lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): c1cncc(-c2cc3c(CNC4CCNCC4)n[nH]c3cn2)c1
InChIKey: UHWGTBKFBOBROH-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 = 3800 nM (pChEMBL 5.420000076293945)
Descriptors: MW 308.39, ALogP 1.86, HBA 5, HBD 3, PSA 78.52, QED 0.69
Compound (SMILES): COC(=O)c1ccc2c(c1)CCN(Cc1ccc(C(C)NC(C)=O)cc1)C2
InChIKey: QEUWJQXZXZICOI-UHFFFAOYSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.Each assay microtiter plate contains wells with vehicle instead of compound as controls for the non-inhibited enzyme (100% CTL; HIGH) and wells without acetyl-CoA as controls for non-specific NADH degradation (0% CTL; LOW).
Measurement: IC50 = 524 nM (pChEMBL 6.28000020980835)
Descriptors: MW 366.46, ALogP 3.23, HBA 4, HBD 1, PSA 58.64, QED 0.83
Compound (SMILES): Cc1cc(C)c(CN2CCc3ccc(OC(C)C)c(Cl)c3C2=O)c(=O)[nH]1
InChIKey: SNBYUGZWDXWLGO-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition of wild-type EZH2 (unknown origin) expressed in baculovirus infected SF9 cells co-expressing SUZ12/EED/RbAp48 complex using HeLa cells derived oligonucleosomes as substrate after 60 mins in presence of [3H] SAM by scintillation counting
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 374.87, ALogP 3.63, HBA 3, HBD 1, PSA 62.4, QED 0.89
Compound (SMILES): Fc1ccc2c(c1Cl)N1CCNCC1CC2
InChIKey: HOZXWTAUGLXWQL-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2C (SINGLE PROTEIN), Homo sapiens
UniProt: P28335
Assay: Agonist activity at unedited 5-HT2C receptor (unknown origin) expressed in HEK293 cells assessed as [3H]inositol phosphate accumulation after 2 hrs by scintillation counting
Measurement: EC50 = 6.31 nM (pChEMBL 8.199999809265137)
Descriptors: MW 240.71, ALogP 2.2, HBA 2, HBD 1, PSA 15.27, QED 0.75
Compound (SMILES): COc1cc(N2CCC(N3CCCC3)CC2)ccc1Nc1nc2c(c(Nc3ccccc3S(=O)(=O)NC(C)C)n1)CCN2
InChIKey: SYUPTFQHURQCGQ-UHFFFAOYSA-N
Target: Focal adhesion kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05397
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 606.8, ALogP 4.69, HBA 10, HBD 4, PSA 123.75, QED 0.26
Compound (SMILES): O=C1c2c(F)cc(C3CC3)cc2OCCN1c1cccc(-c2ccnc3[nH]c(-c4ccc(CN5CCN(C6COC6)CC5)cn4)cc23)c1CO
InChIKey: FSSSDOHMQDOWGP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Recombinant Human BTK Inhibition Assay: The inhibition assays were performed using the generic tyrosine kinase assay kit from C is Bio (HTRF KinEASE-TK #62TK0PEB). The assay is based on the principle of fluorescence resonance energy transfer (FRET) between the Europium Cryptate labeled anti-phosphotyrosine antibody and streptavidin labeled fluorophore, XL-665. In an uninhibited kinase reaction the biotinylated peptide substrate (Tk-Biotin) gets phosphorylated at an intermediate tyrosine site which is captured by anti-phosphotyrosine antibody which eventually acts as a FRET donor. The streptavidin labeled XL-665 binds strongly to the Tk-Biotin substrate and act as a FRET acceptor. The resultant FRET signal is measured at 665 nm. A ratio of this specific fluorescence (665 nm) to the background fluorescence (620 nm), conferred by the free antibody, is directly proportional to the kinase activity. Inhibition of the kinase activity by a pharmacological intervention such as a Btk inhibitor of the present disclosure will result into decreased FRET signal.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 674.78, ALogP 5.36, HBA 8, HBD 2, PSA 107.05, QED 0.23
Compound (SMILES): c1ccc2c(N3CCC4(CCCCC4)CC3)cncc2c1
InChIKey: XYRRTQOHZRVRRZ-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibition of human CYP11B2-CLE9 expressed in chinese hamster V79 cells using 11-deoxycorticosterone as substrate preincubated for 1 hr followed by substrate addition measured for 3 hrs by HTRF assay
Measurement: IC50 = 554 nM (pChEMBL 6.260000228881836)
Descriptors: MW 280.42, ALogP 4.79, HBA 2, HBD 0, PSA 16.13, QED 0.75
Compound (SMILES): COc1cc(OC)cc(N(Cc2ncc[nH]2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: GWCJUEKQORIUTJ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 7.762 nM (pChEMBL 8.109999656677246)
Descriptors: MW 441.5, ALogP 4.11, HBA 8, HBD 1, PSA 93.98, QED 0.41
Compound (SMILES): Cn1ncc(NC(=O)c2nc(-c3c(F)cccc3F)sc2N)c1C1CC(F)C(N)CCO1
InChIKey: ZNKBCQQYCPIUSJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Assay: LC3K assay: PIM-1, -2, and -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. PIM reactions were carried out in a final volume of 10 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000® (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of −500V, an upstream voltage of −2150V, and a screening pressure of −1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm.
