lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C=CC(=O)N1CC[C@]2(C1)C[C@H](n1c(NC(=O)c3ccc(-c4cnco4)s3)nc3cc(N(C)C(C)=O)ccc31)C2
InChIKey: GFXJEHXKSRGUJJ-LBLBPIOVSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Inhibition Assay: The in vitro kinase assays were performed at Nanosyn (Santa Clara, Calif.) utilizing microfluidic detection technology. The test compounds were serially pre-diluted in DMSO and added, by the acoustic dispensing (Labcyte 550), directly to 384 well assay plates into 10 uL of a buffer with enzyme (ITK, TXK, BTK or TEC) comprising: 100 mM HEPES, pH7.5, 5 mM MgCl2, 0.1% bovine serum albumin, 1 mM DTT, 0.01% Triton X-100 and the enzyme. Final DMSO concentration was maintained at 1% in all samples, including the controls. The reactions were initiated by addition of ATP (1 mM final concentration) and the fluorescently labeled peptide substrate to a final concentration of 1 uM, and incubated for 3 hours at 25° C. Following incubation, the reactions were quenched by addition of 40 μL of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 50 mM EDTA). Terminated plates were analyzed using Caliper LabChip 3000 microfluidic electrophoresis instrument (Caliper Life Sciences/Perkin Elmer). The enzymatic modification of the peptide substrate (phosphorylation) results in a change of net charge enabling electrophoretic separation of product from substrate. As substrate and product are separated by electrophoresis, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks was the parameter measured, reflecting enzyme activity. In the presence of inhibitor, the ratio between product and substrate is altered: signal of the product decreases, while the signal of the substrate increases. Activity in each test sample was determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product and S is the peak height of the FAM-cAMP substrate. For each compound, enzyme activity was measured at 12 concentrations spaced by 3× dilution intervals. Negative control samples (0%-inhibition in the absence of inhibitor, DMSO only) and positive control samples (100%-inhibition, in the absence of enzyme or in the presence of control inhibitor) were assembled in replicates of four and were used to calculate %-inhibition values in the presence of compounds. Percent inhibition (Pinh) was determined using the following equation: Pinh=(PSR0%−PSRinh)/(PSR0%−PSR100%)*100, where PSRinh is the product sum ratio in the presence of inhibitor, PSR0% is the product sum ratio in the absence of inhibitor and PSR100% is the product sum ratio in 100%-inhibition control samples. To determine IC50 values, the inhibition curves (Pinh versus inhibitor concentration) were fitted by 4 parameter sigmoid dose-response model using XLfit software (IDBS).
Measurement: IC50 = 0.64 nM (pChEMBL 9.1899995803833)
Descriptors: MW 544.64, ALogP 4.73, HBA 8, HBD 1, PSA 113.57, QED 0.35
Compound (SMILES): NCC1(c2cccc(-c3ncnc4[nH]ccc34)c2)Cc2ccc(CN3CCC[C@@H]3C(N)=O)cc2C1
InChIKey: LICNRIXPNQJNNF-RIBGEGAISA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: TAM Kinase 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 LUMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl (see Axl autophosphorylation assay above), 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 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 466.59, ALogP 3.07, HBA 5, HBD 3, PSA 113.92, QED 0.4
Compound (SMILES): Nc1nc2cc(-c3ccc4ncc(N5CCNC(=O)C5)nc4c3)ccc2o1
InChIKey: NDZYUDOUBFCGCX-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI 3-Kinase HTRF Assay: 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 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM 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 360.38, ALogP 1.96, HBA 7, HBD 2, PSA 110.17, QED 0.56
Compound (SMILES): Cc1ccoc1C(=O)Nc1cccc(C#Cc2cncc(-c3nnn(CCCO)n3)c2)c1
