lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(Cl)ccc1S(=O)(=O)Nc1noc2cc(Cn3cccn3)cc(OC)c12
InChIKey: VCCGVZAZGCNZHI-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7Z6
Assay: Kat8 Assay: TBDA. 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:   KAT8 15 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 = 1440 nM (pChEMBL 5.840000152587891)
Descriptors: MW 448.89, ALogP 3.54, HBA 8, HBD 1, PSA 108.48, QED 0.46
Compound (SMILES): COC1CC(CC#N)(n2ccc(-c3nc(-c4cnn(CCO)c4)cc4ncccc34)n2)C1
InChIKey: RUZWZSKJNBFIMH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% 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 JAK 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% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 5924 nM (pChEMBL 5.230000019073486)
Descriptors: MW 429.48, ALogP 2.77, HBA 9, HBD 1, PSA 114.67, QED 0.48
Compound (SMILES): COC(=O)c1cc2c(cc1C)/C(=C(/Nc1ccc(S(=O)(=O)NOCCN3CCN(C)CC3)cc1)c1ccccc1)C(=O)N2
InChIKey: CLWKPYABKROLCK-ZIADKAODSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Enzyme Inhibition: The inhibitory activities of compounds of the invention against VEGFR2 (VEGFR2 Kinase Enzyme System: Promega), were evaluated by mixing the VEGFR2 protein (1.56 ng/mL, 2 μL), substrate (Poly (4:1 Glu4, Tyr1), 100 ng/mL, 2 μL) with the test compound (2 μL at either 3 μM, 0.67 μM, 0.15 μM, 0.033 μM, 0.0073 μM, 0.0016 μM, 0.0036 μM or 0.00008 μM) for 90 min at 25° C. The kinase reaction was initiated by adding ATP (50 μM, 2 μL) and the mixture was incubated for 1 hr at 25° C. ADP-Glo reagent was added for 40 min (8 μL) then development reagent (16 μL) was added for 40 min prior to detection in a microplate reader (EnVision® Multilabel Reader, Perkin Elmer).
Measurement: Ki = 36.4 nM (pChEMBL 7.440000057220459)
Descriptors: MW 605.72, ALogP 3.17, HBA 9, HBD 3, PSA 129.31, QED 0.14
Compound (SMILES): O=C(c1cnc2ccc(-c3ccc(Cl)c(NS(=O)(=O)c4ccc(F)cc4F)c3)cn12)N1CCOCC1
InChIKey: RTIMCBHDKAJODS-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
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 < 10000 nM (pChEMBL 5)
Descriptors: MW 532.96, ALogP 4.21, HBA 6, HBD 1, PSA 93.01, QED 0.42
Compound (SMILES): C=CC(=O)N1CC(Nc2ncnc3[nH]ccc23)CCC1CO
InChIKey: DVFKFBAEIOVUBS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay: 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 JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. 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 > 10000 nM (pChEMBL 5)
Descriptors: MW 301.35, ALogP 0.91, HBA 5, HBD 3, PSA 94.14, QED 0.73
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@H](C)[C@H](OC)c2nc(C)c(C)s2)nnc1-c1cncc(C)c1
InChIKey: LVTQOZKWKDGPMC-VGSWGCGISA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding Assay: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.)