Measurement: Ki = 0.469 nM (pChEMBL 9.329999923706055)
Descriptors: MW 466.49, ALogP 3.17, HBA 8, HBD 3, PSA 121.08, QED 0.54
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-n1ccn2nc(-c3cccnc3)cc12
InChIKey: SZWYZMFLSCAXOT-UHFFFAOYSA-N
Target: Angiopoietin-1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q02763
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: For the assay, 50 nl of a 100-fold concentrated solution of the respective test substance in DMSO were pipetted into a black low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany) 2 μl of a solution of the Tie2 fusion protein in assay buffer [50 mM HEPES/HCl pH 7, mM MgCl2, 2.5 mM MnCl2, 1 mM dithiothreitol, 0.01% (v/v) Nonidet P-40, 0.1% (w/v) bovine serum albumin (BSA), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added, and the mixture was incubated for 15 min to allow pre-binding of the substance to the enzyme prior to the kinase reaction. The kinase reaction was then started by adding 3 μl of a solution of adenosine triphosphate (ATP, 1.67 mM→final concentration in 5 μl assay volume=1 mM) and substrate (1.67 μM→final concentration in 5 μl assay volume=1 μM) in assay buffer, and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of the Tie2 fusion protein was adapted to the respective activity of the enzyme and adjusted such that the assay was carried out in the linear range. Typical concentrations were in the range of 30 ng/ml. The reaction was stopped by addition of 5 μl of a solution of TR-FRET detection reagents [200 nM streptavidin-XL665 and 2 nM PT66-Eu chelate, a europium chelate-labelled anti-phosphotyrosine antibody (Perkin-Elmer); alternatively, it is also possible to use PT66-Tb cryptate (Cisbio Bioassays, Codolet, France)] in aqueous EDTA solution [90 mM EDTA, 0.28% (w/v) bovine serum albumin (BSA) in 50 mM HEPES pH 7.5]. The resulting mixture was incubated at 22° C. for 1 h to allow formation of the complex of the biotinylated phosphorylated substrate and the detection reagents. The amount of phosphorylated substrate was then determined by measuring the resonance energy transfer from PT66-Eu chelate to streptavidin-XL665. To this end, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET measuring instrument (for example Viewlux, Perkin-Elmer).
Measurement: IC50 = 320 nM (pChEMBL 6.5)
Descriptors: MW 461.45, ALogP 5.77, HBA 5, HBD 1, PSA 64.22, QED 0.37
Compound (SMILES): COc1cc2c(Nc3ccc(Br)c(Cl)c3F)ncnc2cc1OC[C@@H]1CN2CCOC[C@H]2CO1
InChIKey: CMTRUERFSJKMJQ-KBPBESRZSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 553.82, ALogP 4.42, HBA 8, HBD 1, PSA 77.97, QED 0.45
Compound (SMILES): Cc1cc(C#N)cnc1C(=O)Nc1cc(C)c(F)c([C@]2(CF)C[C@@H](C(F)(F)F)OC(N)=N2)c1
InChIKey: IHCNTVZLTALTSC-MGPUTAFESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 467.4, ALogP 3.79, HBA 6, HBD 2, PSA 113.39, QED 0.66
Compound (SMILES): O=C(c1ccc2cc(Oc3cnc(C(F)(F)F)cn3)ccc2n1)N1CCNCC1
InChIKey: GUXGKEDVVNKUKU-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 4630 nM (pChEMBL 5.329999923706055)
Descriptors: MW 403.36, ALogP 2.88, HBA 6, HBD 1, PSA 80.24, QED 0.72
Compound (SMILES): COc1ccc(Oc2nc3ccccc3cc2C(=O)Nc2cccc(S(N)(=O)=O)c2)c(OC)c1
InChIKey: UTMAVNADNBZGJF-UHFFFAOYSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q62968
