lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(O)[C@@]1(C)Cc2cc(NC(=O)c3cnn4cccnc34)c(O[C@H]3CC[C@@H](O)CC3)cc2O1
InChIKey: KRFTVOULWMYDOE-LEHBWIKQSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 490 nM (pChEMBL 6.309999942779541)
Descriptors: MW 466.54, ALogP 3.13, HBA 8, HBD 3, PSA 118.21, QED 0.53
Compound (SMILES): O=C(NCc1cccc(-c2c[nH]c3ncccc23)c1)c1cccn(Cc2ccc(F)c(F)c2)c1=O
InChIKey: SXLISROJDDMSGE-UHFFFAOYSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: Kinase Assay: A PDK1 kinase assay was performed as follows. PDK1 (amino acids 51-360) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to greater than 90% homogeneity. PDK1 protein was divided into two fractions, one of which was subsequently dephosphorylated. To generate dephosphorylated PDK1, the PDK1 was reacted with GST-tagged lambda-phosphatase in vitro. GST was subsequently cleaved proteolytically for both phosphorylated and dephosphorylated PDK1. Protein preparations were run on glutathione Sepharose columns to remove GST and GST-tagged lambda-phosphatase, if present. Phosphorylated PDK1 and dephosphorylated PDK1 were verified by mass-spectrometry. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay using either phosphorylated or unphosphorylated PDK1 and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 3-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide. (final concentrations: 25 nM un-phosphorylated PDK1 or 0.5 nM phosphorylated PDK1, 30 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 24 μM for both forms of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 470.48, ALogP 4.65, HBA 4, HBD 2, PSA 79.78, QED 0.38
Compound (SMILES): CC(=O)N1CCC(CNc2cc3nc(CS[C@H]4CC[C@H](O)CC4)[nH]c(=O)c3c(F)c2F)CC1
InChIKey: NCHMXPYKLYCMGD-WKILWMFISA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Inhibition Assay: Enzymatic inhibition of PARP14 was measured using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) monitoring the auto-modification of PARP14 by biotinylated nicotinamide adenine dinucleotide (biotin-NAD). 1 μL of a dose response curve of each test compound was spotted in 384-well nickel-coated white microplates (Thermo) using a Mosquito (TTP Labtech). Reactions were performed in a 50 μL volume by adding 40 μL of PARP14 in assay buffer (20 mM HEPES pH==8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%0 Tween20), incubating with test compound at 25° C. for 30 min, then adding 10 μL of biotin-NAD (Biolog). The final concentrations of PARP14 and biotin-NAD are 50 nM and 3 μM, respectively. Reactions proceeded at 25° C. for 3 h, then were quenched with 5 μL of 10 mM unmodified nicotinamide adenine dinucleotide (Sigma-Aldrich). The quenched reactions were washed 3 times with 100 μL of TBST wash buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween20). Next, to the washed and dried plate was added 25 μL of DELFIA Europium-N1 streptavidin (Perkin Elmer) diluted in DELFIA assay buffer (Perkin Elmer). After a 30 min incubation at 25° C., the plate was washed 5 times with TBST wash buffer. Finally, 25 μL of DELFIA enhancement solution was added. After a 5 min incubation the plate was read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 340 nm and emission of 615 nm to measure the amount of Europium present in each well, informing on the amount of biotin-NAD that was transferred in the automodification reaction.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 480.58, ALogP 3.41, HBA 6, HBD 3, PSA 98.32, QED 0.59
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1CNC(=O)c1cc(Cl)c(CN2CCC[C@@H](NC)C2)c(C(F)(F)F)c1
InChIKey: TWGWKQNKYRFPRW-GOSISDBHSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Eu Kinase Binding Assay: DDR1 binding activity was measured by the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). A test compound and the Alexa Fluor (Registered trademark) 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After reacting at room temperature for one hour, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 9.9 nM (pChEMBL 8)
Descriptors: MW 566.47, ALogP 4.92, HBA 5, HBD 2, PSA 78.51, QED 0.47
Compound (SMILES): CN(C)c1cnc(C2=C(c3nnn[nH]3)C(=O)N[C@@](c3ccc(OCCCCC(F)(F)F)cc3)(C(F)(F)F)C2)s1
InChIKey: PLVSVXWTJFBXTK-NRFANRHFSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT LCMS Assay: The MGAT enzyme reactions were performed in Corning FALCON® 96-well polypropylene plates, in a total volume of 60 μL of 50 mM potassium phosphate buffer pH 7.4, containing a final concentration of 100 μM 2-oleoylglycerol, 15 μM oleoyl-coenzyme A and 0.0013 μg/μL human or mouse MGAT-2 or 0.0026 μg/μL rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 μL of ice cold methanol containing 1 μg/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters SYMMETRY C8, 50×2.1 mm column, was used for the chromatography of enzyme products. The buffer system consists of 0.1% formic acid in water with a mobile phase consisting 0.1% formic acid in methanol. The shallow gradient is 90-100% mobile phase in 0.2 min with a total run time of 2.3 min. The first 0.5 minutes of each injection was diverted to waste to eliminate the presence of phosphate buffer in the enzymatic reaction. The column was run at 0.6 mL/min and a temperature of 65° C. Mass spectrometry analysis of the samples was performed on a ThermoFisher Quantum Triple quad utilizing APCI (+) as the mode of ionization. Data was acquired in Single Ion Monitoring (SIM) mode analyzing Diolein=m/z 603.6 (PRODUCT) and 1,2-distearoyl-rac-glycerol (IS)=m/z 607.6. The ratio of Diolein to internal standard (Peak Area Ratio) is utilized to calculate IC50 values.