Measurement: EC50 = 7.3 nM (pChEMBL 8.140000343322754)
Descriptors: MW 558.69, ALogP 4.25, HBA 11, HBD 1, PSA 130.35, QED 0.3
Compound (SMILES): Cc1ncc(-c2cc(-c3cc(S(=O)(=O)NC4CCC(O)C4)ccc3C)cnc2N)o1
InChIKey: PSVUWFQVQAHYRE-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: Kinase Glo Luminescent Kinase Assay (Kglo) for PI 3-Kinase Alpha (A), PI 3-Kinase Beta (B), Vps34 (C), PI 4-Kinase Beta (D): The luminescence-based ATP detection reagent KinaseGlo was obtained from Promega, (Cat. No. V6714, Lot No. 236161) through Catalys, Wallisellen, Switzerland. L-alpha-phosphatidylinositol (PI, liver, bovine) was obtained from Avanti Polar Lipid (Cat. No. 840042C, Lot#LPI-274), phosphatidylinositol-4,5-bisphosphate (PIP(4,5)2) was obtained from Avanti Polar Lipid (Cat. No. 840046X). L-α-phosphatidylserine (PS) was obtained from Avanti Polar Lipid (Cat. No. 840032C), n-octylglucoside from Avanti Polar Lipid (Cat. No. 10634425001). Luminescence is a well established readout to determine ATP concentrations and can thus be used to follow the activity of many kinases regardless of their substrate. The Kinase Glo Luminescent Kinase Assay (Promega, Madison, Wis., USA) is a homogeneous HTS method of measuring kinase activity by quantifying the amount of ATP remaining in solution following a kinase reaction.50 nL of compound dilutions were dispensed onto black 384-well low volume Non Binding Styrene (NBS) plates (Costar Cat. No. NBS#3676). L-α-phosphatidylinositol (PI), provided as 10 mg/ml solution in methanol, was transferred into a glass tube and dried under a nitrogen beam. It was then resuspended in 3% OctylGlucoside (1-O-n-octyl-beta-D-glucopyranoside) by vortexing and stored at 4° C. 5 μl of a mix of PI/OctylGlucoside with the PI 3-kinase alpha and PI 3-kinase beta subtypes, or Vps34 or PI 4-kinase beta were added. Kinase reactions were started by the addition of 5 μl of an ATP-mix containing in a final volume 10 μl 10 mM TRIS-HCl pH 7.5, 3 mM MgCl2, 50 mM NaCl, 0.05% CHAPS, 1 mM DTT and 1 μM ATP at room temperature. Reactions were stopped with 10 μl of KinaseGlo and plates were read 10 mins later in a Synergy2 reader using an integration time of 0.1 seconds per well. 2.5 μM of NVP-BGT226 (1-(3-(trifluoromethyl)-4-(piperazin-1-yl)phenyl)-8-(6-methoxypyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-one) was added to the assay plates to generate the 100% inhibition of the kinase reaction, and the 0% inhibition was given by the solvent vehicle (90% DMSO in water). (1-(3-(trifluoromethyl)-4-(piperazin-1-yl)phenyl)-8-(6-methoxypyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-one) was used as a reference compound and included in all assay plates in the form of 16 dilution points in duplicate.IC50 values of the percentage inhibition of each compound at 8 concentrations (10, 3.0, 1.0, 0.3, 0.1, 0.030, 0.010 and 0.003 μM) n=2 were derived by fitting a sigmoidal dose-response curve to a plot of assay readout over inhibitor concentration as described. All fits were performed with the program XLfit4 (ID Business Solutions, Guildford, UK).
Measurement: IC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 428.51, ALogP 2.79, HBA 7, HBD 3, PSA 131.34, QED 0.57
Compound (SMILES): CC(C)(OCc1cc(-c2ccc(Cl)cc2)n(Cc2ccccc2Cl)n1)C(=O)O
InChIKey: HMGBBPKGJHXSQG-UHFFFAOYSA-N
Target: Monocarboxylate transporter 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15427
Assay: MCT4-Mediated Lactate Transport in NCI-H358 Lung Adenocarcinoma Cell: NCI-H358 lung adenocarcinoma cells may be used to measure MCT4 activity in cells with high native levels of MCT4 and low levels of MCT1 and are known to those with skill in the art. Preparation of BCECF-loaded cells and lactate transport activity may be determined as described for Assay 1.