InChIKey: OGVARLFWIBVKET-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: Kinase Assay: PDGFRβ: Biochemical PDGFRβ kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 36 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain PDGFR-b protein (Millipore). Following a 60 minute incubation at 300° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm. IC50 values for compound inhibition were calculated directly from graphs of optical density (arbitrary units) versus compound concentration following subtraction of blank values.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 428.45, ALogP 2.67, HBA 8, HBD 2, PSA 118.96, QED 0.45
Compound (SMILES): C[C@@H]1COCCN1C1CN(c2cnc3ccc(-c4ccc5oc(N)nc5c4)nc3n2)C1
InChIKey: AFUARVRQFCIDAJ-CYBMUJFWSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI 3-Kinase HTRF Assay: 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 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM 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 417.47, ALogP 2.32, HBA 9, HBD 1, PSA 106.43, QED 0.54
Compound (SMILES): COC(=O)N1c2ccc3c(nc(C(C)Cc4ccccc4)n3C3CCNCC3)c2CC[C@@H]1C
InChIKey: ZZOYJWJFSQANTA-GGYWPGCISA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: HTRF Biochemical Assay for CBP and BRD4 Activity: The assay was performed in a final volume of 6 μL in assay buffer containing 50 mM Hepes (pH 7.5, (0.5M Hepes, pH 7.5 solution; Teknova H1575)), 0.5 mM GSH, 0.01% BGG (0.22 μM filtered, Sigma, G7516-25G), 0.005% BSA (0.22 μM filtered, EMD Millipore Corporation, 126575) and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 33 μM to 1.7 nM, top to lowest dose, respectively. 3 of 2× Protein and 3 μL of 2× Peptide Ligand were added to assay plates (pre-stamped with compound). Plates were incubated for varying times at room temperature prior to measuring the signal. TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) was measured on the PHERAstar (BMG, equipped with HTRF optic module [337/520/490]) or on the Envision (PerkinElmer, equipped with the TRF Laser unit, TRF dual mirror D400/D505 and emission filters M520 and M495). Data were reported as percent inhibition compared with control wells based on the following equation: % inh=1−((TR-FRET ratio−AveLow)/(AveHigh−AveLow)) where TR-FRET ratio=(Fluorescence at 520 nm/Fluorescence at 490 nm)*10000), AveLow=average TR-FRET ratio of no enzyme control (n=32), and AveHigh=average TR-FRET ratio of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm. For all assay formats data were reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured Fluorescence, AveLow=average Fluorescence of no enzyme control (n=32), and AveHigh=average Fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 = 50500 nM (pChEMBL 4.300000190734863)
Descriptors: MW 446.6, ALogP 5.21, HBA 5, HBD 1, PSA 59.39, QED 0.6
Compound (SMILES): O=C1CCc2c(Oc3ccc4c(c3)[C@H]3[C@H](NC(=O)c5ccc(CN6CCNCC6)c(C(F)(F)F)c5)[C@H]3O4)ccnc2N1
InChIKey: VCCJAEXPICPGMM-QKDODKLFSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: TBD: TBD
Measurement: IC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 579.58, ALogP 3.84, HBA 7, HBD 3, PSA 104.82, QED 0.41
Compound (SMILES): CCCCc1cc(Cl)ccc1-c1cccc(C(O)/C=C/[C@H]2CCC(=O)N2CCCc2ccc(C(=O)O)cc2)c1
InChIKey: OUJVIJXEGVYSFE-DQOFJDJGSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: cAMP Assay: A 384-well drug plate was prepared to contain 6 test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 microliters. The reaction was initiated by mixing 5 microliters drug dilutions with 5 microliters of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 microliters anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH 7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 microliters biotinylated-cAMP/streptavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH 7.4) for 45 min at room temperature. Fluorescence changes were read using a Fusion-alpha microplate reader.
