lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nc(C)n(-c2cc([C@@H]3C[C@H]3c3nc4ccccc4n3C)nc(OCC3(C)CC3)n2)n1
InChIKey: HOLSBQIXIHIHJA-IAGOWNOFSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: In a typical experiment the PDE10 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. PDE10A2 was amplified from human fetal brain cDNA (Clontech, Mountain View, Calif.) using a forward primer corresponding to nucleotides 56-77 of human PDE10A2 (Accession No. AF127480, Genbank Identifier 4894716), containing a Kozak consensus sequence, and a reverse primer corresponding to nucleotides 2406-2413 of human PDE10A2 (Accession No. AF127480, Genbank Identifier 4894716). Amplification with Easy-A polymerase (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.2-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. AD293 cells with 70-80% confluency were transiently transfected with human PDE10A2.
Measurement: Ki = 0.84 nM (pChEMBL 9.079999923706055)
Descriptors: MW 429.53, ALogP 4.01, HBA 8, HBD 0, PSA 83.54, QED 0.46
Compound (SMILES): COc1cc(C(=O)NC[C@@H](C)c2ccccc2)ccc1-c1cn[nH]c1
InChIKey: ZSRGQQNNHDFZKU-CQSZACIVSA-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 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 335.41, ALogP 3.62, HBA 3, HBD 2, PSA 67.01, QED 0.72
Compound (SMILES): COc1cc(N=S2(=O)CCC2)cc2ncnc(Nc3ccc(F)cc3O[C@H](C)C(=O)NCC#N)c12
InChIKey: KULZBPBHSDBCPL-CQSZACIVSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: In vitro Kinase Assay: The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled eIF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 498.54, ALogP 3.43, HBA 9, HBD 2, PSA 138.59, QED 0.45
Compound (SMILES): CC(Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1nc(-c2cccc(OC(F)(F)F)c2)no1
InChIKey: ZJFRSZUNHMBIIV-MDLDFJCZSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 97 nM (pChEMBL 7.010000228881836)
Descriptors: MW 480.4, ALogP 3.3, HBA 10, HBD 2, PSA 135.73, QED 0.52
Compound (SMILES): CCNCc1cccc(-c2ccc(S(=O)(=O)Nc3c(CC(C)C)nn(C)c3C)cc2)c1
InChIKey: GROWGBVQGVDZRY-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
UniProt: Q388H8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 440.61, ALogP 4.5, HBA 5, HBD 2, PSA 76.02, QED 0.51
Compound (SMILES): CN(C)[C@@H]1CN(c2nc3ccccc3n([C@H]3C[C@H]4CCC[C@@H](C3)N4[C@H]3C[C@@H]4CCC[C@@H](C4)C3)c2=O)[C@@H]1C(=O)O
InChIKey: LDOAWQYBTPLFBU-FTKDUGCWSA-N
Target: Opioid growth factor receptor-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5TC84
Assay: In Vitro ORL-1 Receptor Binding Assay: Radioligand binding assays (screening and dose-displacement) used 0.1 nM [3H]-nociceptin (NEN; 87.7 Ci/mmole) with 10-20 μg membrane protein in a final volume of 500 μL binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Non-specific binding was determined in the presence of 10 nM unlabeled nociceptin (American Peptide Company). All reactions were performed in 96-deep well polypropylene plates for 1 h at about 25 °C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard) presoaked in 0.5% polyethylenimine (Sigma-Aldrich). Harvesting was performed using a 96-well tissue harvester (Packard) followed by three filtration washes with 500 μL ice-cold binding buffer. Filter plates were subsequently dried at 50 °C. for 2-3 hours. Fifty μL/well scintillation cocktail (BetaScint; Wallac) was added and plates were counted in a Packard Top-Count for 1 min/well.
