lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(C)n1nc(-c2[nH]c3cc(C(=O)N4CCCC4)ccc3c2Cl)c2c(N)ncnc21
InChIKey: UTHPECSQYAWGLE-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Enzyme Assay: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer conditions were used for KDR with the addition of 2 mM MnCl2. RETV804M buffer used 1× CisBio enzymatic buffer supplemented with a final concentration of 2 mM MgCl2, 1 mM DTT, 20 nM SEB and 0.01% Triton X100.
Measurement: IC50 = 355 nM (pChEMBL 6.449999809265137)
Descriptors: MW 437.94, ALogP 4.2, HBA 6, HBD 2, PSA 105.72, QED 0.49
Compound (SMILES): Cc1nnc(NC(=O)c2cc(C3CC3NC3CCC(F)(F)CC3)ccc2F)s1
InChIKey: ONWSNDOWFQNNLV-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 Inhibition Assay: A test compound dissolved in 2.5% DMSO was added by 4 μL to 3 μL reaction solution (50 mM Tris-HCl (pH 8.0), 0.1% BSA, 1 mM DTT) containing 7.5 ng of LSD1, and the mixture was reacted at room temperature for 60 min. Biotin-histone H3 mono methylated K4 peptide solution (NH2-ART(me-K)QTARKSTGGKAPRKQLAGGK(Biotin)-CONH2) (3.3 μM) was added by 3 μL to start the reaction. After reaction at room temperature for 5 min, 1 mM 2-PCPA solution was added 5 μL to terminate the reaction. A detection solution (BOO mM potassium fluoride, 0.1% BSA) containing europium-labeled anti-histone H3 antibody (Wako Pure Chemical Industries, Ltd.) and Streptavidin-XL665 (Cisbio) was further added by 5 μL, and the mixture was left standing for 60 min. A time-resolved fluorescence (excitation 320 nm, emission 615 nm, 665 nm) was measured by Envision (PerkinElmer). The LSD1 inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate(%)=(1−(test compound count−blank)÷(control−blank))×100
Measurement: IC50 = 450 nM (pChEMBL 6.349999904632568)
Descriptors: MW 410.47, ALogP 4.26, HBA 5, HBD 2, PSA 66.91, QED 0.77
Compound (SMILES): COc1ccc([C@@H](C)NC(=O)c2cc(=O)[nH]c(-c3ccccc3)n2)cc1
InChIKey: CNYHJZNCGMWTHX-CYBMUJFWSA-N
Target: GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (SINGLE PROTEIN), Macaca mulatta
UniProt: B3VTJ2
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° 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 pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 349.39, ALogP 2.94, HBA 4, HBD 2, PSA 84.08, QED 0.74
Compound (SMILES): COC(=O)N[C@@H](CC/C=C/C(=O)N(C)C)C(=O)Nc1cccn(Cc2nc3cccc(Oc4ccccc4)c3[nH]2)c1=O
InChIKey: WJVVTQGFJBDPOD-NSKACUHBSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 μl/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 572.62, ALogP 3.65, HBA 8, HBD 3, PSA 147.65, QED 0.23
Compound (SMILES): Cc1ccc(CNC(=O)C2CN(c3ncnn4cc(-c5ccc(C(C)(O)C(F)(F)F)cc5)cc34)C2)cc1
InChIKey: KVBXLUFTELNHOP-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit Wild Type Assay: In each well of a 384-well plate, 0.2 ng/ul final (2 nM) of wild type Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 509.53, ALogP 4.23, HBA 6, HBD 2, PSA 82.76, QED 0.41
Compound (SMILES): O=C(NCc1ncc[nH]1)c1ccc2c(c1)OCc1cnccc1-2
InChIKey: INBWALBDNLRSDY-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