Measurement: IC50 = 22000 nM (pChEMBL 4.659999847412109)
Descriptors: MW 419.31, ALogP 5.29, HBA 4, HBD 1, PSA 64.35, QED 0.57
Compound (SMILES): N#C[C@H]1CC[C@@H](c2cc(-c3ccc(NC(=O)c4cccn(-c5ccccc5)c4=O)cc3F)c3c(N)ncnn32)CC1
InChIKey: PHPKMXFRPJBGSR-BGYRXZFFSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Enzymatic Assay: Tyro3, and Mer:The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LUMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 547.59, ALogP 5.32, HBA 8, HBD 2, PSA 131.1, QED 0.31
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3ccc(OCCN4CCCC4=O)nc3)ncc2F)c1
InChIKey: XCCATKZDNIWAER-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Omnia Assay: EGFR (WT) and EGFR (T790M/L858R): The Omnia Assay Protocol for potency assessment against EGFR is performed as described in Example 251 above except that the EGFR-WT- and EGFR T790M/L858R-modified optimized reagent conditions are:[EGFR-WT]=5 nM, [ATP]=15 mM, [Y12-Sox]=5 mM (ATP KMapp˜12 mM); and [EGFR-T790M/L858R]=3 nM, [ATP]=50 mM, [Y12-Sox]=5 mM (ATP KMapp˜45 mM).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 477.5, ALogP 3.62, HBA 8, HBD 3, PSA 121.37, QED 0.38
Compound (SMILES): CCOc1cc2c(=O)oc(-c3ccccc3SC)nc2cc1OC
InChIKey: JTQBMTFVGGUDPN-UHFFFAOYSA-N
Target: Serine protease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P07477
Assay: Trypsin Assay: Materials: Trypsin, 100 ug (Roche, sequence grade, Mw 23500), Substrate S-2288 (Chromogenics, cat. no. 820852). Trypsin activity was determined at 37° C. in 10 mM Na phosphate pH 7.2, 0.5 M NaCl, with final concentrations of 0.8 ug/ml (34 nM) of Trypsin, 1 mM substrate, 5% DMSO in the presence of 0 uM, 0.1 uM, 1.0 uM and 10 uM of inhibitor, in 96 well plates by measuring absorbance at 405 nm in a plate reader (Spectramax).
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 343.4, ALogP 3.98, HBA 6, HBD 0, PSA 61.56, QED 0.65
Compound (SMILES): Nc1ncc(-c2ccc([C@]34C[C@H]3CN(C3CCC(F)(F)CC3)C4)cc2)cc1C(=O)N[C@H]1CC[C@H](O)CC1
InChIKey: WCTODNJGIAJWJX-WFEWNJSDSA-N
Target: Activin receptor type-1-like (SINGLE PROTEIN), Homo sapiens
UniProt: P37023
Assay: Caliper Assay: In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) 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 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
Measurement: IC50 = 135 nM (pChEMBL 6.869999885559082)
Descriptors: MW 510.63, ALogP 4.52, HBA 5, HBD 3, PSA 91.48, QED 0.55
Compound (SMILES): NC(=O)c1ncc(N2CCC[C@@H](NC(=O)C3CC3)C2)nc1Nc1ccc(C2CCN(C3CCCC3)CC2)cc1
InChIKey: AYPQJCUSHCNNTD-XMMPIXPASA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: HotSpot kinase Assay: The degree of inhibition of a panel of kinases is determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 uM ATP).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 531.71, ALogP 3.94, HBA 7, HBD 3, PSA 116.48, QED 0.47
Compound (SMILES): CNC[C@@H](c1ccc(Cl)c(Cl)c1)[C@@H](O)c1cccc(SC)c1
InChIKey: YDSDFUFLGLKXSJ-YOEHRIQHSA-N
Target: Sodium-dependent noradrenaline transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P23975
Assay: Uptake Assay Protocol: NET: This protocol was designed to measure inhibition of uptake by the human norepinephrine transporter. The reagents were human NET (HEK293F) cells, desipramine (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 μL polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1×HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human norepinephrine transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.
