.005 mM or 0.0005 mM of inhibitor. 15 ul of SHMT1 or 2 were added to initiate the reaction. The plate was incubated for 60 minutes at room temperature and the reaction was quenched by the addition of 30 ul of 10% trichloroacetic acid. Serine produced was analyzed using Rapidfire 360 and API4000+ in the positive ion mode.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 449.36, ALogP 5.31, HBA 4, HBD 2, PSA 79.29, QED 0.54
Compound (SMILES): NC(=O)Cc1ccc(Nc2nc(C3CCCC3)nc3c2S(=O)(=O)CCC3)cc1
InChIKey: MKKFHYIRLYFQIK-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Kinetic Assay: Assays are performed in 96-well plates in a total volume of 200 μl/well. Compounds are dissolved in dimethylsulfoxide (DMSO) and added to plates in a volume of 10 μl followed by addition of 165 μl of assay mix. Plates are pre-incubated at 25° C. for 15 min and the reactions are initiated by the addition of 25 μl of cAMP followed by thorough mixing. Reaction rates are measured by monitoring the decrease in fluorescence using excitation at 355 nm and emission at 460 nm for a period of 10 min in a fluorescence plate reader. Initial rates (slopes) are determined from linear portions of the progress curves. Final concentrations of assay components are as follows: 50 mM Tris, pH 8, 10 mM MgCl2, 50 mM KCl, 2% DMSO, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.4 mM phosphenolpyruvate (PEP), 0.01 mM NADH, 0.04 mM adenosine triphosphate (ATP), 0.004 mM cAMP, 7.5 units myokinase from yeast, 1.6 units pyruvate kinase, 2 units lactate dehydrogenase, and either 0.5 nM human PDE4D7 or 10 nM human PDE4B1. All data are percent normalized relative to controls and are presented as percent inhibition. An inhibitory concentration 50% (IC50) value is calculated by fitting of a sigmoidal dose response curve. Human PDE4D7 contained a mutation of serine 54 to aspartic acid to mimic activation by cAMP-dependent protein kinase A (PKA). Human PDE4B1 contained a corresponding mutation of serine 133 to aspartic acid to also mimic PKA activation.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 400.5, ALogP 2.63, HBA 6, HBD 2, PSA 115.04, QED 0.8
Compound (SMILES): CCNc1cc2c(cn1)c(-c1cnn(C)c1)nn2C1CCC(Oc2cccc(C(=O)NC)c2C)CC1
InChIKey: IDBPBYMGBNZBKM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: AXL: Compounds of Formula I were screened for their ability to inhibit AXL kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. His-tagged recombinant human AXL cytoplasmic domain was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A), 2 nM biotinylated anti-His (Cat. No. M4408), and 2 nM europium-labeled Streptavidin (Cat. No. PV5899) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO. Compounds were typically prepared in a threefold 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 was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using a concentration of control compound that completely inhibits the enzyme. The POC values are fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 487.61, ALogP 4.49, HBA 8, HBD 2, PSA 98.89, QED 0.4
Compound (SMILES): COc1cc(C2=NC(C)(C)CO2)ccc1Nc1nc2ccc(-c3ccc(NC(=O)Cc4ccc(F)cc4)cc3)cn2n1
InChIKey: HPWDEHSFYIBWAA-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Mps-1 Kinase Assay: For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Mps-1 in assay buffer [0.1 mM sodium-ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μl of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μl assay volume is 10 μM) and peptide substrate (1.67 μM=>final conc. in the 5 μl assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 μl assay volume). The reaction was stopped by the addition of 3 μl of a solution of HTRF detection reagents (100 mM Hepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nM anti-phospho(Ser/Thr)-Europium-antibody [#AD0180, PerkinElmer LAS, Rodgau-Jügesheim, Germany].The resulting mixture was incubated 1 h at 22° C. to allow the binding of the phosphorylated peptide to the anti-phospho(Ser/Thr)-Europium-antibody. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Europium-labelled anti-phospho(Ser/Thr) antibody to the Streptavidin-XLent. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a Viewlux TR-FRET reader (PerkinElmer LAS, Rodgau-Jügesheim, Germany). The “blank-corrected normalized ratio” (a Viewlux specific readout, similar to the traditional ratio of the emissions at 665 nm and at 622 nm, in which blank and Eu-donor crosstalk are subtracted from the 665 nm signal before the ratio is calculated) was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition).