Measurement: Ki = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 533.72, ALogP 4.13, HBA 7, HBD 1, PSA 81.91, QED 0.62
Compound (SMILES): CNC(=O)C[C@@H](NC(=O)c1ccc(-c2cn[nH]c2)c(C)c1)c1ccccc1
InChIKey: ZZOUOZQSRJYAMC-LJQANCHMSA-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 = 41.1 nM (pChEMBL 7.389999866485596)
Descriptors: MW 362.43, ALogP 2.99, HBA 3, HBD 3, PSA 86.88, QED 0.63
Compound (SMILES): CNC(=O)[C@@H](NC(=O)c1ccc(Br)c(OCC2CCCO2)n1)C(C)(C)C
InChIKey: HXRPRKYDBDQLFE-SBXXRYSUSA-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 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 428.33, ALogP 2.29, HBA 5, HBD 2, PSA 89.55, QED 0.73
Compound (SMILES): NC(=O)[C@@H](O)[C@H](O)c1cccc(-c2ccc(Oc3ccc(C(F)(F)F)cn3)cc2)n1
InChIKey: VETIEAKCCZYWEU-MSOLQXFVSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR TETRA Sodium Dye Assay: FLIPR or FLIPRTETRA Sodium Dye Assay with KCl and Test Article Pre-Incubation: Cells were prepared by plating the recombinant HEK293 cells or other host cells expressing either recombinant or non-recombinant, native, NaV1.7 alpha subunit, alone or in combination with various beta and gamma subunits at a density of 40,000 cells/well into a 96-well black, clear-bottom, PDL-coated plate. The assay can be adapted to 384-well or 1,536-well format, if desired, using proportionately less cells and media. The plate was then incubated in growth media, with or without selective antibiotic, overnight at 37° C. at 5% CO2, 95% humidity, in preparation for the assay. For counter-screens of other voltage-gated sodium channels, the procedure was very similar, though optimal densities of cells, media and subsequent assay components can be fine-tuned for the particular cell line or isoform.The next day, at the start of the assay, the media was flicked from the cells and the wells were washed once.
Measurement: IC50 = 711 nM (pChEMBL 6.150000095367432)
Descriptors: MW 419.36, ALogP 2.83, HBA 6, HBD 3, PSA 118.56, QED 0.57
Compound (SMILES): CCCCOc1ccc(CN2C[C@H](O)[C@@H](O)[C@H](O)[C@H]2CO)cc1
InChIKey: KRANEQWPXTUBEZ-YYIAUSFCSA-N
Target: Lysosomal alpha-glucosidase (SINGLE PROTEIN), Mus musculus
UniProt: P70699
Assay: Enzyme Activity Assay: Cells were washed two times with 200 μL dPBS followed by the addition of 70 μL of substrate (2.11 mM 3 mM 4-MU-α-D-glu) in citrate-phosphate buffer (30 mM sodium citrate, 40 mM sodium phosphate dibasic, pH 4.0), and 2.5% DMSO to rows 1-12. Following incubation at 37° C. with 5% CO2 for about 3 h, 70 μL of stop buffer (0.4 M glycine pH 10.8) was added to rows 1-12. The plate was read in a Victor2 multilabel counter-Wallac fluorescent plate reader and the fluorescence at F460 nm was determined b at an excitation of 355 nm and emission of 460 nm using 1 second read time per well. Enzyme activity per μg of protein in the supernatant was calculated from the amount of fluorescence emitted, which is directly proportional to the amount of substrate hydrolyzed, and hence, the amount of Gaa activity in the lysate.