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. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 1080 nM (pChEMBL 5.96999979019165)
Descriptors: MW 306.32, ALogP 2.29, HBA 4, HBD 2, PSA 79.9, QED 0.78
Compound (SMILES): CC(C)(COc1ncc(C#N)c(-n2cnc3ccccc32)n1)c1ccnc(F)c1
InChIKey: RPLFIOOYCGUEFD-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: Inhibition Assay: The synthesized compounds then were tested for inhibition of the kinase activity (IC50) of Mnk1 and/or Mnk2 using the ADP monitoring assay for kinases described in Zegzouti, H.; Zdanovskaia, M.; Hsiao, K.; Goueli, S. A., ADP-Glo: A Bioluminescent and homogeneous ADP monitoring assay for kinases.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 388.41, ALogP 3.58, HBA 7, HBD 0, PSA 89.51, QED 0.49
Compound (SMILES): Cc1nc(OCc2cn(-c3cccc(F)c3F)nn2)ncc1F
InChIKey: JWBKTOPCJJMRNG-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P19793
Assay: NR2B standard assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ¿EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 15900 nM (pChEMBL 4.800000190734863)
Descriptors: MW 321.26, ALogP 2.36, HBA 6, HBD 0, PSA 65.72, QED 0.74
Compound (SMILES): COCCC(=O)N1CCC[C@@H](c2nc(-c3ccc(C(=O)Nc4ccccn4)cc3C(F)(F)F)c3c(N)nccn23)C1
InChIKey: AKSJYRSZWLCCSW-GOSISDBHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay A: Btk enzyme activity is measured using the IMAP (immobilized metal ion affinity-based fluorescence polarization) assay as outlined below. Btk enzyme (His-Btk (Millipore catalog #14-552), is diluted to 0.4 U/mL in KR buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% NaN3, 1 mM DTT, 2 mM MnCl2, pH 7.2).Serial dilution log 10 from 2 mM to 63.2 nM of test compounds are made in 100% DMSO. The dilutions in DMSO are then diluted 50-fold in KR-buffer. Final compound concentration range in the assay from 10 μM to 0.316 nM. 5 μL/well of test compound in KR buffer (final DMSO concentration in the assay is 1%) is mixed with 5 μl/well of 0.4 U/mL Btk enzyme (final concentration in the assay is 0.1 U/mL). Test compounds and Btk enzyme are pre-incubated 60 minutes at room temperature, before adding 5 μL/well of 200 nM Fluorescin labeled substrate peptide (Blk/Lyntide substrate, e.g. #R7188/#R7233, Molecular Devices) in KR-buffer. Final peptide substrate concentration in assay is 50 nM. The kinase assay is started by adding 5 μL/well of 20 μM ATP in KR-buffer (final ATP concentration is 5 μM ATP, Km ATP in Btk IMAP assay). Following incubation for 2 h at room temperature the enzyme reaction is stopped by adding 40 μL/well IMAP Progressive Binding Solution (according to suppliers (Molecular Devices) protocol using 75% 1× buffer A and 25% 1x buffer B with 1:600 Progressive Binding Solution). After 60 min incubation at room temperature in the dark the FP signal is read. Fluorescence at 535 nm is measured using parallel and perpendicular filters to determine differences in rotation due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmPi) of the controls with and without ATP. EC50 values are determined by curve fitting of the experimental results using Activity Base.