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 575.54, ALogP 4.72, HBA 8, HBD 2, PSA 108.92, QED 0.28
Compound (SMILES): C[C@H](CN[C@H](C(=O)Nc1ccc(-c2cnn(C(C)(C)C#N)c2)cn1)c1ccccc1)c1ccc(C#N)cc1
InChIKey: BNPVMGWFRZYAKK-PIKZIKFNSA-N
Target: Histone acetyltransferase p300 (SINGLE PROTEIN), Homo sapiens
UniProt: Q09472
Assay: Biochemical Assay: The full length p300 SPA assay was run following the same protocol as p300 HAT SPA assay, but used 6 nM purified full length p300 (purchased from Active Motif) in place of the purified p300-HAT domain.Select compounds were also evaluated in a H3K18Ac MSD cellular assay that measures the ability of compounds to inhibit the the acetylation of chromatin at H3K18, a process catalyzed by p300 and CBP. In a typical experiment the p300 HAT inhibitory activity inside cells of the compounds described herein was determined in accordance with the following experimental method. 20 k HCT-116 cells per well are plated in 75 μL RPMI+10% FBS media the night before treatment. Compounds plated in DMSO at 4× final concentration are resuspended in 30 μL RPMI+10% FBS, then 25 μL is combined with corresponding wells containing cells. Treated cells are incubated for 2 hr at 37° C., then lysed in 500 μL final volume and frozen at −80° C. MSD plates (Meso Scale Discovery) are coated overnight at 4° C. with 60 μL 1:500 α-total histone antibody (Millipore MAB3422) in PBS. Plates are then blocked with 5% BSA in TBST shaking at RT for 1 hr, washed, and 30 μL lysate added to each well for 2 hr shaking at RT. Plates are washed and 25 μL 1:216 α-H3K18ac antibody (CST 9675) in PBS added, then incubated for 1 hr shaking at RT. Plates are washed again, then 25 μL 1:1000 Sulfo-Tag goat α-rabbit antibody (Meso Scale Discovery R32Ab-1) in PBS is added for 1 hr shaking at RT. Plates are washed once more, then 150 μL 1× Read Buffer (MSD #R92TD-3) is added to all wells and read on MSD SECTOR Imager 2400 using the conventional read setup.
Measurement: IC50 = 425 nM (pChEMBL 6.369999885559082)
Descriptors: MW 503.61, ALogP 5.15, HBA 7, HBD 2, PSA 119.42, QED 0.33
Compound (SMILES): Cn1nccc1-c1cc(C#N)ccc1Oc1ccc(-c2cccc(C(N)=O)n2)cc1
InChIKey: OERJPSNMNKKPAG-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: The assay buffer was formulated by removing 120 mL from a 1 L bottle of fresh, sterile dH2O (Mediatech, Herndon, Va.) and adding 100 mL of 10×HBSS that does not contain Ca++ or Mg++ (Gibco, Invitrogen, Grand Island, N.Y.) followed by 20 mL of 1.0 M Hepes, pH 7.3 (Fisher Scientific, BP299-100). The final buffer consisted of 20 mM Hepes, pH 7.3, 1.261 mM CaCl2, 0.493 mM MgCl2, 0.407 mM Mg(SO)4, 5.33 mM KCl, 0.441 mM KH2PO4, 137 mM NaCl, 0.336 mM Na2HPO4 and 0.556 mM D-glucose (Hanks et al., Proc. Soc. Exp. Biol. Med. 71:196 (1949)), and the simple formulation was typically the basic buffer throughout the assay (i.e., all wash and addition steps).