Measurement: EC50 = 17300 nM (pChEMBL 4.760000228881836)
Descriptors: MW 325.41, ALogP 0.12, HBA 6, HBD 4, PSA 93.39, QED 0.53
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N2CCN(c3ccccn3)CC2)n2c1COCC2)c1ccccc1
InChIKey: DVESKSPCCGNQPR-JOCHJYFZSA-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 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 473.58, ALogP 3.26, HBA 6, HBD 1, PSA 79.7, QED 0.59
Compound (SMILES): C[C@@H](O)[C@@H](NC(=O)Nc1cc2[nH]nc(OCCOC(F)F)c2c(CO)n1)c1ccccc1
InChIKey: HDJNNLOPPRKSMK-PIGZYNQJSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 451.43, ALogP 2.31, HBA 7, HBD 5, PSA 141.62, QED 0.3
Compound (SMILES): CN(C)CCCOc1ccc(-c2ccc3ncc4c(c3c2)n([C@@H]2CCCOC2)c(=O)n4C)c(F)n1
InChIKey: SBWOIJRPFUUCGS-GOSISDBHSA-N
Target: Serine-protein kinase ATM (SINGLE PROTEIN), Homo sapiens
UniProt: Q13315
Assay: Cellular Assay: Assay a) HT29 cells (ECACC #85061109) were seeded into 384 well assay plates (Costar #3712) at a density of 3500 cells/well in 40 μl EMEM medium containing 1% L glutamine and 10% FBS and allowed to adhere overnight. The following morning compounds of Formula (I) in 100% DMSO were added to assay plates by acoustic dispensing. After 1 h incubation at 37° C. and 5% CO2 plates (up to 6 at a time) were irradiated using the X-RAD 320 instrument (PXi) with equivalent to Ë¿600 cGy. Plates were returned to the incubator for a further 1 h. Then cells were fixed by adding 20 μl of 3.7% formaldehyde in PBS solution and incubating for 20 minutes at r.t. before being washed with 50 μl/well PBS, using a Biotek EL405 plate washer. Then 20 μ 0.1% Triton X100 in PBS was added and incubated for 20 minutes at r.t., to permeabalise cells. Then the plates were washed once with 50 μl/well PBS, using a Biotek EL405 plate washer. Phospho-ATM Ser1981 antibody (Millipore #MAB3806) was diluted 10000 fold in PBS containing 0.05% polysorbate/Tween and 3% BSA and 20 μl was added to each well and incubated over night at r.t. The next morning plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and then 20 μl of secondary Ab solution, containing 500 fold diluted Alexa Fluor® 488 Goat anti-rabbit IgG (Life Technologies, A11001) and 0.002 mg/ml Hoeschst dye (Life technologies #H-3570), in PBS containing 0.05% polysorbate/Tween and 3% BSA, was added. After 1 h incubation at r.t., the plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and plates were sealed and kept in PBS at 4° C. until read. Plates were read using an ArrayScan VTI instrument, using an XF53 filter with 10× objective. A two laser set up was used to analyse nuclear staining with Hoeschst (405 nm) and secondary antibody staining of pSer1981 (488 nm).
Measurement: IC50 = 0.552 nM (pChEMBL 9.260000228881836)
Descriptors: MW 479.56, ALogP 3.77, HBA 8, HBD 0, PSA 74.41, QED 0.3
Compound (SMILES): Cc1ccc([C@H]2C[C@@H]2COc2nc(C)ncc2-c2ccn(CC#N)c(=O)c2)nc1
InChIKey: JCYQSIGNSDOJHL-MSOLQXFVSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: PDE10 Fluorescence Polarization (FP) Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAPU 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 uL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 uL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate.
Measurement: Ki = 1.06 nM (pChEMBL 8.970000267028809)
Descriptors: MW 387.44, ALogP 3.02, HBA 7, HBD 0, PSA 93.69, QED 0.65
Compound (SMILES): Cc1ncc(-c2cc(=O)n(C)cc2C)c(OC[C@H]2C[C@@H]2c2ccccn2)n1
InChIKey: USKPESBECQCDFE-WBVHZDCISA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: PDE10 Fluorescence Polarization (FP) Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAPU 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 uL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 uL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate.
