lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[S+]([O-])CCCOc1ccc2ncc(-c3cc4ccccc4o3)n2n1
InChIKey: OARQRIONHZACAE-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: MKNK1 Kinase Assay: For the assay 50 mL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM magnesium chloride, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22 °C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μL assay volume is 10 μM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 45 min at 22 °C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.05 μg/ml. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5). The resulting mixture was incubated for 1 h at 22 °C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET reader, e.g. a Rubystar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 355.42, ALogP 3.29, HBA 6, HBD 0, PSA 75.62, QED 0.39
Compound (SMILES): CCOCCNc1nc(Nc2cccc(OCCN3CCOCC3)c2)nc2c(/C=C3\NC(=O)NC3=O)cnn12
InChIKey: UGYGNIZRTNPUOF-ZHZULCJRSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 537.58, ALogP 1.21, HBA 12, HBD 4, PSA 156.27, QED 0.15
Compound (SMILES): CCCCOC(=O)c1cc(O)c(=O)c2c(O)c(O)c(OC)cc2c1
InChIKey: LHGKFYVCNTXZIJ-UHFFFAOYSA-N
Target: Toll-like receptor 1/2 (PROTEIN COMPLEX), Mus musculus
UniProt: Q9EPQ1
Assay: Antagonist activity at TLR1/2 in mouse RAW264.7 cells assessed as inhibition of Pam3CSK4-induced receptor activation by measuring NO production after 24 hrs
Measurement: IC50 = 2830 nM (pChEMBL 5.550000190734863)
Descriptors: MW 334.32, ALogP 2.28, HBA 7, HBD 3, PSA 113.29, QED 0.44
Compound (SMILES): CCn1cc(-c2cc3c(-c4cccc(N5CCNCC(F)(F)C5)n4)n[nH]c3cn2)cn1
InChIKey: UNMSQVAIBXEGPW-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 0.029 nM (pChEMBL 10.539999961853027)
Descriptors: MW 424.46, ALogP 2.95, HBA 7, HBD 2, PSA 87.55, QED 0.52
Compound (SMILES): Cc1cc(S(N)(=O)=O)ccc1Nc1ncc2c(n1)N(C)c1ccccc1C(=O)N2C
InChIKey: ACQZFXBAXXIVCX-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of human PI3Kgamma (S144 to A1102 residues) expressed in mammalian expression system incubated for 60 mins by ADAPTA assay
Measurement: IC50 = 2400 nM (pChEMBL 5.619999885559082)
Descriptors: MW 424.49, ALogP 2.53, HBA 7, HBD 2, PSA 121.52, QED 0.66
Compound (SMILES): O=C(O)c1ccc(S(=O)(=O)N(Cc2ccccc2)Cc2ccc(F)cc2)cc1
InChIKey: XIPCWXDGCQOODZ-UHFFFAOYSA-N
Target: Taste receptor type 2 member 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYV8
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 8283 nM (pChEMBL 5.079999923706055)
Descriptors: MW 399.44, ALogP 3.92, HBA 3, HBD 1, PSA 74.68, QED 0.65
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N2CCC[C@@H]2C)n2c1COCC2)c1c(F)cccc1F
InChIKey: CXJXEBLPEHWJRS-KBXCAEBGSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 431.48, ALogP 3.8, HBA 4, HBD 1, PSA 63.57, QED 0.78
Compound (SMILES): CC(C)NC(=O)c1cnc(-n2ncc3cc(C#N)cnc32)cc1Nc1cnn(CC(F)F)c1
InChIKey: DXXMEKWNVCWOGJ-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 11.2 nM (pChEMBL 7.949999809265137)
Descriptors: MW 451.44, ALogP 3.03, HBA 9, HBD 2, PSA 126.34, QED 0.44
Compound (SMILES): CC(Oc1ccc(-c2ccccc2)cc1)C(=O)Cn1nnc2ccccc21
InChIKey: GGXOUHDIKLIMQO-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P97612
Assay: Inhibition of FAAH in rat brain microsomes using N-(2-hydroxyethyl)-4-pyren-1-ylbutanamide as substrate after 60 mins by fluorescence-based reversed phase HPLC analysis
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 357.41, ALogP 4.13, HBA 5, HBD 0, PSA 57.01, QED 0.52
Compound (SMILES): O=C(O)COc1ccc(Cl)cc1[C@@H]1c2scnc2CCN1C(=O)OCc1cc(F)ccc1F
InChIKey: BGCUHHUWSRCRFT-HXUWFJFHSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 μl. First, 25 μl of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 μl Binding-Buffer, 50 μl of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 μl CRTH2 membrane fragments, reaching a final concentration of 20 μg/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 μl of Microscint-40 (Packard) was added to each well and the re