Measurement: Ki = 5158 nM (pChEMBL 5.289999961853027)
Descriptors: MW 546.11, ALogP 7.26, HBA 3, HBD 2, PSA 77.84, QED 0.23
Compound (SMILES): C=CC(=O)N1CC[C@]2(C1)C[C@H](n1c(NC(=O)c3cccs3)nc3ccccc31)C2
InChIKey: IBELJEQCPODFNE-VVONHTQRSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Inhibition Assay: The in vitro kinase assays were performed at Nanosyn (Santa Clara, Calif.) utilizing microfluidic detection technology. The test compounds were serially pre-diluted in DMSO and added, by the acoustic dispensing (Labcyte 550), directly to 384 well assay plates into 10 uL of a buffer with enzyme (ITK, TXK, BTK or TEC) comprising: 100 mM HEPES, pH7.5, 5 mM MgCl2, 0.1% bovine serum albumin, 1 mM DTT, 0.01% Triton X-100 and the enzyme. Final DMSO concentration was maintained at 1% in all samples, including the controls. The reactions were initiated by addition of ATP (1 mM final concentration) and the fluorescently labeled peptide substrate to a final concentration of 1 uM, and incubated for 3 hours at 25° C. Following incubation, the reactions were quenched by addition of 40 μL of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 50 mM EDTA). Terminated plates were analyzed using Caliper LabChip 3000 microfluidic electrophoresis instrument (Caliper Life Sciences/Perkin Elmer). The enzymatic modification of the peptide substrate (phosphorylation) results in a change of net charge enabling electrophoretic separation of product from substrate. As substrate and product are separated by electrophoresis, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks was the parameter measured, reflecting enzyme activity. In the presence of inhibitor, the ratio between product and substrate is altered: signal of the product decreases, while the signal of the substrate increases. Activity in each test sample was determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product and S is the peak height of the FAM-cAMP substrate. For each compound, enzyme activity was measured at 12 concentrations spaced by 3× dilution intervals. Negative control samples (0%-inhibition in the absence of inhibitor, DMSO only) and positive control samples (100%-inhibition, in the absence of enzyme or in the presence of control inhibitor) were assembled in replicates of four and were used to calculate %-inhibition values in the presence of compounds. Percent inhibition (Pinh) was determined using the following equation: Pinh=(PSR0%−PSRinh)/(PSR0%−PSR100%)*100, where PSRinh is the product sum ratio in the presence of inhibitor, PSR0% is the product sum ratio in the absence of inhibitor and PSR100% is the product sum ratio in 100%-inhibition control samples. To determine IC50 values, the inhibition curves (Pinh versus inhibitor concentration) were fitted by 4 parameter sigmoid dose-response model using XLfit software (IDBS).
Measurement: IC50 = 33.2 nM (pChEMBL 7.480000019073486)
Descriptors: MW 406.51, ALogP 4.09, HBA 5, HBD 1, PSA 67.23, QED 0.66
Compound (SMILES): Cn1cc(-c2cc(-c3cnn(C4(CC#N)CC(C#N)C4)n3)c3cccnc3c2)c(N)n1
InChIKey: BFWSHTMRWBTCFB-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 = 105 nM (pChEMBL 6.980000019073486)
Descriptors: MW 409.46, ALogP 3.02, HBA 9, HBD 1, PSA 135.02, QED 0.55
Compound (SMILES): O=C1CNCCN1Cc1nc2c(F)c(F)c(F)c(F)c2[nH]1
InChIKey: BAAMPJPJCCDRKI-UHFFFAOYSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1G (SINGLE PROTEIN), Homo sapiens
UniProt: O43497