Assay: Electrophysiology Assay: Patch clamp electrophysiology was used to assess the efficacy and selectivity of sodium channel blockers in dorsal root ganglion neurons. Rat neurons were isolated from the dorsal root ganglions and maintained in culture for 2 to 10 days in the presence of NGF (50 ng/ml) (culture media consisted of NeurobasalA supplemented with B27, glutamine and antibiotics). Small diameter neurons (nociceptors, 8-12 um in diameter) were visually identified and probed with fine tip glass electrodes connected to an amplifier (Axon Instruments). The "voltage clamp" mode was used to assess the compound's IC50 holding the cells at -60 mV. In addition, the "current clamp" mode was employed to test the efficacy of the compounds in blocking action potential generation in response to current injections.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 479.51, ALogP 3.94, HBA 7, HBD 2, PSA 129.84, QED 0.41
Compound (SMILES): COc1ccc(CN2C(=O)N(c3cnn(Cc4c(C)noc4C)c3)C(=O)C2(C)C)cc1C
InChIKey: QMUIYMVMIQWSKU-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
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 = 540 nM (pChEMBL 6.269999980926514)
Descriptors: MW 437.5, ALogP 3.6, HBA 7, HBD 0, PSA 93.7, QED 0.55
Compound (SMILES): Cc1cnc(-c2nc(C)nn2-c2cc(N3CC(c4nc5ccccc5n4C)C3)nc(C)n2)cn1
InChIKey: JOXPFBFNJLVXON-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition is determined using a known PDE10 inhibitor, which can be any compound that is present at 5,000 times its Ki value in the assay described as follows, such as papaverine (see Siuciak, et al. Neuropharmacology (2006) 51:386-396; Becker, et al. Behav Brain Res (2008) 186(2):155-60; Threlfell, et al., J Pharmacol Exp Ther (2009) 328(3):785-795), 2-{4-[pyridin-4-yl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl]phenoxymethyl}quinoline succinic acid or 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]quinoline succinic acid (see Schmidt, et al. J Pharmacol Exp Ther (2008) 325:681-690; Threlfell, et al., J Pharmacol Exp Ther (2009) 328(3): 785-795). 0% of inhibition is determined by using DMSO (1% final concentrations). A Labcyte Echo 555 (Labcyte, Sunnyvale, Calif.) is used to dispense 200 mL from each well of the titration plate to the 384 well assay plate. A solution of enzyme (1/7000 dilution from aliquots; sufficient to produce 20% substrate conversion) and a separate solution of FAM-labeled cAMP PDE from Molecular Devices (product # R7506), at a final concentration of 50 nM are made in the assay buffer (10 mM Tris HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3 0.01% Tween-20, and 1 mM DTT). The enzyme is added to the assay plates by the addition of 10 μL of enzyme solution to each well, shaken to mix and incubated at room temperature for 60 minutes. The substrate is then added to the assay plates by the addition of 10 μÎ¼L of substrate solution to each well, shaken to mix, and incubated at room temperature for 60 minutes. A binding solution is then made from the kit components, comprised of 80% Solution A, 20% Solution B and binding reagent at a volume of 1/600 the total binding solution. The enzymatic reaction is stopped by addition of 60 lit of the binding solution to each well of the assay plates and the plates are sealed and shaken for 10 seconds. The plate was incubated at room temperature for at least one hour prior to determining the fluorescence polarization (FP).