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 494.9, ALogP 4.82, HBA 6, HBD 1, PSA 88.96, QED 0.53
Compound (SMILES): CN(C)C(=O)c1cc(C#N)ccc1C1C2=C(CCC2=O)N(c2ccnc(C(F)(F)F)c2)C(=O)N1C
InChIKey: REPDNZFIWOIWHD-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 = 16.8 nM (pChEMBL 7.78000020980835)
Descriptors: MW 483.45, ALogP 3.9, HBA 5, HBD 0, PSA 97.61, QED 0.66
Compound (SMILES): Cc1ccc(F)c(NC(=O)Nc2ccc(Oc3ccnc4cc(C(=O)NCCCN5CCCC(CO)C5)sc34)cc2)c1
InChIKey: MPCFFXIIMLGUSM-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: FLIPR (Fluorometric Imaging Plate Reader) Assay: Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 96-well fibronectin coated black-walled plates overnight at 37 °C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37 °C. Cells were washed 4 times (Original Cell Wash, Labsystems) to remove extracellular dye. Test compounds were reconstituted in 100% DMSO and added to the cells to give a final DMSO concentration of 0.1%. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 μM) or at concentrations ranging from 0.01 to 10.0 μM followed by VEGF stimulation (5 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 96 wells using a cooled CCD camera.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 591.71, ALogP 6, HBA 7, HBD 4, PSA 115.82, QED 0.17
Compound (SMILES): N=C(N)NCCC[C@H]1N[C@@H](CNC(=O)c2ccc3ccccc3c2)CCN(CC(Oc2ccccc2)c2ccccc2)C1=O
InChIKey: FQJPIRCJJADUTL-KVZOJTFESA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 592.74, ALogP 4.21, HBA 5, HBD 5, PSA 132.57, QED 0.1
Compound (SMILES): CN1C2COCC1CC(NC(=O)c1cn(CC(C)(F)F)c3ncccc13)C2
InChIKey: JKVLHXVFZDDHCU-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: In Vitro Inhibition of Ca Flux Assay: The 5HT3 antagonist activity of the compounds of the invention was determined by measuring the ability of the compounds to inhibit the calcium flux activity of 3HT3a receptor expressed in HEK-293T cells. HEK-293T cells were transfected with the 5HT3a expression construct using Xtreme Gene 9 (Roche) in 150 mm tissue culture treated plates and incubated for 24 hours at 37 °C. Cells were then split and plated at a density of 60K cells/well in poly-lysine coated, black 96-well plates with clear bottoms (BD BioSciences) and incubated overnight at 37 °C. Growth media was removed and cells loaded with 200 uL calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 5 Assay kit, Molecular Devices) and incubated at 37 °C. for 1 hour. While cells were incubating, the 10× antagonist and agonist/antagonist addition plates were made. For 10× antagonist plate: half log serial dilutions (final concentrations range from 10−7 through 10−10 with the bottom well a negative, no ligand control) were made from test compounds in DMSO at a 1000× concentration and then diluted to 10× in HBSS/20 mM HEPES. For addition plate: 5HT was diluted to 100× in HBSS/20 mM HEPES (final concentration in the assay−216 nM) and 15 uL was added to each well of the addition plate, 15 uL of 10× compound was also added to the addition plate, and finally 120 uL of HBSS/20 mM HEPES (for a total of 150 uL). Cells were then removed from the incubator and equilibrated to room temperature for 10 minutes, then 22.5 uL of 10× test compounds were added in triplicate to the plates and incubated at room temperature for 10 minutes (Tropisetron was used as a positive control in every assay). Test plate and addition plate were loaded into the FlexStation III (Molecular Devices), and using the fluidics, 22.5 uL compound additions were made (at t=~17 seconds), and fluorescence was measured for 90 seconds, reading every 2.2 seconds.