Measurement: Ki = 16.62 nM (pChEMBL 7.78000020980835)
Descriptors: MW 362.43, ALogP 3.04, HBA 6, HBD 0, PSA 69.9, QED 0.7
Compound (SMILES): COC(=O)N[C@H](C(=O)NCc1ccc([C@@H](CO)N(Cc2cc3cccnc3[nH]2)S(=O)(=O)c2ccc(N)cc2)s1)C(c1ccccc1)c1ccccc1
InChIKey: WQBHTCGEDBLVTI-LDNVYYCDSA-N
Target: Gag-Pol polyprotein (SINGLE PROTEIN), Human immunodeficiency virus type 1 group M subtype B (isolate HXB2)(HIV-1)
UniProt: P04585
Assay: Inhibition Assay: The inhibition of WT HIV-1 protease was studied using the reaction of the protease (expressed in Eschericia coli) with a peptide substrate [Val-Ser-Gln-Asn-(betanaphthyl)Ala-Pro-Ile-Val (SEQ ID NO:1)]. Compounds were tested using Procedure A. In Procedure A, the test compound was first preincubated with the enzyme in assay buffer (50 mM sodium acetate, pH 5.5, 100 mM NaCl, and 0.1% BSA) for 30 minutes at room temperature. Substrate was then added to 400 micromolar in a total volume of 80 microliters containing 10 picomolar HIV-1 protease and the reaction was incubated for 1 hour at 30° C. The reaction was quenched with the addition of 120 microliters of 10% phosphoric acid. The product formation was determined after separation of product and substrate on a Zorbax Eclipse XDB-C18 column (Agilent Technologies, Santa Clara, Calif.) connected to an Agilent 1100 high performance liquid chromatography system with fluorescence detection (excitation 270 nanometer and emission 330 nanometer).
Measurement: IC50 = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 738.89, ALogP 5.3, HBA 9, HBD 5, PSA 179.74, QED 0.09
Compound (SMILES): O=C1C(N2CC[C@H](c3ccc(O)cc3)[C@@H](F)C2)CCN1c1ccc(OC(F)F)c(Cl)c1
InChIKey: ZINNJNWQBUSWGG-FAZYOCGDSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q00960
Assay: Radioligand Binding Assay: To perform the competition binding assay, thawed membrane homogenate was added to each well of a 96-well plate (20 ug/well). The experimental compounds were serially diluted in 100% DMSO and added to each row of the assay plate to achieve desired compound concentrations, keeping the DMSO concentration in the assay plate at 1.33% of the final reaction volume. Next, 3H Ro 25-6981 (4 nM) was added to the assay plate. After incubation for 1 hr at room temperature, the membrane bound radioligand was harvested on to GF/B filter plates (treated with 0.5% PEI for 1 hr at room temperature). The filter plates were dried at 50° C. for 20 mins, incubated with microscint 20 for 10 minutes and finally, the counts were read on TopCount (Perkin Elmer). Non-specific binding was determined using MK-0657 (the preparation of this compound is described as example 1 in WO 2004 108705 (40 uM).
Measurement: Ki = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 454.88, ALogP 4.58, HBA 4, HBD 1, PSA 53.01, QED 0.72
Compound (SMILES): O=C(O)C[C@H]1C[C@H](c2cccc(Cl)c2)[C@]2(C(=O)Nc3cc(Cl)ccc32)N(CC2CC2)C1=O
InChIKey: BJYAVSZQIJEQQR-ZCLJVBCSSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Homogenous Time-Resolved Fluorescence Assay (HTRF1 Assay): The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1xPBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag-TrxA-6-His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3.
Measurement: IC50 = 90 nM (pChEMBL 7.050000190734863)
Descriptors: MW 473.36, ALogP 4.66, HBA 3, HBD 2, PSA 86.71, QED 0.66
Compound (SMILES): CC(C)OC(=O)N1CC[C@H](N(C(=O)c2ccc(-n3cncn3)c(F)c2)C2CC2)[C@H](F)C1
InChIKey: TWYSIUSXCSWBRV-MJGOQNOKSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: LANCE cAMP Assay: Quantitative detection of cAMP accumulation from cells expressing human GPR119 receptor is achieved using Perkin Elmer's LANCE cAMP-384 Kit (Cat#AD0264) according to the manufacturer's protocol. Briefly, HEK293 cells stably expressing a mutant form of the human GPR119 receptor as assay tool (Methionine 1 replaced with the amino acid sequence MKTIIALSYIFCLVFADYKDDDDA, and T327 & S329 changed to alanines; SEQ ID No. 1) are grown to 50-70% confluency in cell culture media (DMEM, 10% heat inactivated Fetal Bovine Serum, 50 I.U./mL penicillin, 50 ug/mL streptomycin, 10 mM HEPES, 20 ug/mL G418 Sulfate). On the day of the assay, GPR119 stable HEK293 cells are lifted from the tissue culture plate and 1000 cells/well are incubated along with various concentrations of test compounds for 20 min at 37° C. Detection Buffer (50 mM HEPES, 10 mM calcium chloride, 0.35% Triton X-100, 1 mg/mL BSA) containing cAMP-specific antibody is then added to all wells and allowed to equilibrate in the dark for 10 minutes at room temperature. Upon equilibration, Detection Buffer containing europium-labeled cAMP tracer complex is added to all wells and allowed to react for 1 hour at room temperature. After 1 hour, bound europium-labeled cAMP tracer is measured using a Perkin Elmer Envision plate reader. The quantity of cAMP generated in each well is derived from a standard curve. EC50 is determined using nonlinear regression analysis of the cAMP values over a range of agonist concentration (12 points spanning the range from 30 uM to 100 pM).