Assay: High-Throughput Cav3.1 T-Type Fluorescent Assay: Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior to use in the FLIPR assay. 100 μL of cells (2.0×106 cell/mL) containing doxycyline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression) were added to each well using a Multidrop (Thermo Scientific, Waltham, Mass.) and were maintained in 5% CO2 incubator at 37° C. On the morning of the assay, cells were transferred to a 5% CO2 incubator at 29° C. Cells were washed with a wash buffer containing (in mM): 118 NaCl, 18.4 HEPES, 11.7 D-glucose, 0.05 CaCl2, 0.5 MgSO4, 1 KCl, and 1.2 KH2PO4, with the pH adjusted to 7.2 with NaOH. 4.4 μM of the fluorescent indicator dye, Fluo-4 (Invitrogen), prepared in pluronic acid (Sigma-Aldrich), were loaded into the wells and incubated for 45 minutes at 29° C. in 5% CO2. Cells were then rinsed with the following low Ca2+ buffer (in mM): 0.34 Na2HPO4, 4.2 NaHCO3, 0.44 KH2PO4, 0.41 MgSO4, 0.49 MgCl2-6H2O, 20 HEPES, 5.5 D-Glucose, 137 NaCl, 5.3 KCl, and 0.001 CaCl2, with 0.1% BSA and the pH adjusted to 7.2 with NaOH. Concentration-dependent response curves were generated from 5 mM stock solutions prepared in DMSO (Sigma-Aldrich) and diluted in the buffer containing low Ca2+ and incubated for 20 minutes at 29° C. in 5% CO2 Calcium entry was evoked with an addition of (in mM): 0.34 Na2HPO4, 4.2 NaHCO3, 0.44 KH2PO4, 0.41 MgSO4, 0.49 MgCl2-6H2O, 20 HEPES, 5.5 D-Glucose, 137 NaCl, 5.3 KCl, and 6 CaCl2, with 0.1% BSA and the pH adjusted to 7.2 with NaOH. A change in the Fluo-4 fluorescence signal was assessed using FLIPRTETRA¿ instrument (Molecular Devices, Sunnyvale, Calif.) for 3 minutes following the elevation of extracellular KCl using an illumination wavelength of 470-495 nm with emissions recorded at 515-575 nm.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 302.23, ALogP 1.05, HBA 3, HBD 2, PSA 61.02, QED 0.5
Compound (SMILES): C=CC(=O)N1CC(CCn2c(=O)ccc3cnc(Nc4cccc(C(=O)N5CCCCC5)c4)nc32)C1
InChIKey: IIBYBGNFQITLQU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Biochemical JAK and Tyk2 Kinase Assays: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies. Serially or discretely diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 486.58, ALogP 3.2, HBA 7, HBD 1, PSA 100.43, QED 0.52
Compound (SMILES): CC(C)n1nc(C(=O)Nc2ccc(Oc3ccnc4[nH]nc(N[C@@H](C)CO)c34)c(F)c2)c(=O)n(-c2ccc(F)cc2)c1=O
InChIKey: AAYNQFQFLVLOHH-HNNXBMFYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
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 = 18700 nM (pChEMBL 4.730000019073486)
Descriptors: MW 592.56, ALogP 3.36, HBA 11, HBD 4, PSA 169.05, QED 0.2
Compound (SMILES): O=C(O)C[C@H](NC(=O)CNC(=O)c1cncc(NC2=NCC(O)CN2)c1)c1cc(Br)cc(C(F)(F)F)c1
InChIKey: JXVVEFIKDDVLOS-KRWDZBQOSA-N
Target: Integrin alpha-V/beta-3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Solid Phase Receptor Assay (SPRA) for alphaVbeta3: Recombinant human vitronectin (R& D Systems, 2308-VN) diluted to 1 μg/mL in TBS+ buffer (25 mM Tris pH 7.4, 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2) was added to wells (50 μL/well) of a 96-well half-well transparent microtiter plate (Greiner 675061) and incubated overnight at 4° C. Wells were washed 3 times with 150 μL TBS+ and 150 μL of blocking buffer (TBS+ with 1% bovine serum albumin, Sigma A7906) were added. The plate was incubated for 1 hr at 37° C. and then washed 3× with TBS+ buffer. Recombinant human integrin αvβ3 (R&D Systems, 3050-AV) was diluted to 1 μg/mL in TBS+/0.1% bovine serum albumin. Compounds were diluted 1:100 into the integrin solution and then 50 μL added to empty wells of the washed vitronectin-coated plate according to a standard template with each sample repeated in triplicate. After incubation for two hours at room temperature, the plate was washed 3× with 150 μL of TBS+ buffer. To each well, 50 μL of biotinylated anti-αv antibody (R&D Systems, BAF1219) at 0.5 μg/mL in