Measurement: IC50 = 1.05 nM (pChEMBL 8.979999542236328)
Descriptors: MW 378.42, ALogP 2.28, HBA 5, HBD 1, PSA 59.39, QED 0.89
Compound (SMILES): NCc1ccc(CNC(=O)c2ccc(NC(=O)N3Cc4ccc(F)cc4C3)cc2)cc1
InChIKey: WVQSPARLAONXET-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 2.78 nM (pChEMBL 8.5600004196167)
Descriptors: MW 418.47, ALogP 3.76, HBA 3, HBD 3, PSA 87.46, QED 0.59
Compound (SMILES): O=C(Nc1ccc(C2CCNC2)cc1)N1CCOCC1
InChIKey: OCCAXLZULXSHFT-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 359.1 nM (pChEMBL 6.449999809265137)
Descriptors: MW 275.35, ALogP 1.63, HBA 3, HBD 2, PSA 53.6, QED 0.86
Compound (SMILES): COC(=O)CCCCN/C=C1\C(=O)C(O)=C(C(C)C)c2cc(C)c(-c3c(C)cc4c(c3O)/C(=C/NCCCCC(=O)OC)C(=O)C(O)=C4C(C)C)c(O)c21
InChIKey: VLQNBXGESWDKCL-RSSRHXQMSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Inhibition of FAM-Bid binding to human BCL2 expressed in Escherichia coli BL21 after 30 mins by fluorescence polarization assay
Measurement: IC50 = 483 nM (pChEMBL 6.320000171661377)
Descriptors: MW 744.88, ALogP 6.94, HBA 12, HBD 6, PSA 191.72, QED 0.06
Compound (SMILES): O=C(O)c1cccc(NCc2ccc(CNc3ccccc3)cc2)c1
InChIKey: MMPKYACWCJVMHO-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O08565
Assay: Competitive Binding Assay: A synthetic 14-mer peptide, TN14003, was previously reported to block both SDF-1/CXCR4 mediated invasion in vitro and metastasis in vivo with a high specificity by binding competitively with its ligand SDF-1. Aa competitive binding assay using biotin-labeled TN14003 and streptavidin-conjugated rhodamine was developed to determine the binding efficiency of new chemical entities to the SDF-1 binding domain of CXCR4. Cells incubated with high affinity compounds show only blue nuclear staining, whereas compounds with low affinity result in staining CXCR4 (red; rhodamine) as well as the nuclei (blue; cytox blue).
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 332.4, ALogP 4.61, HBA 3, HBD 3, PSA 61.36, QED 0.59
Compound (SMILES): CSc1nc(N)cc(Oc2ccc(-c3nnc(Nc4cccc(C(F)(F)F)c4)[nH]3)cc2)n1
InChIKey: CMYHZFCJPORPHY-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Competitive Binding Assay (KINOMEscan): For the competitive binding assay, test compounds were screened at 10 uM against 70 diverse kinase targets for competitive binding using the KINOMEscan technology (Ambit Biosciences) (Karaman, et al. (2008) Nature biotechnology 26:127-132). 11-point Kd curves were determined for hits identified during the initial screen.