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 567.57, ALogP 4.39, HBA 8, HBD 2, PSA 127.74, QED 0.34
Compound (SMILES): O=C1Nc2cc3[nH]nc(-c4ccnnc4)c3cc2CN1Cc1cc(F)cc(F)c1
InChIKey: BZVPOQDVSBDIEQ-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Z′-LYTE Kinase Assay: The inhibition of ERK activity by the compounds disclosed herein was determined using the Z′-LYTE kinase assay kit (Life Technologies) with a Ser/Thr 3 peptide substrate (Life Technologies) according to manufacturers' instructions. The assay was run with an ERK2 enzyme (Life Technologies) concentration of 0.47 ng/μL at 100 μM ATP (approximately the ATP Km for ERK2). The IC50 values for the compounds were determined with 3-fold serial dilutions in duplicate. The compounds were first diluted in 1:3 dilutions in 100% DMSO at 100× the desired concentration, and then further diluted (1:25) in 20 mM HEPES buffer (Invitrogen) to make 4× solutions prior to adding to the enzyme solution. The final DMSO concentration in the assay was 1%. Final reaction volume was 20 μL/well in 384-well plates. Kinase reactions were conducted or 1 hour followed by the assay development reaction (1 hour) in a 20 ul/well in a 384 well plate format.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 392.37, ALogP 3.85, HBA 4, HBD 2, PSA 86.8, QED 0.55
Compound (SMILES): C=CC(=O)N1CC[C@@H](NC(=O)c2sc3nccc4c3c2NC(=O)N4c2ccc(Oc3ccccc3)c(Cl)c2)C1
InChIKey: WYKPYMXPBIHGEY-MRXNPFEDSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TBD: TBD
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 560.04, ALogP 5.94, HBA 6, HBD 2, PSA 103.87, QED 0.28
Compound (SMILES): CCn1cc(Nc2ncc(C(N)=O)c(Nc3c(C)cccc3C)n2)cn1
InChIKey: UASHYQIXXSVIQQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Kinase Activity Assay: JAK1 (the final concentration was 10 nM); ATP (the final concentration was 10 μM); ULight-labeled JAK-1 (Tyr1023) Peptide (the final concentration was 100 nM); the enzymatic reaction time was 2 hours. The maximum final concentration of compounds was 2.5 μM. After a 3-fold gradient dilution, 11 concentrations were obtained, with the minimum final concentration of 0.042 nM. The final DMSO concentration was 1%.
Measurement: IC50 = 7.07 nM (pChEMBL 8.149999618530273)
Descriptors: MW 351.41, ALogP 2.9, HBA 7, HBD 3, PSA 110.75, QED 0.63
Compound (SMILES): CCc1c(F)c(F)c2[nH]ncc2c1-c1ccc2nc(NC(=O)[C@@H]3C[C@@H]3F)cn2c1
InChIKey: AOEWHMHQKRERQE-YPMHNXCESA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: ADP-Glo kinase assay: HPK1 kinase activity was measured by Promega's ADP-Glo kinase assay. In this assay, 5 ng of recombinant human HPK1 (signalchem) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of MBP (0.5 μg/μl) and 5 μL of ATP (25 μM) in buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 5 μM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30′ C for 40 minutes. After the incubation, 25 uL ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 uL per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software and SigmaPlot13.0.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 399.38, ALogP 4.01, HBA 4, HBD 2, PSA 75.08, QED 0.54
Compound (SMILES): C[C@@]1(O)[C@H](O)[C@@H](COC(Cc2ccccc2)(C(=O)O)c2nn[nH]n2)O[C@H]1n1cnc2c(N)nc(Cl)nc21
InChIKey: YATVUJRJCCEDHK-PFNBLGQVSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Inhibition Enzyme In Vitro Assay: For measurements of soluble CD73 enzyme activity, recombinant CD73 was obtained from R&D Systems, Cat. No. 5795-EN-010. Serial dilutions of test compounds were incubated with recombinant CD73 and AMP in reaction buffer (25 mM Tris HCl pH7.5, 5 mM MgCl2, 50 mM NaCl, 0.25 mM DTT, 0.005% Triton X-100). The final reaction volume was 25 μL and the final concentrations of recombinant CD73 and AMP were 0.5 nM and 50 μM, respectively. Reactions were allowed to proceed for 30 minutes at room temperature before the addition of 100 μL Malachite Green (Cell Signaling Technology, Cat. No. 12776). After 5 minutes at room temperature, absorbance at 630 nm was determined on a microplate spectrophotometer.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 531.92, ALogP -0.18, HBA 13, HBD 5, PSA 220.3, QED 0.19