TBS+/0.1% BSA were added and the plate covered and incubated for 1 hr at room temperature. After washing the plate 3× with 150 μL of TBS+ buffer, 50 μL of streptavidin-conjugated horseradish peroxidase (R&D Systems, DY998) diluted in TBS+ blocking buffer were added to the wells and the plate incubated for 20 min at room temperature. The plate was washed 3× with TBS+ buffer followed by 50 μL of TMB substrate (Sigma, T4444) added to each well and the plate was incubated for 20 min at room temperature. Plates were read by colorimetric detection at 650 nm wavelength using a Tecan Safire II plate reader. Concentration-response curves were constructed by non-linear regression (best fit) analysis, and IC50 values were calculated for each compound.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 587.35, ALogP 1.66, HBA 8, HBD 6, PSA 165.04, QED 0.27
Compound (SMILES): C[C@@H]1CNC(=O)c2cc3ccc(NC(=O)c4cnn(Cc5ccccc5)c4)nc3n21
InChIKey: XYMNFMNOEWBPNB-CQSZACIVSA-N
Target: Ribosomal protein S6 kinase alpha-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P51812
Assay: Kinase Assay: Radioisotope assays were performed for the evaluation of the kinase target profiling and all assays were performed in a designated radioactive working area. The kinase targets were RSK1, RSK2, RSK3, RSK4 and MK2. The kinase assays (in duplicate) were performed at 30° C. for 15 minutes in a final volume of 25 μL according to the following assay reaction recipe:  Component 1: 5 μL of diluted active kinase target (100 ng per reaction)  Component 2: 5 μL of peptide substrate (0.5 μg per reaction) (for RSK1, RSK2, RSK3 and RSK4, RSK S6K substrate was used; for MK2, HSP27tide was used)  Component 3: 5 μL of kinase assay buffer  Component 4: 5 μL of compound described herein (various concentrations: 0, 0.1, 1, 10, 100 or 1000 nM or 1, 3, 10, 30, 100, 300 nM)  Component 5: 5 μL of 33P-ATP (5 μM stock solution, 0.8 μCi; 20 μM final concentration)The assay was initiated by the addition of 33P-ATP and the reaction mixture incubated at 30° C. for 15 minutes. After the incubation period, the assay was terminated by spotting 10 μL of the reaction mixture onto Multiscreen phosphocellulose P81 plate. The Multiscreen phosphocellulose P81 plate was washed 3 times for approximately 15 minutes each in a 1% phosphoric acid solution. The radioactivity on the P81 plate was counted in the presence of scintillation fluid in a Trilux scintillation counter.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 400.44, ALogP 2.84, HBA 6, HBD 2, PSA 93.84, QED 0.55
Compound (SMILES): COC/C=C/C(=O)N1CC[C@@H](n2nc(-c3ccc(Oc4ccccc4)cc3)c(C(N)=O)c2N)C1
InChIKey: HXVTZYMAJRTBSQ-KRZKXXONSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro Inhibitory Activities on ITK: In vitro activity in inhibiting ITK kinase was determined similar to that in respect of BTK kinase.The inhibitory activity of the compound of Example 9 on BTK is very high with an IC50 value of 1.9 nM, comparable to the literature compound, Ibrutinib/PCI-32765, which has been approved for clinical application. The platform for the enzymatic assay on ITK kinase (another Tec Kinase, mainly expressed in T cells) was establish with the same method, and the inhibitory ability of the compound of example 9 on ITK was tested. The results showed that IC50 value of this inhibition of ITK was more than 1000 nM. The selectivity of the compound of example 9 on BTK vs ITK was calculated as more than 1000 folds, while the selectivity of the literature compound, PCI-32765, was reported as approximately 100 folds. Accordingly, the selectivity of the compound of example 9 is significantly higher than that of PCI-32765.