Measurement: IC50 = 285 nM (pChEMBL 6.539999961853027)
Descriptors: MW 395.42, ALogP 3.91, HBA 6, HBD 1, PSA 106.82, QED 0.55
Compound (SMILES): COc1cc(-c2cccc(F)c2)c(Cl)cc1-c1ncnc2cc(S(=O)(=O)Nc3cnsn3)ccc12
InChIKey: XZERGQMLWPTXGK-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to â¿¿10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 51.5 nM (pChEMBL 7.289999961853027)
Descriptors: MW 527.99, ALogP 5.42, HBA 8, HBD 1, PSA 106.96, QED 0.31
Compound (SMILES): COC[C@@H](C)Oc1nc(C(=O)N2CC(F)(F)C2)cc(N2CCC(c3c[nH]c4nccc(OC)c34)CC2)n1
InChIKey: UKCSRTKFPOZTEF-OAHLLOKOSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: AXL enzyme inhibition (% inhibition, Kiapp and Ki values) by small molecule inhibitors was evaluated using a fluorescence-based microfluidic mobility shift assay. AXL catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-30 (5-FAM-KKKKEEIYFFF-CONH2, CPC Scientific, Sunnyvale, Calif.). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Human wild-type receptor tyrosine kinase protein Axl comprising residues 505-811 was produced in-house using the baculoviral expression vector system that incorporated a hexahistidine affinity tag into the protein (LJIC-1916B1.1). The enzyme was preactivated by auto-phosphorylation of 34 uM non-activated enzyme in the presence of 2 mM ATP, 4 mM MgCl2, 50 mM NaCl and 1 mM TCEP in 20 mM HEPES, pH 7.3 at 4° C. for 30 minutes. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 120 μM ATP (ATP Km=70.4 μM), 0.01% Tween-20, 3 μM FL-Peptide-30, and 0.5 nM phosphorylated AXL enzyme in 100 mM HEPES buffer at pH 7.3. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 30 minutes at 25° C. by the addition of 50 μL of 200 mM EDTA, pH 7.5. The Ki values were determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable. (See, Morrison, J. F. (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors, Biochimica et biophysica acta 185, 269-286; and Murphy, D. J. (2004) Determination of accurate KI values for tight-binding enzyme inhibitors: an in silico study of experimental error and assay design, Analytical biochemistry 327, 61-67.)
Measurement: Ki = 0.018 nM (pChEMBL 10.739999771118164)
Descriptors: MW 516.55, ALogP 3.25, HBA 8, HBD 1, PSA 105.7, QED 0.49
Compound (SMILES): CNCc1cc(C)c(-c2cc3c(-c4ccc(NC(C)=O)cc4)n[nH]c3cc2C#N)c(F)c1
InChIKey: ZCKXGPJNWOCSJD-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 Î¼L of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 Î¼l of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 Î¼l of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25Â° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 427.48, ALogP 4.89, HBA 4, HBD 3, PSA 93.6, QED 0.42
Compound (SMILES): CCc1nc(C(=O)NC[C@]2(O)CC[C@@H](S(C)(=O)=O)CC2)c(C#N)n1-c1ccc(CCC(F)(F)F)cc1F
InChIKey: KMUDNKVHNQFDCH-PAVFIEEWSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Fluorescence Based Assay: ThermoFluor is a fluorescence based assay that estimates ligand binding affinities by measuring the effect of a ligand on protein thermal stability (Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R. (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6, 429-40, and Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258-66). This approach is applicable to a wide variety of systems, and rigorous in theoretical interpretation through quantitation of equilibrium binding constants (KD).