Compound (SMILES): Cc1ncoc1C(=O)N1CCN(c2nc(C3CC3)c3c(c2C=N)CC(C)(C)OC3)C[C@H]1C(C)C
InChIKey: PDDIRLZEWOZURM-NRFANRHFSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Inhibition Assay: Assays were conducted in a volume of 76 μl assay buffer (150 mM NaCl, 10 mM MgCl2, 20 mM Tris pH 7.5, 0.03% bovine serum albumin) as follows in a standard 384-well plate: To 25 ul of substrate mix (8 uM NADPH, 2 mM aKG), 1 μl of test compound was added in DMSO. The plate was centrifuged briefly, and then 25 μl of enzyme mix was added (0.2 μg/ml IDH1 R132H) followed by a brief centrifugation and shake at 100 RPM. The reaction was incubated for 50 minutes at room temperature, then 25 μl of detection mix (30 μM resazurin, 36 μg/ml) was added and the mixture further incubated for 5 minutes at room temperature. The conversion of resazurin to resorufin was detected by fluorescent spectroscopy at Ex544 Em590 c/o 590.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 465.6, ALogP 4.09, HBA 7, HBD 1, PSA 95.55, QED 0.67
Compound (SMILES): Clc1cnc(N2CC3CC2CN3)nc1Nc1ccc2[nH]ncc2c1
InChIKey: XSLFUSVQDXPOPE-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Determination of Compound's ROCK Inhibitory Activity In Vitro (Z'Lyte Assay): Recombinant ROCK1 (amino acids 1-535) and ROCK2 (amino acids 1-552) proteins were purchased from ThermoFisher Scientific. Compound's activities were measured by Z′-lyte kinase kit (ThermoFisher Scientific) and IC50 was calculated
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 341.81, ALogP 2.3, HBA 6, HBD 3, PSA 81.76, QED 0.68
Compound (SMILES): OCCc1nccn1Cc1cn(-c2cccc(C3CC3)c2)nn1
InChIKey: QUXRRGHMYGOPOY-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition Assay: Cells were split 2-3 times weekly between 1:3 and 1:4. For binding assays and membrane preparations the cell culture medium was removed, cells were washed with PBS. Crude membranes for radioligand binding experiments were prepared by scraping the cells off the dishes in ice cold 20 mM HEPES/0.1 mM KCl/pH 7.2. The cell suspension was homogenized on ice (Ultra turrax, 3×20 sec.) and the homogenate was spun for 10 min (1° C., 1000 g, OPTIMA, SW28, 2800 U/min). The supernatant was than centrifuged for 40 min at 100000 g (1° C., OPTIMA, SW28, 23000 U/min). The membrane pellet was resuspended in 20 mM HEPES/0.1 mM KCl/pH 7.2, frozen and stored at −80° C.After thawing on the day of the assay, the membrane suspension was diluted further with 20 mM HEPES/0.1 mM KCl/pH 7.2.The incubation mixture of 200 μl contained 1.5 nmol/l 3H-Dofetilide, optimized amount of membrane preparation, 20 mM HEPES/0.1 mM KCl/(pH 7.2) and inhibitor in 1% DMSO. Nonspecific binding was estimated in the presence of 10 M Dofetilide. The samples were incubated for 90 min. at RT.Binding was terminated by filtration of the incubated membrane preparations using Filtermat B (Pharmacia, Uppsala Sweden) and a Micro Cell Harvester (Skatron, Lier, Norway). The Filtermat B had been presoaked with 1% polyethylenimine and carefully washed with 0.05 M Tris/HCl-buffer pH=7.7 after the filtration to separate free and bound radioactivity. The filters were counted in a scintillation counter (Betaplate 1205, Berthold, Wildbad, Germany) in order to determine the specific binding of [3H]-Dofetilide.