Measurement: Ki = 37.1 nM (pChEMBL 7.429999828338623)
Descriptors: MW 356.32, ALogP 4.75, HBA 3, HBD 2, PSA 32.26, QED 0.73
Compound (SMILES): C[C@H](CO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4ccnn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: OLQRMVXQMXDNNY-CQSZACIVSA-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 = 19.7 nM (pChEMBL 7.710000038146973)
Descriptors: MW 533.5, ALogP 3.62, HBA 9, HBD 4, PSA 147.05, QED 0.24
Compound (SMILES): CCc1cncc2ccc(NC(=O)[C@@H]3C[C@H]3c3ccc(S(=O)(=O)Nc4ncc(C5CCNCC5)cn4)cc3)cc12
InChIKey: VQGOXHJDTULAPP-WUFINQPMSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: ROCK Kinase Inhibition Assays: All compounds were initially prepared as 10 mM stocks in anhydrous dimethylsulfoxide (DMSO). A 20 μl aliquot of the 10 mM solutions was transferred to individual wells in column 1 of a 96-well polypropylene microtiter plate (Corning #3363) and diluted with DMSO to give a final compound concentration of 4 mM. Test compounds were then serially diluted 1:5 in DMSO for an 11-point concentration response and further diluted in the assay buffer bringing all compound concentrations to a final range of 100 μM to 10 pM in 2.5% DMSO. The assay was performed in white 96-well, flat-bottom, half-area, non-binding assay plate (Corning #3642) in assay buffer consisting of 20 mM HEPES (pH 7.5), 10 mM MgCl2*6H2O, 100 μM sodium orthovanadate, 0.05% CHAPS and 0.1% bovine serum albumin. A 10 μL aliquot of compound from each well of the intermediate dilution plate and 20 μL of a 2× substrate/enzyme solution containing acceptor substrate (800 nM RSK2 peptide KRRRLSSLRA (SEQ ID NO: 1)), ROCK2 enzyme (10 nM), or ROCK1 enzyme, and 1,4-Dithiothreitol (DTT, 2 μM) were added to all wells. The reaction was initiated by the addition of 10 μL of 4× stock solution ATP (2 ∥M). Reactions were thoroughly mixed manually, covered and allowed to incubate at room temperature for 75 min. Protein kinase activity was quantitated using Promega's KINASE-GLO luminescent Kinase Assay Kit according to the manufacturer's directions. ATP concentrations remaining in Test wells following the termination of the enzymatic reaction were compared against control wells containing equivalent amounts of DMSO containing no inhibitor (CTRL). ATP concentrations in both Test wells and CTRL wells were normalized against background (BKG) ATP concentrations in wells containing concentrations of inhibitor that completely inhibited the protein kinase under investigation (i.e. a concentration that prevented any consumption of ATP over the course of the incubation). Percent of Control (POC) values were determined for each concentration of compound tested according to the equation: POC=((Test well value−BKG)/(CTRL−BKG))*100IC50 values were calculated using the following 4-parameter logistic curve-fitting algorithm: f(x)=(A+((B−A)/(1+((x/C){circumflex over ( )}D))))IC50 values were converted to Ki values using the Cheng-Prusoff Equation: Ki=IC50/(1+([ATP]/Km ATP])).
Measurement: IC50 = 235 nM (pChEMBL 6.630000114440918)
Descriptors: MW 556.69, ALogP 4.6, HBA 7, HBD 3, PSA 125.97, QED 0.29
Compound (SMILES): CC(CC(F)F)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4cn(C)cc(-c5ccc(F)cc5)c4=O)nc3)c12
InChIKey: OSRKFUPTYNLOFP-UHFFFAOYSA-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 = 55.7 nM (pChEMBL 7.25)
Descriptors: MW 563.54, ALogP 5.36, HBA 8, HBD 3, PSA 126.82, QED 0.22
Compound (SMILES): COCCN1C(=O)Cc2cc(-c3cncc(-c4ccc(=O)n(C(C)C)c4)c3)ccc21
InChIKey: LPSPVIAPUVQYGG-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: In Vitro Enzyme Inhibition Using a Biochemical Peptide Phosphorylation Assay- Caliper Assay: Example 54:The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 uM of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 uM Na3VO4, 10 mM ß-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 uM ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reactions were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.