Measurement: Kd = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 459.46, ALogP 5.12, HBA 8, HBD 3, PSA 114.63, QED 0.27
Compound (SMILES): O[C@H]1[C@@H](O)C[C@@]2(n3cnc4c(NCc5cc(F)ccc5F)nc(Cl)nc43)C[C@@H]12
InChIKey: JTESUQCWWMOESR-XFFUPTALSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Radioligand Binding Assay: Radioligand binding assays at A1, A2A, and A3ARs were performed according to the procedures described previously. Each tube in the binding assay contained 100 uL of membrane suspension (20 ug of protein), 50 pt of a stock solution of agonist radioligand, and 50 uL of increasing concentrations of the test ligands in Tris-HCl buffer (50 mM, pH 7.5) containing 10 mM MgCl2. Nonspecific binding was determined using a final concentration of 10 uM NECA, a non-specific agonist, diluted with the buffer.The mixtures were incubated at 25° C. for 60 min. Binding reactions were terminated by filtration through Whatman GF/B filters under a reduced pressure using a MT-24 cell harvester (Brandell, Gaithersburg, Md.). Filters were washed three times with 5 ml of 50 mM ice-cold Tris-HCl buffer (pH 7.5). The radioactive agonists [3H]R-PIA and [3H]CGS21680 were used for the A1 and A2AAR assays, respectively, while [125I]AB-MECA was used for the A3AR assay.
Measurement: Ki = 1260 nM (pChEMBL 5.900000095367432)
Descriptors: MW 407.81, ALogP 2.21, HBA 7, HBD 3, PSA 96.09, QED 0.57
Compound (SMILES): CCN(C(=O)Oc1ccc(F)cc1)[C@@H]1CN(C(=O)C2CCN(C(=O)C3(C)CC3)CC2)C[C@H]1c1ccc(F)c(F)c1
InChIKey: MLWPXWLOQRKWON-JYFHCDHNSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: [3H]SR142801 Competition Binding Assay: hNK3 receptor binding experiment were performed using [3H]SR142801 (Catalog No. TRK1035, specific activity: 74.0 Ci/mmol, Amersham, GE Healthcare UK limited, Buckinghamshire, UK) and membrane isolated from HEK293 cells transiently expressing recombinant human NK3 receptor. After thawing, the membrane homogenates were centrifuged at 48,000×g for 10 mM at 4° C., the pellets were resuspended in the 50 mM Tris-HCl, 4 mM MnCl2, 1 μM phosphoramidon, 0.1% BSA binding buffer at pH 7.4 to a final assay concentration of 5 μg protein/well. For inhibition experiments, membranes were incubated with [3H]SR142801 at a concentration equal to KD value of radioligand and 10 concentrations of the inhibitory compound (0.0003-10 μM) (in a total reaction volume of 500 μl) for 75 min at room temperature (RT). At the end of the incubation, membranes were filtered onto unitfilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.3% PEI+0.3% BSA, Packard BioScience, Meriden, Conn.) with a Filtermate 196 harvester (Packard BioScience) and washed 4 times with ice-cold 50 mM Tris-HCl, pH 7.4 buffer. Nonspecific binding was measured in the presence of 10 μM SB222200 for both radioligands. The radioactivity on the filter was counted (5 min) on a Packard Top-count microplate scintillation counter with quenching correction after addition of 45 μl of microscint 40 (Canberra Packard S.A., Zürich, Switzerland) and shaking for 1 h.
Measurement: Ki = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 557.61, ALogP 4.96, HBA 4, HBD 0, PSA 70.16, QED 0.5
Compound (SMILES): O=C(O)C(CN1CCC(c2cc(-c3ccc(F)cc3)nn2-c2ncccn2)CC1)c1ccccc1
InChIKey: ASWVEQWRTMUNCF-UHFFFAOYSA-N
Target: Lysosomal Pro-X carboxypeptidase (SINGLE PROTEIN), Homo sapiens
UniProt: P42785
Assay: Enzyme Activity Assay: The assay was run in a 384-well microliter plate at 37° C. with a total volume of 50 uL. Final assay concentrations were 0.13 nM human PrCP (CHO) or 0.09 nM mouse PrCP (CHO) enzyme, 15 uM substrate and varying concentrations of inhibitor in buffer containing 10 mM NaOAc, 100 mM NaCl and 19.5 ug/mL BSA at pH 5.5. The assay also contained 2% DMSO used to solubilize the substrate and inhibitor. Inhibitors were prepared in 100% DMSO and serial diluted (in 100% DMSO) to generate 11 point titration curves. Either 39 uL of human or mouse PrCP enzyme was added to the wells of the assay plate, followed by a 1 uL addition of the serially diluted inhibitor and mixed three times using a 30 uL mix volume. The reaction was initiated by the addition of 10 uL substrate and mixed three times using a 30 μL mix volume. The reactions were monitored continuously over 25 min at 37° C. to obtain initial velocities.