Measurement: IC50 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 461.52, ALogP 3, HBA 7, HBD 2, PSA 125.7, QED 0.5
Compound (SMILES): Cn1nccc1Nc1nccc(-c2cc3n4c(nnc4c2)[C@@H](Cc2ccc(F)cc2)CCO3)n1
InChIKey: VJJWYAULXXNGLG-MRXNPFEDSA-N
Target: Mitogen-activated protein kinase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16659
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 2 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm. The data collected from each well was fit to a straight line, and the resulting rates were used to calculate percent of control. Percent of control was plotted against compound concentration, and IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.311 nM (pChEMBL 9.510000228881836)
Descriptors: MW 456.49, ALogP 3.91, HBA 9, HBD 1, PSA 95.05, QED 0.43
Compound (SMILES): CN(C)Cc1cccc(-c2cc(C(=O)/N=c3\[nH]c4ccccc4n3CC(C)(C)O)ccn2)c1
InChIKey: SCNSTJJOCSEKLM-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Biochemical EGFR Inhibition Assays (5 mM ATP): 5 μL of EGFR enzyme form in assay buffer (50 mM HEPES pH 7.3, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween 20, 2 mM DTT) are dispensed into columns 1-23, than 5 μL of ATP and ULight-poly-GT substrate (PerkinElmer; TRF0100-M) mix in assay buffer is added to all wells (final assay concentration of the ULight-poly-GT substrate 200 nM). Each of the different EGFR enzyme form assays is available at low ATP (final assay concentration 5 μM). After 90 minutes incubation at room temperature 5 μL EDTA (final assay concentration 50 mM) and LANCE Eu-anti-P-Tyr (PT66) antibody (PerkinElmer, AD0069) (final assay concentration 6 nM) mix are added to stop the reaction and start the binding of the antibody. After additional 60 minutes incubation at room temperature the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the TR-FRET LANCE Ultra specs of PerkinElmer (used wavelengths: excitation 320 nm, emission 1 665 nm, emission 2 615 nm). Each plate contains 16 wells of a negative control (diluted DMSO instead of test compound; w EGFR enzyme form; column 23) and 16 wells of a positive control (diluted DMSO instead of test compound; w/o EGFR enzyme form; column 24). Negative and positive control values are used for normalization and IC50 values are calculated and analysed using a 4 parametric logistic model.
Measurement: IC50 = 213 nM (pChEMBL 6.670000076293945)
Descriptors: MW 443.55, ALogP 3.61, HBA 5, HBD 2, PSA 86.51, QED 0.48
Compound (SMILES): CCCCNc1ncc2c(-c3ccc(N4CCN(C)CC4)cc3)cn(C3CCC(O)CC3)c2n1
InChIKey: QDCNDOVOAIZYTL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Inhibition Assay: Inhibition constants of MerTK, Flt3, Tyro3 and Axl kinase activity by an active compound as described herein is determined at the Km for ATP using a microfluidic capillary electrophoresis (MCE) assay in which phosphorylated and unphosphorylated substrate peptides were separated and analyzed using a LabChip EZ Reader.Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μl, of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 mM incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 462.64, ALogP 4.54, HBA 7, HBD 2, PSA 69.45, QED 0.5
Compound (SMILES): COc1cc(F)ccc1C1=C(c2ccc(O[C@H]3CCN(CCCF)C3)cc2)c2ccc(O)cc2CCC1
InChIKey: KEZQAOPYJUSQSS-SANMLTNESA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Estrogen Receptor Coactivator Assay: It is a competition assay, where binding of a test compound to a complex comprised of (i) His6-ERα298-554 protein representing ERα ligand-binding domain, (ii) Tb-labeled His6 antibody, (iii) a fluorescein-labeled PGC1α coactivator peptide (EAEEPSLLKKLLLAPANTQ), and (iv) estradiol, results in a decrease of the TR-FRET signal due to dissociation of the coactivator peptide. His6-ERα298-554 proteins were expressed as WT or D538G or Y537S mutants in E. coli and purified by affinity chromatography. The assay works in a homogeneous mix-and-read format. In a typical experiment, a 4 μL mixture of 0.5 nM His6-ERα298-554, 0.5 nM Tb-labeled His6 antibody, 250 nM PGC1a peptide, and 3 nM estradiol in 100 mM potassium phosphate, pH 7.4, 0.01% Tween-20, 0.02% NaN3, 5 mM DTT, was added to 40 nL test compound in DMSO and incubated overnight at room temperature. The TR-FRET 520:495 nm emission ratio was calculated and used to determine the IC50 value from a dose response curve fit to the 4-parameter logistic equation. The antagonist activity with respect to estrogen receptors in this test is given by the concentration which inhibits 50% of the estrogen receptor activity (or IC50) in nM.