Measurement: IC50 = 41.1 nM (pChEMBL 7.389999866485596)
Descriptors: MW 309.37, ALogP 1.92, HBA 6, HBD 1, PSA 68.76, QED 0.76
Compound (SMILES): CCCC(=O)Nc1cnc(N2CCC(NC3CCCCC3)CC2)c(C(=O)OC)c1
InChIKey: UAMAEHXHDGMPPG-UHFFFAOYSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: OPRK1 antagonist assay: The cell line for the OPRK1 antagonist assay stably expresses the following elements. The carboxy terminus of the OPRK1 receptor has a 7 amino acid linker, followed by the TEV protease cleavage site and a GAL4-VP16 fusion protein. The cell line also expresses a b-arrestin-2-TEV protease fusion protein and contains a reporter construct consisting of the UAS response element and the b-lactamase (bla) reporter gene. Upon activation of the receptor, g-protein receptor kinase (GRK) phosphorylates specific intracellular residues of OPKR1 and this induces recruitment of B-arrestin2-TEV protease fusion protein. The TEV protease recognizes and cleaves the TEV site, releasing the GAL4-VP16 fusion protein, which then translocates to the nucleus. The GAL4-V16 binds to the UAS element, driving expressing of the b-lactamase gene. B-lactamase expression is detected with the cell permeable, fluorescent substrate, CCF4-AM. This substrate consists of coumarin tethered to fluoroscein via a b-lactam ring. In the absence of b-lactamase, excitation of the dye with 405 nm light results in FRET from the coumarin to fluoroscein and emission of green (525 nm maximum) light. B-lactamase cleavage of the substrate separates the courmarin fluorophore from the fluorscein, and 405 nm excitation results in blue (460 nm maximum) emission. The assay is monitored by the blue/green emission ratio. The antagonist assay is performed by seeding the cells into 384 well plates and incubating them 16-24 hours at 37° C. Test antagonist compounds are added and incubated for 30 minutes at 37° C. Next an EC80 challenge of U-50488 (OKRK1 agonist) is added and the samples are incubated for 4 hours at 37° C., followed by addition of CCF4-AM substrate. The plates are then incubated 2 hours at room temperature in the dark and the blue/green ratio determined on a fluorescent plate reader. See J Biomol Screen April 2009, vol. 14 no. 4, pp 381-394.
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 402.54, ALogP 3.5, HBA 6, HBD 2, PSA 83.56, QED 0.68
Compound (SMILES): O=C(CN1CCN(CC(O)CN2c3ccccc3Sc3ccc(C(F)(F)F)cc32)CC1)NC[C@H](O)CO
InChIKey: QCEZBURVLBSXKA-OYKVQYDMSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Fluorescence Polarization Assay: The competition binding assay was performed in a 384-well plate format and in 20 μl reactions. Serial dilutions (0.001-100 μM) of a test compound were incubated with CaM in the presence of 16B05 at room temperature for 30 min. The final binding reagent contained 50 nM of CaM and 5 nM of 16B05 in the assay buffer (50 mM HEPES pH 7.5, 0.01% (w/v) Triton X-100 and 100 μM CaCl2). Each plate contained two types of control wells, containing either 5 nM fluorescent probe bound to 50 nM CaM (bound state, maximum read) or 5 nM unbound probe alone (free state, minimum read) in the assay buffer. Reference compounds Trifluoperazine (CP-Chengdu, Barcode 20008599), A-3 hydrochloride (Sigma, Cat. No. A1980), and A-7 (Tocris Bioscience, Cat. No. 0378) were also included. Upon completion of the incubation, fluorescence polarization degrees (excitation at 531 nm, emission at 595 nm) were measured on the EnVision Microplate Reader (PerkinElmer). Data analysis was performed according to Audran et al., Biochim Biophys Acta. 1833 (2013) 1720-1731. Fluorescence decay was plotted against log concentration of the compound and half maximal inhibitory concentration (IC50) was calculated by XLfit version 4.3.1 (ID Business Solutions) with a 4-parameter logistic model or sigmoidal dose-response model.