Measurement: Kd = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 544.57, ALogP 3.39, HBA 7, HBD 2, PSA 125.08, QED 0.49
Compound (SMILES): COc1cc2c(Oc3ccc(N(/N=C/c4ccc(F)cc4)C(N)=O)cc3F)ccnc2cc1OCCCN1CCC(C)CC1
InChIKey: OPKCGECTCOQTEI-BBUXEPKYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Kinase Assay: c-Met Kinase Activity was Measured with an ELISA Reader. The Specific Operation as Follows:To the plate filled with 0.25 mg/mL PGT, the compounds, 50 pM c-Met (His-tagged recombinant human Met (Amino acids 974-ends), by baculovirus expression) and 5 μM ATP in buffer solution (25 mM MOPS, pH 7.4, 5 mM MgCl2, 0.5 raM MnCl2, 100 μM sodium orthovanadate, 0.01% Triton X-100, 1 mM DTT, 1% of DMSO1% (v/v)) was added, the solution was incubated for 20 min. The reaction mixture was removed by washing with 0.2 μg/mL conjugated horseradish peroxidase (HRP) monoclonal antibody specific for phosphotyrosine (PY20) detecting phosphorylation of the substrate polymer. 1M phosphoric acid was added to terminate the color, the chromogenic substrate (TMB) was tested by spectrophotometry at 450 nm.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 603.67, ALogP 6.73, HBA 7, HBD 1, PSA 102.51, QED 0.12
Compound (SMILES): COc1cc(-c2cnc(N)c(-c3ccc(NC(=O)c4cn(CC5(OC)CCOCC5)cc(-c5ccc(C)cc5)c4=O)cc3)c2)ccc1OC[C@H]1COCCO1
InChIKey: YQDQJTHWHPDCGQ-UUWRZZSWSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Cell-Free Axl Kinase Inhibitory Activity: A kinase dilution solution containing 170 ng/ml AXL (the 464th to 885th amino acids of the intracellular domain of human AXL expressed as a fusion protein with glutathione transferase in a baculovirus expression system and purified by glutathione Sepharose chromatography; Carna Biosciences, Inc., catalog No. 08-107) was prepared using a kinase reaction buffer solution (100 mM HEPES (pH 7.4), 0.003% Brij-35, 0.004% Tween-20, 1 mM DTT, and 10 mM MgCl2) and added at 19 μl/well to a 384-well plate.Next, each test compound was diluted with DMSO, and this dilution solution was added at 1 μl/well to the plate.After preincubation at room temperature for 30 minutes, a solution containing a substrate peptide (FL-Peptide 30 (5FAM-KKKKEEIYFFF-CONH2), Caliper Life Sciences, catalog No. 760430) and ATP at 1.5 μM and 10 μM, respectively, was prepared and added at 5 μl/well to the plate to start kinase reaction. The plate was incubated at 28° C. for 1.5 hours, and the reaction was terminated by the addition of a termination buffer solution (100 mM HEPES (pH 7.4), 0.015% Brij-35, 40 mM EDTA, and 0.1% Coating Reagent 3) at 40 μl/well.The substrate peptide and the phosphorylated peptide in the reaction mixture were separated and quantified using EZ Reader II (Caliper Life Sciences).The kinase reaction was evaluated on the basis of a product ratio (P/(P+S)) calculated from the peak height (S) of the substrate peptide and the peak height (P) of the phosphorylated peptide.The rate of inhibition (Inhibition) was determined according to the following expression (automatically calculated using the software of the EZ Reader II system).
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 746.86, ALogP 6.39, HBA 11, HBD 2, PSA 145.39, QED 0.15
Compound (SMILES): CN1CCC(CC(=O)N[C@H]2CC[C@@H](Nc3ncnc4[nH]cc(C(=O)c5ccccc5Cl)c34)CC2)CC1
InChIKey: MHYLLMKMZCSKRY-KDURUIRLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Mobility Shift Assay (MSA) Type A: Full length unphosphorylated form of BTK expressed in Sf9 cells was employed to test inhibitory activity in the inactive BTK assay. The assay was measured in buffer solution containing 100 mM HEPES pH7.5, 0.01% Triton X-100, 0.1% BSA, 5 mM MgCl2, 1 mM DTT. The enzyme and increasing concentrations of inhibitor was incubated at room temperature for 30 minutes and the kinase reaction was initiated by the addition an activation mixture diluted in assay buffer containing Srctide peptide substrate, DOPS/DOPC, PtdIns(3,4,5)P3, and ATP for final concentrations of 1 μM Srctide, 5.5 μM DOPS, 5.5 μM DOPC, 0.5 μM PtdIns(3,4,5)P3, and 16 μM ATP. The plates were incubated for 60 minutes at room temperature, and then the reaction stopped with 100 mM HEPES buffer containing 0.01% Triton and 40 mM EDTA and read on Caliper Life Sciences Labchip EZ Reader II instrument.