Measurement: EC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 433.46, ALogP 2.97, HBA 6, HBD 0, PSA 80.56, QED 0.72
Compound (SMILES): CCNC(=O)C(C)c1ccc(O[C@@H]2CCN(c3ccnc(OC(C)C)c3)C2)cc1
InChIKey: YSFXWJRKSSZMMT-FBLFFUNLSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Malonyl CoA formation by acetyl CoA carboxylases is stoichometrically linked to the consumption of ATP. ACC2 activity is measured in a NADH-linked kinetic method measuring ADP generated during the ACC reaction using a coupled lactate dehydrogenase/pyruvate kinase reaction. 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, acetylCoenzyme 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.
Measurement: IC50 = 375 nM (pChEMBL 6.429999828338623)
Descriptors: MW 397.52, ALogP 3.77, HBA 5, HBD 1, PSA 63.69, QED 0.73
Compound (SMILES): Cc1c(CC(=O)N[C@H](CO)C(=O)O)cc(-c2ccc(S(C)(=O)=O)cc2)n1-c1ccc(F)cc1
InChIKey: LYJNRGFCMLOFNV-HXUWFJFHSA-N
Target: Prostaglandin G/H synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35354
Assay: In Vitro Inhibition Assay: The murine monocyte/macrophage cell line J774 is grown in Dulbecco's modified Eagle's medium (DMEM), enriched with glutamine (2 mM), HEPES (25 mM), penicillin (100 ug/mL), streptomycin (100 ug/mL), 10% of fetal bovine serum (FBS) and 1.2% of sodium pyruvate. The cells are distributed in 24-well plates at a density of 2.5x105 cells/mL or in culture dishes with a diameter of 10 cm (1x107 cells/10 mL/dish) and kept for 2 hours at 37° C. in a CO2 (5%)/O2 (95%) atmosphere. Just before the experiments, the culture medium is replaced with fresh medium without FBS to avoid interference during the radio-immunological phase and the cells are stimulated as described below.Evaluation of COX-1 Activity.The cells are pretreated with the test compounds for 15 minutes and then incubated for 30 minutes with arachidonic acid (15x10-6 m). At the end of incubation the supernatants are collected for evaluating, by means of radio-immunological assays, the amount of PGE2 produced.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 474.51, ALogP 2.1, HBA 6, HBD 3, PSA 125.7, QED 0.46
Compound (SMILES): C[C@@H](NC(=O)Nc1ncc2c(OCc3cccnc3)n[nH]c2c1F)c1ccccc1
InChIKey: HSJPRAWOSKXJOP-CYBMUJFWSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 1.89 nM (pChEMBL 8.720000267028809)
Descriptors: MW 406.42, ALogP 3.95, HBA 5, HBD 3, PSA 104.82, QED 0.45
Compound (SMILES): Cc1nn(C)c(C)c1NS(=O)(=O)c1ccc(-c2cc(CN3CCN(C)CC3)cc(OC(C)C)c2)cc1
InChIKey: GWQHGEKEPLMGJC-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
UniProt: Q388H8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 480 nM (pChEMBL 6.320000171661377)
Descriptors: MW 511.69, ALogP 4.04, HBA 7, HBD 1, PSA 79.7, QED 0.49
Compound (SMILES): COc1cc2c(N)nc(N3CCN(C(=O)C[C@@H](N)c4ccc(F)cc4)CC3)nc2c(F)c1OC
InChIKey: JIBSJZSTMKHSTK-MRXNPFEDSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: ELISA Assay: CVF-Bb complex prepared from purified cobra venom factor (1 μM), recombinant human complement factor B (expressed in drosophila cells and purified using standard methods) and human complement factor D (expressed in E. Coli, refolded and purified using standard methods). CVF-Bb complex at 3 nM concentration was incubated with test compound at various concentrations for 1 hour at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human complement C3 substrate purified from plasma was added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction was stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, was quantified by means of an enzyme-linked-immunosorbent assay.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 472.5, ALogP 2.25, HBA 8, HBD 2, PSA 119.83, QED 0.56