Measurement: IC50 = 13910 nM (pChEMBL 4.860000133514404)
Descriptors: MW 540.61, ALogP 1.76, HBA 8, HBD 4, PSA 99.51, QED 0.38
Compound (SMILES): O=C(O)C[C@H](NC(=O)CNC(=O)c1cc(O)cc(NC2=NCC(O)CN2)c1)c1cc(Cl)cc(C(F)F)c1
InChIKey: GDEQKRGKHZOXGD-SFHVURJKSA-N
Target: Integrin alpha-5/beta-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P05556
Assay: Solid Phase Receptor Assay (SPRA) for alpha5beta5: Purified human fibronectin (R&D Systems, 1918-FN) diluted to 2 μg/mL in TBS+ buffer (25 mM Tris pH 7.4, 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2) was added to wells (50 μL/well) of a 96-well half-well transparent microtiter plate (Greiner 675061) and incubated overnight at 4° C. Wells were washed 3 times with 150 μL TBS+ and 150 μL of blocking buffer (TBS+ with 1% bovine serum albumin, Sigma A7906) were added. The plate was incubated for 1 hr at 37° C. and then washed 3× with TBS+ buffer. Recombinant human integrin α5β1 (R&D Systems, 3230-A5) was diluted to 0.1 μg/mL in TBS+/0.1% bovine serum albumin. Compounds were diluted 1:100 into the integrin solution and then 50 μL added to empty wells of the washed fibronectin-coated plate according to a standard template with each sample repeated in triplicate. After incubation for two hours at room temperature, the plate was washed 3× with 150 μL of TBS+ buffer. To each well, 50 μl of biotinylated anti-α5 antibody (R&D Systems, BAF1864) at 0.5 μg/mL in TBS+/0.1% BSA were added and the plate covered and incubated for 1 hr at room temperature. After washing the plate 3× with 150 μL of TBS+ buffer, 50 μL of streptavidin-conjugated horseradish peroxidase (R&D Systems, DY998) diluted in TBS+ blocking buffer were added to the wells and the plate incubated for 20 min at room temperature. The plate was washed 3× with TBS+ buffer followed by 50 μL of room temperature TMB substrate (Sigma, T444) added to each well and the plate incubated for 20 min at room temperature. Plates were read by colorimetric detection at 650 nm wavelength using a Tecan Safire II plate reader. Concentration-response curves were constructed by non-linear regression (best fit) analysis, and IC50 values were calculated for each compound.
Measurement: IC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 539.92, ALogP 1.78, HBA 8, HBD 7, PSA 172.38, QED 0.25
Compound (SMILES): Cc1cc(-c2cc(Cl)ccc2OCCn2c(C)nc3cc(Cl)c(C4CC4)c(C#N)c3c2=O)c2scc(C(=O)O)c2n1
InChIKey: OTRWUNZJAMCBGX-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 19.5 nM (pChEMBL 7.710000038146973)
Descriptors: MW 605.5, ALogP 7.12, HBA 8, HBD 1, PSA 118.1, QED 0.21
Compound (SMILES): Cc1c(-c2cc3cc(NC(=O)OC4CN(C)C4)ncc3c(N)c2F)cnc2c1NCCO2
InChIKey: GSKQDBMULILTPD-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBD: TBD
Measurement: Ki = 0.11 nM (pChEMBL 9.960000038146973)
Descriptors: MW 438.46, ALogP 2.99, HBA 8, HBD 3, PSA 114.63, QED 0.53
Compound (SMILES): Cc1nc(S(=O)(=O)Nc2ccsn2)ccc1NCc1c(F)cccc1CN1CCC1
InChIKey: ZSEYDVSVDIEUOY-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 25020 nM (pChEMBL 4.599999904632568)
Descriptors: MW 447.56, ALogP 3.6, HBA 7, HBD 2, PSA 87.22, QED 0.55
Compound (SMILES): CC/C=C\CNc1nc(Nc2cnn(C(C)(C)C)c2)ncc1C(N)=O
InChIKey: PFYOMCKWSCABBE-SREVYHEPSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Kinase Activity Assay: JAK1 (the final concentration was 10 nM); ATP (the final concentration was 10 μM); ULight-labeled JAK-1 (Tyr1023) Peptide (the final concentration was 100 nM); the enzymatic reaction time was 2 hours. The maximum final concentration of compounds was 2.5 μM. After a 3-fold gradient dilution, 11 concentrations were obtained, with the minimum final concentration of 0.042 nM. The final DMSO concentration was 1%.