Measurement: IC50 = 6.1 nM (pChEMBL 8.210000038146973)
Descriptors: MW 509.05, ALogP 4.41, HBA 6, HBD 3, PSA 103.01, QED 0.41
Compound (SMILES): CN(C)c1nc(-n2cc(C(=O)O)cn2)nc2ccc(-c3ccccc3)cc12
InChIKey: DRJPKCAEJCBBIN-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: PHD enzymatic assay: The PHD enzymatic assay was performed in 0.5 ml of reaction mixture containing the following: purified PHD2181-417 polypeptide (3 μg), synthetic HIF-1α peptide comprising residues [KNPFSTGDTDLDLEMLAPYIPMDDDFQLRSFDQLS] (10 μM, California Peptide Research Inc., Napa, Calif.), and [5-14C]-2-oxoglutaric acid (50 mCi/mmol, Moravek Chemicals, Brea, Calif.) in reaction buffer (40 mM Tris-HCl, pH 7.5, 0.4 mg/ml catalase, 0.5 mM DTT, 1 mM ascorbate) for 10 minutes. Compounds were pre-incubated for 30 min before starting the reaction (all test compounds were dissolved at 10 mM in 100% DMSO (w/v) and were tested with final compound concentrations at 100 μM in 1% DMSO (w/v)). The reaction was stopped by addition of 50 μl of 70 mM H3PO4 and 50 μl of 500 mM NaH2PO4, pH 3.2. Detection of [14C]-succinic acid was achieved by separating from [5-14C]-2-oxoglutaric acid by incubating the reaction mixture with 100 μl of 0.16 M DNP prepared in 30% perchloric acid. Next, 50 μl of unlabeled 20 mM 2-oxoglutaric acid/20 mM succinic acid, serving as carrier for the radioactivity, was added to the mixture, and was allowed to proceed for 30 minutes at room temperature. The reaction was then incubated with 50 μl of 1 M 2-oxoglutaric acid for 30 additional minutes at room temperature to precipitate the excess DNP. The reaction was then centrifuged at 2800×g for 10 minutes at room temperature to separate [14C]-succinic acid in the supernatant from the precipitated [14C]-dinitrophenylhydrazone. Fractions of the supernatant (400 μI) were counted using a beta counter (Beckman Coulter, Fullerton, Calif.). Inhibition of PHD2181-417 activity was measured as a decrease in succinic acid production. The IC50 values were estimated by fitting the data to a three-parameter logistic function using GraphPad Prism, version 4.02 (Graph Pad Software, San Diego, Calif.).
Measurement: IC50 = 63.1 nM (pChEMBL 7.199999809265137)
Descriptors: MW 359.39, ALogP 3.25, HBA 6, HBD 1, PSA 84.14, QED 0.6
Compound (SMILES): C/C=C\C=C/C(=O)[C@@H](O)[C@@H](O)C/C=C/c1cc(OC)cc(OC)c1C(=O)OC
InChIKey: VOQPOIOVAHLXEH-RPSNWOEYSA-N
Target: Mitogen-activated protein kinase kinase kinase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: O43318
Assay: TaK1-TaB1 Inhibitor Assays: The assay was performed at BSP Biosience Inc. (5Z)-7-oxozeaenol was used as a positive control. Analogue 3 was not tested due to the short-term stability as indicated by the sub 90% purity detected by UPLC following prep HPLC purification. The only synthesized analogue that was found to be relatively active was difluoro (5Z)-7-oxozeaenol 5.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 390.43, ALogP 2.32, HBA 7, HBD 2, PSA 102.29, QED 0.36
Compound (SMILES): O=C(Cc1cc(Cl)ccc1O)Nc1cccc(C(=O)NC2CCOCC2)c1
InChIKey: GZRBRTKEDPJYBO-UHFFFAOYSA-N
Target: Anoctamin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5XXA6
Assay: Automated Whole-Cell Patch Clamp Assay to Detect TMEM16A Activity in Recombinant Cells: Compound activity was quantified by measuring the increase in current upon compound addition and expressing this as a percentage increase of baseline TMEM16A current level. Percentage increases in current were determined for each concentration and the data plotted as a function of concentration using either the Qpatch software or Graphpad Prism v6.05 providing the concentration which gave 50% of its maximal effect (EC50) and maximum efficacy (percentage of baseline increase). The method of calculating the results is illustrated in FIG. 1, which shows an example trace from the Qpatch TMEM16A assay. In FIG. 1, IBL equals baseline current, I[#1] equals the peak current during test compound concentration 1 incubation period and so on.