Measurement: Ki = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 452.53, ALogP 2.93, HBA 10, HBD 0, PSA 103.33, QED 0.41
Compound (SMILES): CC(C)(C)N1CCN(c2cc(Cc3n[nH]c(=O)c4c3CCCC4)ccc2F)C1=O
InChIKey: VOENODSCQBOKGH-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 1630 nM (pChEMBL 5.789999961853027)
Descriptors: MW 398.48, ALogP 3.42, HBA 3, HBD 1, PSA 69.3, QED 0.86
Compound (SMILES): O=C(Nc1cnc(Oc2ccc(-c3cnc(N4CCCCC4=O)s3)cc2)nc1)Nc1cc(C(F)(F)F)cnc1-c1ccc(C(F)(F)F)nc1
InChIKey: YHGCQMMSHUBAMT-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: LanthaScreen Kinase Assay: TrkA-inhibiting activity was measured according to the following method using LanthaScreen kinase assay reagents (Invitrogen; Fluorescein-Poly GT, LanthaScreen Tb-PY20 and TR-FRET Dilution Buffer), TrkA (Invitrogen), ATP (Sigma-Aldrich), a kinase reaction buffer (50 mM HEPES buffer (Sigma-Aldrich) (pH 7.5) containing 0.01% Brij35 (Sigma-Aldrich), 10 mM MgCl2 (Sigma-Aldrich) and 1 mM EGTA (Sigma-Aldrich)) and 0.5 M EDTA (pH 8.0) (Invitrogen).The present compound was dissolved in dimethylsulfoxide (hereinafter abbreviated as DMSO) to prepare a 10 mM solution. The test compound at 10 mM was distributed into a 96-well plate and serially diluted with DMSO with the geometrical ratio of 3. The serial dilutions (DMSO solutions) were diluted with the kinase reaction buffer to 20-fold to prepare solutions of the present compound with 5-times concentrations (DMSO concentration: 5%). TrkA was diluted with the kinase reaction buffer to prepare a solution at 38.5 ng/mL (enzyme solution). Adenosine
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 700.63, ALogP 8.05, HBA 9, HBD 2, PSA 135.12, QED 0.16
Compound (SMILES): N#Cc1c(N)nc(N)nc1N[C@H](c1nc2c(F)ccc(Cl)c2c(=O)n1-c1cccnc1)C1CC1
InChIKey: XDSXYMOZKDUASY-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy transfer (TR-FRET) Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoforms: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 10lps delay and 500 μs read window).
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 477.89, ALogP 2.96, HBA 10, HBD 3, PSA 161.42, QED 0.39
Compound (SMILES): CS(=O)(=O)N1C(=O)N(c2cccc(C(F)(F)F)c2)C2=C(C(=O)CC2)C1c1ccc(C#N)cc1
InChIKey: ZSGUFCFIQTTZBC-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available. 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 uL of these compound dilutions were mixed with 10 ul 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 uL substrate solution in assay buffer were added (250 uM 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. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 475.45, ALogP 4.14, HBA 5, HBD 0, PSA 98.55, QED 0.66
Compound (SMILES): Cc1cc(F)cc(C(=O)Nc2cccc(CN3CCC(C(=O)NC4CCCCC4)CC3)c2)c1
InChIKey: OZPOVHQQLDALNV-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 451.59, ALogP 5.05, HBA 3, HBD 2, PSA 61.44, QED 0.65
Compound (SMILES): CC(C)(C)CN1CCC2(CC1)CN(c1ccccc1NC(=O)Nc1ccc(C(C)(C)C)cc1)c1c2ccc(F)c1O
InChIKey: ZOWXRUSQGZUGEA-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 uL containing 15 fmol of P2Y1 receptor (1.7 ug of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 uM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium Chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 ug of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 uL of the aqueous soluttion was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKERSM (Amersham) Image Reader. Dose-response curves (IC50) were fit by non-linear regression (Toolset an in house data processing program) and binding constants (Ki) calculated using the Cheng-Prusoff relationship (Ki=IC50/(1+L/Kd) in which a Kd for 2MeS-ADP to the P2Y1 receptor was determined to be 14 nM.
Measurement: Ki = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 558.74, ALogP 8, HBA 4, HBD 3, PSA 67.84, QED 0.3
Compound (SMILES): CS(=O)(=O)c1ccc2c(c1)-c1cc(Cl)c3cccnc3c1NS2(=O)=O
InChIKey: ZCCNBQAQDOCKKT-UHFFFAOYSA-N
Target: Ferroportin (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP59
Assay: Antagonist activity at C-terminal halo-tag fused human ferroportin expressed in MDCK cells assessed as inhibition of hepcidin-induced ferroportin internalization after overnight incubation by Draq5 staining-based fluorescence assay
Measurement: IC50 = 25000 nM (pChEMBL 4.599999904632568)
Descriptors: MW 394.86, ALogP 3.07, HBA 5, HBD 1, PSA 93.2, QED 0.68
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1COc2ccccc2N(Cc2c(Cl)n(-c3ccccc3C#N)c3ccccc23)C1=O
InChIKey: IHVOEWKFQHMHHV-AVRDEDQJSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6x Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(8-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 528.01, ALogP 4.17, HBA 6, HBD 2, PSA 99.39, QED 0.39
Compound (SMILES): O=C(O)CC(c1cccc(-c2ccc3c(c2)OCCO3)c1)c1nccs1
InChIKey: CUHRKYMYZMTULM-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature, as substrate then bradykinin carrying an N-terminal® Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) label (JPT Peptide Technologies GmbH; dissolved in assay buffer) was added to the mixture. The final concentration of cathepsin A was 833 ng/ml and the final concentration of labeled bradykinin 2 μM. After incubation for 15 min at room temperature the reaction was stopped by the addition of stop buffer (130 mM 2-(4-(2-hydroxy-ethyl)-piperazin-1-yl)-ethanesulfonic acid, pH 7.4, containing (v/v) 0.013%° Triton X-100, 0.13% Coating Reagent 3 (Caliper Life Sciences), 6.5% DMSO and 20 μM ebelactone B (Sigma, #E0886)).