Compound (SMILES): O=C([C@@H]1CCCN(c2ccc3nc(-c4cnn(-c5ccccc5)c4)[nH]c3n2)C1)N1CCCC1
InChIKey: RUZQAFJKBDRWIM-GOSISDBHSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: In Vitro DGAT2 Assay: For determination of IC50 values, the reactions were carried out in 384-well white Polyplates (Perkin Elmer) in a total volume of 20 uL. To 1 uL of compounds dissolved in 100% DMSO and spotted at the bottom of each well, 5 uL of 0.04% bovine serum albumin (BSA) (fatty acid free, Sigma Aldrich) was added and the mixture was incubated at room temperature for 20 minutes. To this mixture, 10 uL of hDGAT2 membrane fraction (0.01 mg/mL) diluted in 100 mM Hepes-NaOH, pH 7.4, 20 mM MgCl2 containing 200 nM methyl arachidonyl fluorophosphonate (Cayman Chemical; dried from ethyl acetate stock solution under argon gas and dissolved in DMSO as 5 mM stock) was added. After this mixture was preincubated at room temperature for 2 hours, DGAT2 reactions were initiated by the addition of 4 uL of substrates containing 30 uM [1-14C]decanoyl-CoA (custom-synthesized by Perkin Elmer, 50 mCi/mmol) and 125 uM 1,2-didecanoyl-sn-glycerol (Avanti Polar Lipids) dissolved in 12.5% acetone. The reaction mixtures were incubated at room temperature for 40 min and the reactions were stopped by addition of 5 uL of 1% H3PO4. After the addition of 45 uL MicroScint-E (Perkin-Elmer), plates were sealed with Top Seal-A covers (Perkin-Elmer) and phase partitioning of substrates and products was achieved using a HT-91100 microplate orbital shaker (Big Bear Automation, Santa Clara, Calif.). Plates were centrifuged at 2,000×g for 1 min in an Allegra 6R Centrifuge (Beckman Coulter) and then were sealed again with fresh covers before reading in a 1450 Microbeta Wallac Trilux Scintillation Counter (Perkin Elmer). DGAT2 activity was measured by quantifying the generated product [14C]tridecanoylglycerol in the upper organic phase.
Measurement: IC50 = 22.1 nM (pChEMBL 7.659999847412109)
Descriptors: MW 441.54, ALogP 3.65, HBA 6, HBD 1, PSA 82.94, QED 0.52
Compound (SMILES): CO[C@]12CC[C@@]3(C[C@@H]1COCc1ccccc1)[C@H]1Cc4ccc(OCC(=O)O)c5c4[C@@]3(CCN1CC1CC1)[C@H]2O5
InChIKey: HAAGAXZZVIBWSV-SMHPFREUSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Radioligand Dose-Displacement Binding Assay: Radioligand dose-displacement binding assays for t-opioid receptors used 0.3 nM [3H]-diprenorphine (Perkin Elmer, Shelton, Conn.), with 5 mg membrane protein/well in a final volume of 500 ul binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 2 hr at room temperature. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.), presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by performing three filtration washes with 500 ul of ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 ul/well), and plates were counted using a Packard Top-Count.