Measurement: IC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 403.48, ALogP 3.69, HBA 5, HBD 0, PSA 64.43, QED 0.63
Compound (SMILES): Cc1cc(-c2cc3ncccc3c(-c3ccn(C4(CC#N)CC(C#N)C4)n3)n2)cc(N)n1
InChIKey: CUQHZDCHJCJOFI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% 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 JAK 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% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 420.48, ALogP 3.99, HBA 8, HBD 1, PSA 130.09, QED 0.53
Compound (SMILES): Cc1c(-c2ccn(C)n2)cc(C(=O)Nc2ccc(-c3cc(C4CCN(C(=O)C(C)C)CC4)n4ncnc(N)c34)cc2)c(=O)n1-c1cccnc1
InChIKey: GUBJZSVCAQTUBA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: TAM Enzymatic Assay: The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LLIMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech). Percentage of inhibition was calculated for each concentration and IC50 value was generated from curve fitting with GraphPad Prism software.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 670.78, ALogP 4.85, HBA 11, HBD 2, PSA 158.33, QED 0.24
Compound (SMILES): OC(c1cnc(F)c(-c2ncnc3cc(N4CCOCC4)ccc23)c1)c1nccs1
InChIKey: HORBNTVKYAVYTP-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Patch Clamp Assay: The patch-clamp measurement was carried out at room temperature in whole-cell configuration on human embryonic kidney cells (HEK293) which have been transfected in a stable manner with the hERG gene.The whole-cell configurations were carried out using an automated patch clamp device (Patchliner, Nanion Technologies, Munich). This is a glass chip-based system with which automated whole-cell measurements on up to 8 cells simultaneously are possible. The glass chip has a hole of defined size to which the cell is transferred into the Gigaseal by application of a reduced pressure and brought into the whole-cell configuration. Buffer, cell suspension and test substances were added to microchannels of the chip using a Teflon-coated pipette. The cells were clamped to a holding potential of -80 mV. For measurement of substance-promoted inhibition of the Kv11.1 channel, the following voltage protocol was applied at 10-second intervals: 51 ms/-80 mV, 500 ms/+40 mV, 500 ms/-40 mV, 200 ms/-80 mV. The leakage current is subtracted by means of the P4 method. The cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cell had been collected, the seal was improved by addition of a seal enhancer buffer. As soon as the whole-cell configuration had been reached, the seal enhancer buffer was washed out and replaced by extracellular buffer. The measurement started in EC for 1.5 min. DMSO (vehicle control, 0.1% of DMSO) was then applied, and the control current was recorded for 3 min. The test substance was subsequently added twice in the same concentration, and the potassium current was measured for 3.5 min in each case.
Measurement: Ki = 20000 nM (pChEMBL 4.699999809265137)
Descriptors: MW 423.47, ALogP 3.21, HBA 8, HBD 1, PSA 84.26, QED 0.5
Compound (SMILES): Cc1ccc2c(n1)C(=O)N(CCN1CCC(c3noc4ccccc34)CC1)CC2
InChIKey: CRSNRKDXRDGEMB-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Binding Activity Assay: Binding affinity of the present compound for human 5-HT1A receptor, human 5-HT2A receptor, and human D2 receptor was measured by the following procedures.CHO cell membrane fraction in which human 5-HT1A receptor, human 5-HT2A receptor, and human D2 receptor were expressed was purchased from PerkinElmer, Inc. In a test for evaluating binding affinity, a test compound dissolved in dimethylsulfoxide (DMSO) and each receptor membrane sample diluted in buffer were mixed with [3H]8-OH-DPAT, [3H]Ketanserin, or [3H]Spiperone (all purchased from PerkinElmer, Inc.) for 5-HT1A receptor, 5-HT2A receptor, and D2 receptor, respectively. Each mixture was incubated at room temperature for 60 minutes. Then, the mixture was added quickly on a glassfiber filter plate (Multiscreen FB, Millipore, Inc.) coated with 0.3% aqueous polyethylenimine, and vacuum-filtered. Radioactivity remaining on the filter was measured with a liquid scintillation counter (PerkinElmer, Inc.). Binding inhibition rate was calculated from the following formula.
Measurement: Ki = 1.59 nM (pChEMBL 8.800000190734863)
Descriptors: MW 390.49, ALogP 3.41, HBA 5, HBD 0, PSA 62.47, QED 0.68
Compound (SMILES): C=CC(=O)N1CCC(Oc2cc3c(Nc4ccc(Cl)cc4Cl)ncnc3cc2OC)C1
InChIKey: VJZFBNUADOMMRR-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Cell Viability Assay and IC50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described. Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone at a final volume of 40 μL per well. After 3 days hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes, and bioluminescence was determined using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO treated cells, and normalized values were plotted in GraphPad Prism using non-linear regression fit to normalized data with a variable slope. IC50 values were calculated by GraphPad Prism at 50% inhibition.