Measurement: IC50 = 224 nM (pChEMBL 6.650000095367432)
Descriptors: MW 505.61, ALogP 6.65, HBA 4, HBD 1, PSA 41.93, QED 0.37
Compound (SMILES): Nc1nnc(N2CCC(N3C[C@H](Cn4cccn4)OC[C@@H]3Cc3ccc(Cl)cc3)CC2)[nH]1
InChIKey: NQWHSRRBFUUUAC-PMACEKPBSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: ERG Channel Binding Assay: Fluorescence polarization assay based on the principle of fluorescence polarization where a red-shifted fluorescent tracer is displaced from the hERG channel by compounds that bind to the channel was used to establish the binding of the compounds to hERG channel. Briefly, the compounds in serial dilutions were incubated with a membrane fraction containing hERG channel protein and a high-affinity red fluorescent hERG channel ligand in black 384-well plate. The reaction was incubated for 4 h at room temperature followed by the measurement of the fluorescence polarization using Tecan Spark multimode plate reader at excitation wavelength of 535 nm and emission wavelength 590 nm. The IC50 values were calculated using GraphPad Prism.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 456.98, ALogP 2.22, HBA 8, HBD 2, PSA 101.12, QED 0.59
Compound (SMILES): C=CC(=O)NC1CN(C(=O)N(C)[C@H](C(=O)N[C@H]2Cc3nc(cs3)-c3ccc4c(c3)c(c(-c3cccnc3[C@H](C)OC)n4CC)CC(C)(C)COC(=O)[C@@H]3CCCN(N3)C2=O)C(C)C)C1
InChIKey: KQKSMSGFARQVOU-YSTKZGGWSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 896.13, ALogP 5.27, HBA 12, HBD 3, PSA 180.33, QED 0.14
Compound (SMILES): CC(C)[C@H](N)C(=O)N(C)[C@H]1CN[C@@](CCCCB(O)O)(C(=O)O)C1
InChIKey: GLKHEHMZDJSINZ-TYNCELHUSA-N
Target: Arginase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P05089
Assay: Inhibition of Arg1 (unknown origin)
Measurement: IC50 = 2420 nM (pChEMBL 5.619999885559082)
Descriptors: MW 343.23
Compound (SMILES): CCC(=O)Nc1ccccc1Nc1nc(Nc2ccc3c(c2)OCCOCCOCCO3)nc2[nH]cnc12
InChIKey: MQSCGRDOQBVIMP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibitive Activities Against BTK wt, BTK(C481S), BMX, FAK, ITK, EGFR wt Kinases: The inhibiting activities of each of Compound-1 to Compound-24 against BTK wt, BTK(C481S), BMX, FAK, ITK, and EGFR wt kinases were determined using 33P ATP HotSpot kinase assay platform. (See, e.g., Ma, et al, 2008 Expert Opin Drug Discov. 3(6): 607-621.)Each of Compound-1 to Compound-24 were prepared as 10 mM DMSO solution, and then 1:3 serially diluted to a concentration of 1 μM to 0.05 nM. Then, Kinase assays were performed using the HotSpot assay platform.Briefly, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer; 20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO. Compounds were delivered into the reaction, followed about 20 minutes later by addition of a mixture of ATP (Sigma, St. Louis Mo.) and 33P ATP (Perkin Elmer, Waltham Mass.) to a final concentration of 10 μM.Reactions were carried out at room temperature for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper (Whatman Inc., Piscataway, N.J.). Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data was expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions. IC50 values and curve fits were obtained using Prism (GraphPad Software).