Measurement: IC50 = 987 nM (pChEMBL 6.010000228881836)
Descriptors: MW 367.43, ALogP 4.19, HBA 5, HBD 1, PSA 68.65, QED 0.73
Compound (SMILES): CC(C)(CN1CCOCC1)Nc1nc(NC2CC2)n2ncc(/C=C3\NC(=O)NC3=O)c2n1
InChIKey: QGTVKOSHHRGQDN-ZROIWOOFSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 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; 75 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 the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 441.5, ALogP 0.4, HBA 10, HBD 4, PSA 137.81, QED 0.36
Compound (SMILES): COc1ccc2sc(Nc3nc(N[C@@H]4CCCC[C@@H]4N)nnc3C(N)=O)c(C)c2c1
InChIKey: VVRDDABYTUFNHQ-UONOGXRCSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Syk Kinase Inhibition Assay: The in vitro inhibitory activity of the compound of the present invention against Syk kinase activity was assayed under the following conditions: the purified human Syk protein used in the test were purchased from Carna Biosciences, Inc. For the inhibitory activity assay on the compound, the compound of the present invention was first serially diluted with dimethyl sulfoxide (DMSO). Next, the purified human Syk protein FL-Peptide 22 (final concentration: 1 uM) (Caliper Life Sciences, Inc.), magnesium chloride (final concentration: 5 mM), ATP (final concentration: 30 uM), and each DMSO solution of the compound of the present invention (final concentration of DMSO: 5%) were added into a buffer solution for reaction (20 mM tris-HCl pH 7.5, 20 mM NaCl, 2.5 mM DTT, 0.02% Tween-20, 1% glycerol, 0.01 mM Pefabloc), and the mixture was then incubated at 25° C. for 30 minutes to perform kinase reaction. The kinase reaction was stopped by the addition thereto of EDTA.
Measurement: IC50 = 0.27 nM (pChEMBL 9.569999694824219)
Descriptors: MW 427.53, ALogP 2.93, HBA 9, HBD 4, PSA 141.07, QED 0.47
Compound (SMILES): CCc1ccc(OCCNc2nc(Nc3cc(C)[nH]n3)cc(C)c2C#N)cn1
InChIKey: NKWGBOOLJXYPQR-UHFFFAOYSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Inhibition of recombinant Aurora A kinase derived from human Hela cells using kemptide as substrate in presence of [c-32P]ATP
Measurement: Ki = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 377.45, ALogP 3.49, HBA 7, HBD 3, PSA 111.54, QED 0.52
Compound (SMILES): N#Cc1ccc(Nc2ncc(Br)c(Nc3cc(C4CC4)n[nH]3)n2)cc1
InChIKey: ZYZVKVWSZPLFEL-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 396.25, ALogP 4.2, HBA 6, HBD 3, PSA 102.31, QED 0.6
Compound (SMILES): c1nc(NC2=NC[C@@]3(CN4CCC3CC4)O2)c2sccc2n1
InChIKey: WQURRNDPPVYVAR-HNNXBMFYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Homo sapiens
UniProt: P36544
Assay: Agonist activity at alpha7 nACh receptor (unknown origin) expressed in HEK293 cells assessed as increase in Ca2+ flux by Fluo-4-AM dye based FLIPR assay
Measurement: EC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 315.4, ALogP 1.95, HBA 7, HBD 1, PSA 62.64, QED 0.87
Compound (SMILES): Cc1c(Nc2c(CO)nc3c(N)cc(N4CCOC(CO)C4)nn23)cccc1C(F)(F)F
InChIKey: KHWGXDFXYVESQN-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Binding affinity to human PI3Kalpha (108 to 1068 residues) expressed in mammalian expression system by KINOMEscan assay
Measurement: Kd > 30200 nM (pChEMBL 4.519999980926514)
Descriptors: MW 452.44, ALogP 2.07, HBA 9, HBD 4, PSA 121.17, QED 0.46
Compound (SMILES): CC(NC(=O)c1ccc(-c2cn[nH]c2)c(C=O)c1)c1ccccc1F
InChIKey: VBKDAAYYAUWVCA-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 983 nM (pChEMBL 6.010000228881836)
Descriptors: MW 337.35, ALogP 3.52, HBA 3, HBD 2, PSA 74.85, QED 0.7