Measurement: IC50 = 0.51 nM (pChEMBL 9.289999961853027)
Descriptors: MW 471.54, ALogP 4.52, HBA 6, HBD 1, PSA 84.14, QED 0.43
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(c3)-c3[nH]c5ccccc5c3C3(CCCC3)O4)cc12
InChIKey: XAZHFYGWRKRGSG-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: ELISA-Based Assay: Measurement of inhibition by compounds was performed using the HCV replicon system. Several different replicons encoding different HCV genotypes or mutations were used. In addition, potency measurements were made using different formats of the replicon assay, including different ways of measurements and different plating formats. See Jan M. Vrolijk et al., A replicons-based bioassay for the measurement of interferons in patients with chronic hepatitis C, 110 J. VIROLOGICAL METHODS 201 (2003); Steven S. Carroll et al., Inhibition of Hepatitis C Virus RNA Replication by 2'-Modified Nucleoside Analogs, 278(14) J. BIOLOGICAL CHEMISTRY 11979 (2003). However, the underlying principles are common to all of these determinations, and are outlined below.Stable neomycin phosphotransferase encoding replicons-harboring cell lines were used, so all cell lines were maintained under G418 selection prior to the assay. Potency was determined using a cell ELISA assay with an antibody to the replicons encoded NS3/4a protease. See Caterina Trozzi et al., In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor, 77(6) J. Virol. 3669 (2003).
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 649.74, ALogP 7.97, HBA 5, HBD 2, PSA 104.64, QED 0.2
Compound (SMILES): CC1C(O)CC[C@@]2(C)C1CC[C@@H]1[C@@H]2CC[C@]2(C)C(c3cccnc3)=CC[C@@H]12
InChIKey: JSOBPDOKYJKCFC-FFCNATMCSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human testicular microsomes using 17alpha-hydroxypregnenolone as substrate assessed as decrease in DHEA levels after 90 mins by ELISA
Measurement: IC50 <= 11 nM (pChEMBL 7.959000110626221)
Descriptors: MW 365.56, ALogP 5.72, HBA 2, HBD 1, PSA 33.12, QED 0.69
Compound (SMILES): Cc1cc(C(N)=O)c2[nH]c3cc(C(C)(C)O)ccc3c2c1-c1cccc(-n2c(=O)cc3c(Cl)cccn3c2=O)c1C
InChIKey: BIXZQQNECLOHBK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Assay: To V-bottom 384-well plates were added test compounds, human recombinant Btk (1 nM, Invitrogen Corporation), fluoresceinated peptide (1.5 μM), ATP (20 μM), and assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij 35 surfactant and 4 mM DTT in 1.6% DMSO), with a final volume of 30 μL. After incubating at room temperature for 60 min, the reaction was terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product.