Measurement: IC50 = 320 nM (pChEMBL 6.5)
Descriptors: MW 459.33, ALogP 4.85, HBA 6, HBD 1, PSA 76.58, QED 0.53
Compound (SMILES): C[C@H](NC(=O)c1c(N)nn2cccnc12)c1cc2cccc(C#Cc3ccnn3C)c2c(=O)n1-c1ccccc1
InChIKey: SUKUGYAKJGLYNW-IBGZPJMESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition Assays: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/mL). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration of 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 μL 1N HCl followed by 160 μL CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 528.58, ALogP 3.24, HBA 9, HBD 2, PSA 125.13, QED 0.34
Compound (SMILES): CCCCOC[C@@H]1COc2cc(C3CCC(N)(COP(=O)(O)O)C3)ccc2O1
InChIKey: KLAYGTVYXLUWAC-KOHRHEQBSA-N
Target: Sphingosine-1-phosphate lyase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95470
Assay: hS1P1 ERK Phosphorylation (S1P1 pERK): hS1P1/CHO cells were plated into BD Amine 384-well plates the day before the assay. On the day of the assay, growth medium was removed and replaced with serum-free medium (Ham's F-12 Invitrogen) and incubated for 2 hours. Test compounds pre-diluted in HBSS (Gibco)/20 mM HEPES (Gibco) were transferred to the cell plates and incubated for 7 minutes at 37° C. Cells were lysed in lysis buffer (Perkin Elmer) and phospho-ERK was measured using the SureFire pERK kit (Perkin Elmer) as described by the manufacturer. Data was plotted as percentage activation of the test compound relative to the efficacy of 10 μM S1P. The EC50 is defined as the concentration of test compound which produces 50% of the maximal response and was quantified using the 4 parameter logistic equation to fit the data.
Measurement: EC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 415.42, ALogP 2.72, HBA 6, HBD 3, PSA 120.47, QED 0.42
Compound (SMILES): COc1cc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)C)nn4-c4ccc(C)cc4)c4ccccc34)n2)cc(C(=O)NCCN2CCOCC2)c1
InChIKey: YARHUYMJLJCURZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Syk Enzyme Inhibition 2: The inhibitory activities of compounds of the invention against Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (2000 ng/mL, 2.5 μL) is incubated with the test compound (either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL, 2.5 μL each) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solutions (2.5 μL, 60 μM ATP) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 224 nM (pChEMBL 6.650000095367432)
Descriptors: MW 755.88, ALogP 7.5, HBA 11, HBD 4, PSA 156.79, QED 0.09
Compound (SMILES): Cc1nc2c(F)cc(-c3nc(Nc4ccc5c(n4)CCN(C(=O)CCN(C)C)C5)ncc3F)cc2n1C(C)(C)C
InChIKey: AKDZNPFQMDRCMU-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: LANCE Ultra ULight Assay: 1. Take 10 mM stock solution of the test compound, in 96-well compound plate, DMSO was used to dilute the compound to an initial concentration of 100x, then this concentration was used as the first concentration, 3-fold diluted to make 10 serial concentrations; 1 μL each serial dilution was then added to 19 μL 1x reaction buffer to prepare 5x compound for use; 2 μL 5x compound was transferred from 96-well plate to 384-well plate; compound-free control well was added with 2 μL the following liquid: 1x reaction buffer with the addition of 1 μL DMSO; 2 μL 250 mM EDTA was added to the Min control well.2.1x reaction buffer was used to formulate the kinase, substrate and ATP into a 2.5xenzyme/substrate mixture and 2.5xATP solution respectively. In the experiment, the final concentration of CDK4/CycD3 kinase is 0.76 ng/μL, the final concentration of ATP is 8 μM; the final concentration of CDK6/CycD3 kinase is 0.5 ng/μL, the final concentration of ATP is 50M; the final concentration of CDK2/CycA2 kinase is 0.86 ng/μL, the final concentration of ATP is 1 μM; the final concentration of CDK2/CycE1 kinase is 1.016 ng/μL, the final concentration of ATP is 20M; 2.5xenzyme/substrate mixture was added to a 384-well plate, incubated at room temperature for 5 minutes; then added with 2.5xATP solution, reacted at room temperature for 30 minutes.3. LANCE Detection Buffer was used, 1xto prepare 2xLANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) for use. After the enzymatic reaction was continued for 30 minutes, 10 mM EDTA was added to 384-well plate and the mixture was reacted at room temperature for 5 minutes. Then LANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) was added, reacted at room temperature for 1 hour.4. The 384-well plate was placed in HERAEUS Multifuge X1R centrifuge, centrifuged at 2000 rpm for 2 minutes; data were measured on EnVision, 337 nM wavelength laser was used as the excitation light, test at RFU665 nM and RFU615 nM, and RFU665 nM/RFU615 nM x10000 was used as the final data for analysis.