Measurement: IC50 = 14.1 nM (pChEMBL 7.849999904632568)
Descriptors: MW 519.56, ALogP 3.99, HBA 10, HBD 4, PSA 144.54, QED 0.29
Compound (SMILES): CN1CC2CCC(C1=O)N2c1ncc2[nH]nc(-c3cnn(-c4ccc(N(C)C)nc4)c3)c2n1
InChIKey: STFFFMLZTKDEOR-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Enzymatic Assay: un-Phosphorylated (UP): The inhibitor potency of the exemplified compounds was determined in an enzyme discontinuous assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.2 μL was transferred to the wells of a 384-well plate. For the isoforms of FGFR (−1, −2, −3 wild-type and mutant isoforms, −4) including un-phosphorylated (UP)proteins, a 5 μL/well volume of enzyme diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated with inhibitor for 5 to 15 minutes at ambient temperature. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The reaction was initiated by the addition of a 5 μL/well volume containing both biotinylated EQEDEPEGDYFEWLE (SEQ ID: 1) peptide substrate and ATP in assay buffer. The 10 μL/well reaction concentration of the peptide substrate was 500 nM whereas the ATP concentration was maintained near or below the ATP Km for each FGFR isoform. The ATP Km values were pre-determined for each FGFR isoform in a separate series of experiments. The reaction plate was incubated at 25° C. for 1 hr and the reactions were ended with the addition of 5 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 45 mM EDTA, 600 nM staurosporin, with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for −10 minutes at ambient temperature before scanning on a PheraStar plate reader (BMG Labtech) instrument.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 444.5, ALogP 1.48, HBA 9, HBD 1, PSA 111.96, QED 0.5
Compound (SMILES): Nc1ncc(N2CCN(c3cccc(C4CCOCC4)c3)CC2)c(N)n1
InChIKey: JFIWMIVDCNPRTN-UHFFFAOYSA-N
Target: Bifunctional dihydrofolate reductase-thymidylate synthase (SINGLE PROTEIN), Leishmania major
UniProt: P07382
Assay: TBD: TBD
Measurement: IC50 = 7.94 nM (pChEMBL 8.100000381469727)
Descriptors: MW 354.46, ALogP 1.86, HBA 7, HBD 2, PSA 93.53, QED 0.87
Compound (SMILES): CC(C)n1cc(C(=O)Nc2ccc(Oc3ccnc4[nH]nc(N[C@H](C)CO)c34)c(F)c2)c(=O)n(-c2ccc(Cl)cc2)c1=O
InChIKey: UQHBMBBDESEXNW-MRXNPFEDSA-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 = 4.1 nM (pChEMBL 8.390000343322754)
Descriptors: MW 608.03, ALogP 4.48, HBA 10, HBD 4, PSA 156.16, QED 0.19
Compound (SMILES): COc1cc(OC)cc(N(CCCO)c2ccc3ncc(-c4cnn(C)c4)cc3n2)c1
InChIKey: OYHDEFFNZNIKPS-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR4 (Enzymatic Assay): In a final reaction volume of 30 μL, FGFR4 (h) (60 ng/ml) was incubated with 50 mM HEPES pH 7.5, 6 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 0.01% Triton-X-100, 500 nM Btn-Flt3 and 5 μM ATP in the presence of compound (1% DMSO final). After incubation for 60 minutes at room temperature the reaction was stopped with 2.27 nM EU-anti P-Tyr, 7 mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA which was present for 60 minutes at room temperature. Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) signal (ex340 nm. Em 620 nm, em 655 nm) was measured afterwards and results are expressed in RFU (Relative Fluorescence Units). In this assay, the inhibitory effect of different compound concentrations (range 10 μM to 0.1 nM) was determined and used to calculate an IC50 (M) and pIC50 (−log IC50) value.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 419.49, ALogP 3.57, HBA 8, HBD 1, PSA 85.53, QED 0.47
Compound (SMILES): CCn1cc(Nc2ncc(C(N)=O)c(NCc3cccc(F)c3)n2)cn1
InChIKey: BKMBJAPTYASMEY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Kinase Activity Assay: JAK1 (the final concentration was 10 nM); ATP (the final concentration was 10 μM); ULight-labeled JAK-1 (Tyr1023) Peptide (the final concentration was 100 nM); the enzymatic reaction time was 2 hours. The maximum final concentration of compounds was 2.5 μM. After a 3-fold gradient dilution, 11 concentrations were obtained, with the minimum final concentration of 0.042 nM. The final DMSO concentration was 1%.
Measurement: IC50 = 37.1 nM (pChEMBL 7.429999828338623)
Descriptors: MW 355.38, ALogP 2.29, HBA 7, HBD 3, PSA 110.75, QED 0.6
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCC(N5CC(S(C)(=O)=O)C5)CC4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: AQUVUUSACYGOHY-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 4887 nM (pChEMBL 5.309999942779541)
Descriptors: MW 520.7, ALogP 4.77, HBA 7, HBD 1, PSA 96.25, QED 0.41