Measurement: Ki = 163.53 nM (pChEMBL 6.789999961853027)
Descriptors: MW 545.68, ALogP 4.59, HBA 6, HBD 1, PSA 77.46, QED 0.47
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N[C@H](C)c2ccc(F)cc2)n2c1CSCC2)c1ccccc1
InChIKey: NIIMCDLYCAWLAT-VGOFRKELSA-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 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 465.59, ALogP 5.25, HBA 4, HBD 2, PSA 63.13, QED 0.5
Compound (SMILES): CCNC(=O)C(C)c1ccc(O[C@@H]2CCN(c3ccc(F)c(C(F)(F)F)c3)C2)cc1
InChIKey: FZUPIZUXVJYCHF-XPKAQORNSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Malonyl CoA formation by acetyl CoA carboxylases is stoichometrically linked to the consumption of ATP. ACC2 activity is measured in a NADH-linked kinetic method measuring ADP generated during the ACC reaction using a coupled lactate dehydrogenase/pyruvate kinase reaction. 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, acetylCoenzyme 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.
Measurement: IC50 = 229 nM (pChEMBL 6.639999866485596)
Descriptors: MW 424.44, ALogP 4.74, HBA 3, HBD 1, PSA 41.57, QED 0.68
Compound (SMILES): C[C@@H]1CCCN1CCc1ccc(-c2ccc(CCC(=O)NCC(=O)OC(C)(C)C)cc2)cc1
InChIKey: NTVAMIDNRXYMHB-OAQYLSRUSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: HTRF cAMP Assay: Compounds of the present invention were evaluated using the human H3 receptor (H3R) HTRF cAMP assay. In this assay, HEK293 cells expressing the human H3 receptor were suspended in PBS containing 100 μM IBMX and plated into 384-well assay plates (Perkin Elmer Proxiplate 384-Plus; 15,000 cells per well; 5 μL plating volume) and allowed to equilibrate for an hour. Test compounds were serially diluted in 100% DMSO and then further diluted in PBS containing forskolin (2 μM). Test compounds (5 μL) were then added to the assay plate and the mixture was incubated for 1 hour. HTRF assay reagents (Cisbio, Dynamic 2 cAMP Kit), cAMP-d2 and cryptate-labeled anti-cAMP antibody, are mixed with cell lysis buffer and added to the assay plate. After 1-hour incubation with these reagents, the assay plate was read on an HTRF-compatible microplate reader (Perkin Elmer EnVision or BMG Pherastar).
Measurement: IC50 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 450.62, ALogP 4.77, HBA 4, HBD 1, PSA 58.64, QED 0.56
Compound (SMILES): Nc1nccn2c([C@@H]3CC[C@H]4CCC(=O)N4C3)nc(-c3ccc(C(=O)Nc4nccc(C(F)(F)F)n4)cc3)c12
InChIKey: FFGPCWBZVXIFTH-SJORKVTESA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 uM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 uL) was dispensed, followed by the addition of 7.5 uL of 1x kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/uL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 uL 1x kinase buffer containing 8 uM biotinylated "A5" peptide (Biotin-EQEDEPEGDYFEWLE-NH2) (SEQ ID NO: 1), and 100 uM ATP. The final reaction in each well of 10 uL consists of 100 pM hBTK, 2 uM biotin-A5-peptide, and 25 uM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1x quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 536.52, ALogP 3.91, HBA 8, HBD 2, PSA 131.4, QED 0.4
Compound (SMILES): CC(CF)Nc1cc(-n2ccc3cncnc32)ncc1C(=O)NC[C@@H](F)C(C)(C)O
InChIKey: OGEZLJGKMHUYIX-PVQCJRHBSA-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 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 418.45, ALogP 2.42, HBA 7, HBD 3, PSA 104.96, QED 0.52
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)NC(C)c2ncccn2)n2c1COCC2)c1ccccc1
InChIKey: HOEHRXVHNCXSCQ-LRTDYKAYSA-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 = 560 nM (pChEMBL 6.25)
Descriptors: MW 433.51, ALogP 3.18, HBA 6, HBD 2, PSA 98.14, QED 0.6
Compound (SMILES): Cc1cc([C@](C)(O)C#Cc2ccc3c(c2)-c2nc(C(N)=O)sc2[C@H](O)CO3)no1
InChIKey: AWFVHVCDZZHDQC-ZUOKHONESA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 411.44, ALogP 1.89, HBA 8, HBD 3, PSA 131.7, QED 0.55