Measurement: IC50 = 0.41 nM (pChEMBL 9.390000343322754)
Descriptors: MW 567.05, ALogP 5.35, HBA 6, HBD 3, PSA 122.59, QED 0.27
Compound (SMILES): O=C(Nc1cc(C2CCNCC2)nn1-c1ccc(C2CC2)cn1)c1cnn2cccnc12
InChIKey: YDXGENDDXXNZPG-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 428.5, ALogP 2.91, HBA 8, HBD 2, PSA 102.03, QED 0.51
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(CCC(N)=O)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: LRHYXMKKPWPLDM-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 467.53, ALogP 2.8, HBA 8, HBD 2, PSA 134.71, QED 0.39
Compound (SMILES): CNC(C)C(=O)Nc1cc(-c2cccc3c2c(C)nn3C)cc(NC(=O)c2ccc(Cl)cn2)n1
InChIKey: QMYOVGLYSKKPJK-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: DELFIA Assay: R3 domains of human XIAP (covering amino acids 241 to 356; XIAP BIR3) and cIAPl (covering amino acids 256 to 363; cIAPl BIR3) were expressed and purified from Ecoli as GST-fusion proteins. Peptide AVPIAQKSE-Lys(Biotin), representing the N-terminus of mature human SMAC (SMAC peptide), was used as interaction partner in the protein- peptide interaction assay.BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% TritonXlOO) for one hour at room temperature in the presence of inhibitiory compounds. The assay mixture was transferred to a strepatvidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-Nl- antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes. Time resolved Europium fluoresecence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 391 nM (pChEMBL 6.409999847412109)
Descriptors: MW 477.96, ALogP 3.79, HBA 7, HBD 3, PSA 113.83, QED 0.39
Compound (SMILES): N#CC1=C(N)Nc2[nH]c(=O)[nH]c(=O)c2C1c1ccc(Br)cc1
InChIKey: JPADETQXMCODCI-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Inhibition of N-terminus 6xHis-tagged human IDO1 expressed in Escherichia coli M15 using L-tryptophan as substrate preincubated for 1 hr measured after 15 mins in presence of bovine liver and methylene blue by spectrophotometric analysis
Measurement: IC50 = 942 nM (pChEMBL 6.03000020980835)
Descriptors: MW 360.17, ALogP 1.08, HBA 5, HBD 4, PSA 127.56, QED 0.61
Compound (SMILES): CC(=O)c1c(N(Cc2ccc(F)c(C(F)(F)F)c2)S(=O)(=O)c2ccc(C#N)cc2)sc2ccccc12
InChIKey: KZUDEPUZYNSSMQ-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 532.54, ALogP 6.53, HBA 5, HBD 0, PSA 78.24, QED 0.21
Compound (SMILES): CC(C)(C)C(NC(=O)c1cc(OCC(F)(F)F)c(C2CC2)cn1)c1nccs1
InChIKey: KIYWEZTWWYNULU-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 5.8 nM (pChEMBL 8.239999771118164)
Descriptors: MW 413.47, ALogP 4.87, HBA 5, HBD 1, PSA 64.11, QED 0.73
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)Nc3cccc(C(F)(F)F)c3)CC2)C1
InChIKey: CTRLBBJLDSLVHZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 549.56, ALogP 4.46, HBA 7, HBD 2, PSA 118.76, QED 0.38
Compound (SMILES): CNC(=O)c1cc(Nc2ncnc(N3CC(COc4ccc(Cl)cc4)C3)n2)ccc1Cl
InChIKey: WUPDARNEKJAUOE-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 1260 nM (pChEMBL 5.900000095367432)
Descriptors: MW 459.34, ALogP 3.8, HBA 7, HBD 2, PSA 92.27, QED 0.56
Compound (SMILES): CNC(=O)C1=C(C)NC(Nc2nc3ccccc3o2)=NC1c1nn(C)cc1Cl
InChIKey: LJYIEDZBEIDLQF-UHFFFAOYSA-N
Target: Galactokinase (SINGLE PROTEIN), Homo sapiens
UniProt: P51570
Assay: qHTS Assay: Assay details and protocol: The primary assay monitored ATP depletion using Promega's KinaseGlo technology, where ATP levels are measured through luminescence generated from firefly luciferase, a bioluminescent ATP-dependent enzymes. ATP was held at 35 μM, near its reported KM value, and the KM for galactose was determined under the 1536-well assay conditions to be 50-100M (FIG. 1A). As well, the IC50 for a commercially available CD45 inhibitor (N-(9,10-dioxo-9,10-dihydrophenanthren-2-yl)pivalamide) was confirmed (previously found to inhibit GALK (FIG. 1B)). This was used as the positive control for the assay. The assay used 5 nM GALK and a 1 hr incubation time, which gave sufficient signal:background and stability for the HTS.