Measurement: EC50 = 650 nM (pChEMBL 6.190000057220459)
Descriptors: MW 388.85, ALogP 3.14, HBA 4, HBD 3, PSA 87.66, QED 0.73
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CCC[C@@]2(C)c2nc3c(F)c(F)ccc3[nH]2)n1
InChIKey: GKCWRTQGIQLARU-NRFANRHFSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 149 nM (pChEMBL 6.829999923706055)
Descriptors: MW 423.43, ALogP 3.28, HBA 6, HBD 1, PSA 92.59, QED 0.55
Compound (SMILES): COCc1noc(C)c1-c1cc(N2CCOC[C@H]2C)nc2c(-c3ccnn3C3CCCCO3)nccc12
InChIKey: RLOKBODXCOYFLN-BPNWFJGMSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET Tracer A): To determine of binding activity of the test compounds, full-length human ATR protein was expressed and purified together with ATRIP as described above. Furthermore, a fluorescently labelled compound (tracer A) was used as a tracer molecule. Detection of the binding event of the tracer was achieved by time-resolved fluorescence energy transfer (TR-FRET). We used an anti-GST-Terbium antibody (CisBio) that binds to the GST-tag at the N-terminus of ATR-kinase. Excitation of Terbium with 337 nm light results in emission of fluorescent light with 545 nm. In case a tetrameric complex has formed (antiGST-Tb+GST-ATR+Strp2-ATRIP+tracer), part of the energy will be transferred from the Terbium to the fluorophore that itself emits light of 570 nm. Displacement of the fluorescent tracer by a test compound leads to a reduction of the TR-FRET-signal.For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (MTP, Greiner Bio-One, Frickenhausen, Germany). To prepare the ATR-working solution, ATR/ATRIP stock solution was diluted in assay buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (w/v) Igepal, 0.01% (w/v) BSA] to 4.2 nM protein concentration (concentration may vary from lot to lot of protein preparation). AntiGST-Tb antibody was diluted to 4.2 nM. The ATR-working solution was incubated for 30 min at 22° C. prior to dispensing to pre-form the complex of antiGST-Tb+GST-ATR+ATRIP. Then, 3 μl of the ATR-working solution were added to the test compound and the mixture was incubated for 10 min at 22° C. to allow pre-binding of the test compounds to ATR/ATRIP. Then, 2 μl of a 100 nM solution of either tracer A or B in assay buffer were added to the ATR-working solution. The resulting mixture was incubated for 30 min at 22° C. The measurement of the TR-FRET signal was performed in a standard HTRF-compatible MTP reader instrument (e.g. BMG Pherastar) by recording the fluorescence emissions at 545 nm and 570 nm after excitation at 337-350 nm. The ratio between emission at 570 nm divided by emission at 545 nm was calculated to give the well ratio. The experimental data (well ratios) were normalised by the following way: positive control contained ATR-working solution plus either tracer A or B solution (=0% inhibition), the negative control contained all components except GST-ATR/ATRIP (=100% inhibition).
Measurement: IC50 = 32.9 nM (pChEMBL 7.480000019073486)
Descriptors: MW 504.59, ALogP 4.53, HBA 10, HBD 0, PSA 100.56, QED 0.37
Compound (SMILES): CCOc1c(C(C)n2ccc(=O)c3c(N)ncnc32)cc(Cl)c(C#N)c1-c1cncc(S(C)(=O)=O)c1
InChIKey: MPNWWLXSJKXPKN-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: [γ-33P]ATP (10 mCi/mL) was purchased from PerkinElmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) was purchased from Millipore (Bedford, Mass.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from SigmaAldrich (St. Louis, Mo.). Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from GE healthcare life sciences (Piscataway, N.J.).The kinase reaction was conducted in polystyrene 384-well matrix white plate from Thermo Fisher Scientific in a final volume of 25 μL Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The PI3K assays were carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi [γ-33P] ATP, 4 nM PI31(6. Reactions were incubated for 210 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by scintillation counting on Topcount (PerkinElmer).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 524.99, ALogP 3.37, HBA 10, HBD 1, PSA 153.85, QED 0.4
Compound (SMILES): CCc1ccc(F)c(C(=O)N[C@@H](C(=O)N2CCC3(CC2)C(=O)N(C)C(=O)N3c2ccc3cnc(C)n3c2)C(C)C)c1
InChIKey: YXAWXHAIPIUYAF-RUZDIDTESA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Autotaxin LPC Cascade Assay: The efficacy of the Examples described herein, as inhibitors of ATX are demonstrated and confirmed by pharmacological in vitro assays. The following assays and their respective methods are carried out with the compounds according to the present invention. Activity possessed by the compounds may be demonstrated in vivo. Those skilled in the art will appreciate that a variety of assay formats may be used to determine the activity of the compounds of this invention.Autotaxin LPC Cascade Assay:Assay Buffer:100 mM Tris-HCl, pH=9500 mM NaCl5 mM MgCl25 mM CaCl20.05% Triton X 100Reagents:Enzyme Source Stock conc Working conc. Final conc. Autotaxin X-Chem 4.6 μM 20 nM 5 nM LPC Sigma L5254 8 mM in assay buffer 400 μM 100 μM Choline Oxidase Sigma C5896 50 U/mL in water 0.8 U/mL 0.1 U/mL HRP Sigma P8375 1000 U/mL in water 8 U/mL 1 U/mL Ampliflu Red Sigma 90101 100 mM in DMSO 400 μM 50 μM.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 562.65, ALogP 3.56, HBA 6, HBD 1, PSA 107.33, QED 0.46