Compound (SMILES): COc1cc(Br)c(Cl)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: ARIJPFAQWNKHLP-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 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 -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 = 178 nM (pChEMBL 6.75)
Descriptors: MW 510.75, ALogP 4.2, HBA 7, HBD 1, PSA 103.43, QED 0.43
Compound (SMILES): CC1CN(C(=O)c2ccc3cc(Oc4ccc(C(F)(F)F)cn4)ccc3n2)CC1O
InChIKey: VOCREZZJJXIVFU-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 14800 nM (pChEMBL 4.829999923706055)
Descriptors: MW 417.39, ALogP 3.89, HBA 5, HBD 1, PSA 75.55, QED 0.7
Compound (SMILES): CC1(C)[C@@H]2CC[C@@]1(C)[C@@H](NC(=O)[C@H](CC1CCCCC1)NS(=O)(=O)N[C@@H](CCCN)C(=O)O)C2
InChIKey: UFLJZTNISDJMGN-LJTNYMJHSA-N
Target: Carboxypeptidase B2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96IY4
Assay: Inhibition of activated human plasma TAFI incubated for 15 mins by chromogenic assay
Measurement: IC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 500.71, ALogP 2.27, HBA 5, HBD 5, PSA 150.62, QED 0.28
Compound (SMILES): CNC(=O)[C@H](CC(C)C)NC(=O)c1cccc(-c2cccc(C(F)(F)F)c2)n1
InChIKey: HIRINKHJHLEVAH-KRWDZBQOSA-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.times.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.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul 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 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM 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.
Measurement: EC50 = 57.1 nM (pChEMBL 7.239999771118164)
Descriptors: MW 393.41, ALogP 3.66, HBA 3, HBD 2, PSA 71.09, QED 0.79
Compound (SMILES): O=c1[nH]c2ccc(S(=O)(=O)Nc3ccc(Br)cc3)cc2[nH]c1=O
InChIKey: NESCUKRAVDFFCC-UHFFFAOYSA-N
Target: N-acetyltransferase Eis (SINGLE PROTEIN), Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
UniProt: P9WFK7
Assay: Eis inhibition assay: To investigate the potential of these compounds as Eis inhibitors for use in combination with KAN, their biochemical (IC50 values against purified Eis enzyme) and biological (effect on the MIC values of KAN in KAN-sensitive Mtb H37Rv and in Mtb K204, which is KAN-resistant due to Eis upregulation properties were evaluated in parallel studies (Table 2 and FIG. 1). The freshly synthesized compound 29 displayed robust inhibition of Eis in vitro (IC50=0.08±0.02 μM). When combined with KAN, sulfonamide 29, resulted in a four-fold reduced KAN MIC value (2.5 μg/mL) compared to KAN alone (10 μg/mL) for K204 Mtb. This was a reduction almost to the MIC level of KAN in the KAN-susceptible Mtb H37Rv (1.25 μg/mL) parent strain. To gain insight into the importance of the substitution pattern on the aniline portion of the sulfonamide scaffold, secondary (NHAr) and tertiary (N(Me)Ar) sulfonamides (29-47) were first generated. Eis inhibition assays with the synthesized sulfonamides were carried out in combination with KAN. The non-methylated counterpart of lead compound 29, compound 30, displayed lower Eis inhibitory activity (IC50=6.24±1.31 μM) and, contrary to 29, did not overcome KAN resistance in Mtb K204 (MICKAN=10 μg/mL). Two other non-methylated derivatives, 31 and 32, also resulted in lower Eis inhibitory activity (IC50>200 and 10.6±2.5 μM, respectively) and did not overcome KAN resistance in Mtb K204 (MICKAN=10 and 5 μg/mL, respectively).
Measurement: IC50 = 6200 nM (pChEMBL 5.210000038146973)
Descriptors: MW 396.22, ALogP 1.78, HBA 4, HBD 3, PSA 111.89, QED 0.59