Measurement: IC50 = 7.47 nM (pChEMBL 8.130000114440918)
Descriptors: MW 548.64, ALogP 4.81, HBA 8, HBD 1, PSA 92.07, QED 0.37
Compound (SMILES): COC1CC(CC#N)(n2ccc(-c3nc(-c4cnn(CCO)c4)cc4ncccc34)n2)C1
InChIKey: RUZWZSKJNBFIMH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% 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 JAK 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% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 1233 nM (pChEMBL 5.909999847412109)
Descriptors: MW 429.48, ALogP 2.77, HBA 9, HBD 1, PSA 114.67, QED 0.48
Compound (SMILES): CC(C)(OCc1cc(-c2ccc(Cl)cc2)n(Cc2ccccc2Cl)n1)C(=O)O
InChIKey: HMGBBPKGJHXSQG-UHFFFAOYSA-N
Target: Monocarboxylate transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53985
Assay: MCT1-Mediated Lactate Transport in BT20 Breast Cancer Cells: MCT1 activity may be measured using BT-20 breast cancer cells that express high native levels of MCT1, but do not express MCT4 and are known to those with skill in the art. Preparation of BCECF loaded cells are as described for Assay 1. Lactate transport assay is as described for Assay 1, except 10 mM L-lactate (rather than 50 mM) is added.
Measurement: IC50 = 454 nM (pChEMBL 6.340000152587891)
Descriptors: MW 419.31, ALogP 5.29, HBA 4, HBD 1, PSA 64.35, QED 0.57
Compound (SMILES): Nc1ncc(-c2ccc([C@]34C[C@H]3CN(C3CCC(F)(F)CC3)C4)cc2)cc1C(=O)N[C@H]1CC[C@H](O)CC1
InChIKey: WCTODNJGIAJWJX-WFEWNJSDSA-N
Target: Bone morphogenetic protein receptor type-1B (SINGLE PROTEIN), Homo sapiens
UniProt: O00238
Assay: Caliper Assay: In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) 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 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
Measurement: IC50 = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 510.63, ALogP 4.52, HBA 5, HBD 3, PSA 91.48, QED 0.55
Compound (SMILES): CNC[C@@H](c1ccc(Cl)c(Cl)c1)[C@@H](O)c1cccc(SC)c1
InChIKey: YDSDFUFLGLKXSJ-YOEHRIQHSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Uptake Assay Protocol: DAT: This protocol was designed to measure inhibition of uptake by the human dopamine transporter. The reagents were human DAT (HEK293F) cells, GBR 12909 (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 pt polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1×HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human dopamine transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.12 μL of test compound (10 mM) in DMSO was added to the wells, and nomifensine was used as a control. GBR 12909 (final assay concentration of 10 μM) was used for background signal. The neurotransmitter transporter dye was prepared in AB prior to use.
Measurement: Ki = 40.3 nM (pChEMBL 7.389999866485596)
Descriptors: MW 356.32, ALogP 4.75, HBA 3, HBD 2, PSA 32.26, QED 0.73
Compound (SMILES): C[C@H](CO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4ccnn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: OLQRMVXQMXDNNY-CQSZACIVSA-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 = 5.4 nM (pChEMBL 8.270000457763672)
Descriptors: MW 533.5, ALogP 3.62, HBA 9, HBD 4, PSA 147.05, QED 0.24