Compound (SMILES): CC(C)(C(=O)NC(N)=O)[C@@H]1c2ccc(-c3ccc(C(=O)N4CC[C@@H](F)C4)cc3)nc2Oc2c(F)cccc21
InChIKey: IUOHWOUDFIBXAH-VGSWGCGISA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Receptor Binding Assay: Binding Assay (I):In order to assess the affinity of test compounds for the human glucocorticoid receptor, a commercially available kit was used (Glucocorticoid Receptor Competitor Assay Kit, Invitrogen Part #2893). Briefly, purified human recombinant full-length glucocorticoid receptor (2 nM) was mixed with fluorescently labeled glucocorticoid (1 nM Fluormone GS Red) in the presence or absence of test compound. After two hour incubation at room temperature in the dark, the fluorescence polarization (FP) of the samples was measured. The FP of a mixture of receptor, fluorescent probe (i.e., Fluormone GS Red) and 5 μM dexamethasone represented background fluorescence or 100% inhibition, whereas, the FP of the mixture without dexamethasone (but in the presence of vehicle) was taken to be 100% binding. The percentage inhibition of test compounds were then compared to the sample with 5 μM dexamethasone and expressed as % relative binding activity with dexamethasone being 100% and no inhibition is 0%. Test compounds were analyzed in the concentration range from 8.5E-05 μM to 5 μM. Binding Assay (II):In order to measure the binding of compounds on the glucocorticoid receptor a commercially available kit was used (Glucocorticoid receptor competitor assay kit, PanVera Co., Madison, Wis., P2816). Briefly, a cell lysate containing recombinantly expressed human full-length glucocorticoid receptor was mixed with a fluorescently labeled glucocorticoid (1 nM Fluormone GS1) in the presence or absence of test compound. After one hour at room temperature, the fluorescence polarization (FP) of the samples were measured. The FP of a mixture of receptor, fluorescent probe (i.e., Fluormone GS1) and 1 mM dexamethasone represented background fluorescence or 100% inhibition, whereas, the FP of the mixture without dexamethasone was taken to be 100% binding. The percentage inhibition of test molecules were then compared to the sample with 1 mM dexamethasone and expressed as % relative binding activity with dexamethasone being 100% and no inhibition is 0%. Test molecules were analyzed in the concentration range from 2.4 nM to 40 μM.Site I binding assays for any NHR (Nuclear Hormone Receptor) are conducted similarly to the above. An appropriate cell lysate or purified NHR is used as the source of the NHR. The fluorescent probe and unlabeled competitor are appropriate for the specific NHR, i.e., are ligands for the specific NHR.
Measurement: Ki = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 520.54, ALogP 4.53, HBA 5, HBD 2, PSA 114.62, QED 0.52
Compound (SMILES): Cn1ncc2cc(-c3cccc(S(N)(=O)=O)c3-c3nn[nH]n3)ccc21
InChIKey: HFXJZACIYJVKAW-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 1.99 nM (pChEMBL 8.699999809265137)
Descriptors: MW 355.38, ALogP 1.07, HBA 7, HBD 2, PSA 132.44, QED 0.56
Compound (SMILES): COc1ccccc1Oc1ccc(N2C(=O)Nc3c(C(=O)NC4CCCN(C(=O)/C(C#N)=C/C5CC5)C4)sc4nccc2c34)c(C)c1
InChIKey: XDVVXVVQFPVHJY-OQKWZONESA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 31.6 nM (pChEMBL 7.5)
Descriptors: MW 648.75, ALogP 6.67, HBA 8, HBD 2, PSA 136.89, QED 0.16
Compound (SMILES): OCC1O[C@H](Sc2cc(Cl)ccc2Cl)C(O)[C@@H](n2cc(-c3cc(F)c(F)c(F)c3)nn2)[C@H]1O
InChIKey: PFKACLPVUCUOHD-MKZLOUGKSA-N
Target: Galectin-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P17931
Assay: fluorescence anisotropy assay : The affinity of Example 1-106 for galectins were determined by a fluorescence anisotropy assay where the compound was used as an inhibitor of the interaction between galectin and a fluorescein tagged saccharide probe as described Sörme, P., Kahl-Knutsson, B., Huflejt, M., Nilsson, U. J., and Leffler H. (2004) Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions. Anal. Biochem. 334: 36-47, (Sörme et al., 2004) and Monovalent interactions of Galectin-1 By Salomonsson, Emma; Larumbe, Amaia; Tejler, Johan; Tullberg, Erik; Rydberg, Hanna; Sundin, Anders; Khabut, Areej; Frejd, Torbjorn; Lobsanov, Yuri D.; Rini, James M.; et al, From Biochemistry (2010), 49(44), 9518-9532, (Salomonsson et al., 2010). The assay was also adapted to be able to measure the high affinity of compounds for galectin-3 by using the below probe constructed to have high affinity for galectin-3 which made it possible to use a low concentration of galectin-3 (50 nM). 100 nM albumin was included as a carrier to prevent protein loss at such low concentration of galectin.