Measurement: IC50 = 1.04 nM (pChEMBL 8.979999542236328)
Descriptors: MW 564.62, ALogP 6.02, HBA 8, HBD 2, PSA 102.14, QED 0.24
Compound (SMILES): COc1ccc2c(n1)c(C(=O)NCc1c(C)cc(N)nc1C)cn2Cc1ccc2ncc(Cl)cc2c1
InChIKey: VWMBRYCYRNXPCV-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Enzyme Inhibition: A 10 mM solution of the test compound was made in DMSO. This solution was serially diluted 1:5 in DMSO to yield 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256 and 0.00512 μM compound test solutions. A control tube containing only DMSO is included. 16 μL of each compound test solution was combined with 384 μL of assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Triton X-100) to yield a 4× test compound buffer stock.Separately, a 40 nM solution of human Plasma Kallikrein (Abcam) and a 93.6 μM solution Pro-Phe-Arg-AMC (Bachem) were made using assay buffer. These solutions are hereby referred to as 4×hPK and 2×PFR-AMC, respectively.60 μL of each 4× test compound buffer stock was combined with 60 μL of 4×hPK to yield 120 μL of 2× test compound buffer stock/2×hPK. 50 μL was removed from this mixture and placed into duplicate wells on a Microfluor 1Black U-bottom microtiter plate (Thermo Scientific). This plate was incubated for 5 minutes at 37° C. To each well, 50 μL of pre-warmed 2×PFR-AMC was added to start the enzymatic reaction. Cleavage of PFR-AMC was monitored in a Biotek Synergy H4 reader set at 37° C. Readings are taken every 43 seconds for 1 hour. The highest mean velocity over 20 reads (15 minutes) is used to calculate the IC50. The IC50 is calculated using the Gen5 (Biotek Instruments).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 500.99, ALogP 4.82, HBA 7, HBD 2, PSA 107.95, QED 0.35
Compound (SMILES): N=C(N)c1ccc(OC(=O)c2cnc(N3CCC(C(=O)N[C@@H](CC(=O)O)C(=O)O)CC3)s2)cc1
InChIKey: MSPBXGXHQIAMCD-AWEZNQCLSA-N
Target: Enteropeptidase (SINGLE PROTEIN), Homo sapiens
UniProt: P98073
Assay: Enteropeptidase Inhibition Assay: The inhibitory activity of the enteropeptidase inhibitor synthesized using the purified Recombinant Human Enteropeptidase and the substrate Acetyl-Asp-Asp-Asp-Asp-Lys-AFC (BioVision) was measured. 7.2 ng/mL of Enteropeptidase diluted with a buffer (20 mM Tris, 50 mM NaCl, pH 7.5) in a 96 well plate (Costar), 30 μM of Acetyl-Asp-Asp-Asp-Asp-Lys-AFC, several concentrations of Enteropeptidase inhibitors (1% DMSO concentration) were dispensed such that a final volume was 100 μL, and then the enzyme reaction was carried out at 30° C. for 1 hour. At this time, 1% DMSO, a substrate and Enteropeptidase instead of the compound were dispensed onto positive control wells, and 1% DMSO and a substrate were dispensed onto negative control wells. The enzyme reaction was started using an excitation wavelength of 380 nm and an emission wavelength of 500 nm in a fluorescence spectrometer, and then the rate of increase in fluorescence (milli-units per min) between 20 and 60 minutes was measured.
Measurement: Ki = 4.7 nM (pChEMBL 8.329999923706055)
Descriptors: MW 489.51, ALogP 0.91, HBA 9, HBD 5, PSA 196, QED 0.15
Compound (SMILES): NC(=O)c1cnc2ccc(-c3c(-c4ccc(F)cc4)ncn3CC(O)C(F)(F)F)cn12
InChIKey: INIGOXBLEFDAFT-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 21.3 nM (pChEMBL 7.670000076293945)
Descriptors: MW 433.37, ALogP 3.03, HBA 6, HBD 2, PSA 98.44, QED 0.47