Measurement: IC50 = 16343.3 nM (pChEMBL 4.789999961853027)
Descriptors: MW 399.84, ALogP 2.35, HBA 8, HBD 3, PSA 109.37, QED 0.62
Compound (SMILES): COCCOc1ccc2nc3ccc(=O)cc-3oc2c1
InChIKey: WIDXMQJOBHHXHT-UHFFFAOYSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: In Vitro Competitive Radioligand Binding Assay: To determine the Ki values, fixed brain tissues prepared from 15-month-old Tg2576 mice (N=5) will be incubated for 30 min with 1 uM methoxy-X34 in the presence of a serial dilution of test compounds (10-9-10-6 M, 6 concentrations/compound). Tissue sections (3 sections per reaction) will be then mounted and coverslipped. Photographs of methoxy-X34 binding images will be taken using a Nikon fluorescent microscope. The fluorescent intensity on cerebral amyloid deposits and neuritic plaques will be separately quantified using Image J software.
Measurement: Ki = 347 nM (pChEMBL 6.460000038146973)
Descriptors: MW 271.27, ALogP 2.32, HBA 5, HBD 0, PSA 61.56, QED 0.54
Compound (SMILES): O=C(/C=C/c1cc2ccccc2[nH]1)c1ccc(Br)cc1O
InChIKey: RBRVYSKMHQQFOG-SOFGYWHQSA-N
Target: Pyruvate kinase (SINGLE PROTEIN), Staphylococcus aureus (strain MRSA252)
UniProt: Q6GG09
Assay: Inhibition of recombinant N-terminal His-tagged methicillin-resistant Staphylococcus aureus MRSA252 pyruvate kinase expressed in Escherichia coli BL21(DE3) using PEP as substrate measured after 5 mins by LDH-coupled assay
Measurement: IC50 = 553 nM (pChEMBL 6.260000228881836)
Descriptors: MW 342.19, ALogP 4.53, HBA 2, HBD 2, PSA 53.09, QED 0.54
Compound (SMILES): CC1(C)OC(=O)N(c2ccc(C(=O)Nc3cnccc3O)cc2)[C@H]1c1ccccc1
InChIKey: BDLKJQVEJOWDFJ-FQEVSTJZSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Tankyrase Biochemical Assays: The tankyrase 1 biochemical activity of the compounds was assayed in the following assay buffer (50 mM MOPS pH7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.01% Tween-20, 0.05% BSA, and 1 mM DTT) as follows: 0.25 nM of 6×HIS-tankyrase 1 (1091-1325) was incubated in the presence of compound (DMSO 1.85% final) in a Perkin Elmer 384 well Proxiplate Plus™ (cat. no. 6008289) with 400 nM of NAD for 60 minutes at room temperature. The assay was then stopped with the above assay buffer containing a 0.6 M inhibitor and the following detection components: 0.05 g/mL monoclonal anti-PAR antibody (Trevigen cat. no. 4335-MC-01K-AC) prebound for 60 minutes with 0.63 μg/mL protein G AlphaLisa® acceptor bead (Perkin Elmer cat. no. AL102M) and 5 μg/mL AlphaLisa® nickel chelate donor bead (Perkin Elmer cat. no. AS101M). The assay was incubated for 16 hours at room temperature in the dark and read on a Perkin Elmer Envision® multi label reader using the default program set with laser excitation at 680 nM and emission at 615 nM. The tankyrase 2 biochemical assay was performed using the procedure set forth above with respect to tankyrase 1 except that 4 nM 6×HIS-tankyrase 2 (946-1162) and 250 nM NAD were used.
Measurement: IC50 = 1340 nM (pChEMBL 5.869999885559082)
Descriptors: MW 403.44, ALogP 4.52, HBA 5, HBD 2, PSA 91.76, QED 0.67
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(-c3ccc(Cl)cc3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: RZNHCPVPLNLKLM-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 507, ALogP 5.7, HBA 7, HBD 1, PSA 91.62, QED 0.33