Measurement: Kd = 84 nM (pChEMBL 7.079999923706055)
Descriptors: MW 522.33, ALogP 3.44, HBA 8, HBD 3, PSA 100.63, QED 0.44
Compound (SMILES): O=C(O)c1ccc2c(c1)CCCC(c1ccc(Cl)c(F)c1)=C2c1ccc(O[C@H]2CCN(CCCF)C2)cc1
InChIKey: RNNLCWBLRAXREA-VWLOTQADSA-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 = 244 nM (pChEMBL 6.610000133514404)
Descriptors: MW 538.03, ALogP 7.29, HBA 3, HBD 1, PSA 49.77, QED 0.33
Compound (SMILES): CCOc1ncccc1-c1nnc([C@@H]2CCCN2C(=O)c2ccc(Cl)cc2-n2nccn2)[nH]1
InChIKey: XEWMCRKQHIEWNY-KRWDZBQOSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 464.92, ALogP 3.48, HBA 8, HBD 1, PSA 114.71, QED 0.46
Compound (SMILES): Cc1ccc(S(=O)(=O)C(C)CCN(C)c2nc3c(C)cccc3cc2CNC(=O)c2nccnc2N)cc1
InChIKey: VHRZGJTUKMWSGW-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Luciferase-Coupled Chemiluminescence Assay: Kinase activity is measured as the percent of ATP consumed following the kinase reaction using luciferase-luciferin-coupled chemiluminescence. Reactions were conducted in 384 or 1536-well white medium binding microtiter plates (Greiner). Kinase reactions were initiated by combining test compounds, kinase, and ATP in a 20 μL volume (6 μL volume for 1536-well plate). The reaction mixture was incubated at ambient temperature for 2 h. Following the kinase reaction, a 20 μL (or 3 μL for 1536-well plate) aliquot of KinaseGlo (Promega) was added and the chemiluminescence signal measured using an EnVision plate reader (Perkin Elmer). Total ATP consumption was limited to 25-60% and the IC50 values correlate well with those determined by radiometric assays.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 532.67, ALogP 3.84, HBA 8, HBD 2, PSA 131.17, QED 0.33
Compound (SMILES): CCCCCNc1nc(N)nc2ccn(Cc3cc(CC(=O)OCC(=O)N4CCOCC4)ccc3OC)c12
InChIKey: QLZDGWRAIFYJAV-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Reporter Gene Assay: Human embryonic kidney 293 (HEK 293) cells were stably transfected with human TLR7 and an NF-kB-driven luciferase reporter vector (pNifty-Luciferase). As a control assay, normal Hek293 transfected with pNifty-Luc were used. Cells were cultured in DMEM supplemented with 2 mM L-glutamine, 10% heart inactivated FBS, 1% penicillin and streptomycin, 2 μg/ml puromycin (InvivoGen #ant-pr-5) and 5 μg/ml of blasticidin (Invitrogen #46-1120). Bright-Glo Luciferase assay buffer and substrate were supplied by Promega #E263B and #E264B (assay substrate and buffer respectively). 384 well clear-bottom plates were supplied by Greiner bio-one (#789163-G) and were custom bar-coded plates.Cells were plated at 25,000 cells/well in 384-well plates in a final volume of 50 μl of media. Cells were allowed to adhere to the plates after overnight (18 hours) culture at 37° C. and 5% CO2. Serially diluted experimental and positive control compounds were then dispensed to each well and incubated for 7 hours at 37° C. and 5% CO2. Cells stimulated with DMSO alone also serve as negative controls. After the incubation, 30 μl of the pre-mix assay buffer and substrate buffer were added to each well according to manufacturer's instructions. The luminescence signal was read on a CLIPR machine with an integration time of 20 seconds per plate.Dose response curves are generated for each compound and EC50 values were determined as the concentration that gives 50% of the maximal signal.
Measurement: EC50 = 10 nM (pChEMBL 8)
Descriptors: MW 524.62, ALogP 2.62, HBA 10, HBD 2, PSA 133.83, QED 0.27