Measurement: IC50 = 8.7 nM (pChEMBL 8.0600004196167)
Descriptors: MW 343.44, ALogP 2.65, HBA 7, HBD 3, PSA 110.75, QED 0.67
Compound (SMILES): COc1cc(Nc2cc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)cc(NS(C)(=O)=O)c4OC)c4ccccc34)ccn2)ccc1C(=O)NCCCCN1CCOCC1
InChIKey: CSXDRRLHZJWOOX-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: GSK 3alpha Enzyme Inhibition: The inhibitory activities of compounds of the invention against the GSK 3α enzyme isoform (Invitrogen), are evaluated by determining the level of activation/phosphorylation of the target peptide. The GSK3-α protein (500 ng/mL, 2.5 μL) is mixed with the test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptide (8 μM, 2.5 μL), which is a phosphorylation target for GSK3α, and ATP (40 μM, 2.5 μL) are then added to the enzyme/compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).In all cases, the site-specific protease cleaves non-phosphorylated peptide only and eliminates the FRET signal. Phosphorylation levels of each reaction are calculated using the ratio of coumarin emission (donor) over fluorescein emission (acceptor), for which high ratios indicate high phosphorylation and low ratios indicate low phosphorylation levels. The percentage inhibition of each reaction is calculated relative to non-inhibited control and the 50% inhibitory concentration (IC50 value) is then calculated from the concentration-response curve.Method 2
Measurement: IC50 = 5575 nM (pChEMBL 5.25)
Descriptors: MW 840.02, ALogP 7.94, HBA 11, HBD 5, PSA 181.48, QED 0.06
Compound (SMILES): COc1ccccc1Nc1nnc(-c2nc(-c3ccc(C(=O)N(C)C)cc3)cnc2N)o1
InChIKey: SAHOPFPAIUTJGL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 M [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 45 μL of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 431.46, ALogP 3.23, HBA 9, HBD 2, PSA 132.29, QED 0.47
Compound (SMILES): Cc1c(N2CCC(c3ccccc3)CC2)ncn2c(CC3CC3)nnc12
InChIKey: YWXRKRAQITUPBO-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: MGluR2 cAMP Assay: Clonally expressed human mGluR2 in HEK293 background was glutamate starved overnight in media without glutamine (GIBCO MEM 12360), containing 10% FBS, 1% penicillin streptomycin. Compounds were added to white, standard volume 384 non-binding surface plates (Corning 3574). Cells were resuspended in stimulation buffer consisting of Hanks Balanced Salt Solution (14175-095) pH 7.0, 20 mM HEPES, 2.0 mM CaCl2, 5.0 mM MgCl2, and 1 mM IBMX (Sigma 15879), 1 Î¼M forskolin, and 1 Î¼M LY-341495, for 30 minutes. Buffer without forskolin was used as a negative control. Solutions of D2 and cryptate detection reagents from the CISBIO dynamic cAMP kit (62AM4PEJ) were diluted 1:40 in lysis buffer. Lysis buffer consisted of 50 mM Phosphate Buffer pH 7.0, 800 mM Potassium Fluoride, 0.2% BSA, and 1.0% Triton. Assay reaction was terminated by addition of detection reagents in lysis buffer. One hour later, plates were read on a PE Viewlux.
Measurement: EC50 = 62 nM (pChEMBL 7.210000038146973)
Descriptors: MW 347.47, ALogP 3.77, HBA 5, HBD 0, PSA 46.32, QED 0.72
Compound (SMILES): COc1cc(CO)c(-c2cn3ccc(N)nc3n2)cc1Cl
InChIKey: SKMKQDGYLCVDLG-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase SUV39H2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H5I1
Assay: Suv 39H2 Assay: Assay buffer (4 μL) was added into all the wells, including test compound and control wells (except for Sinefungin control wells), using the Multidrop Combi. The plate was centrifuged at 1000 rpm for 1 min. Substrate mix (8 μL) containing radiolabelled 3H-SAM (final conc: 100 nM) and H3 Histone Peptide (final conc: 350 nM) was added to all wells using Multidrop Combi and centrifuged at 1000 rpm for 1 min. SUV39H2 (8 μL, final conc: optimized for each lot of the enzyme based on specific activity) was added by using the Multidrop Combi. The assay plate was centrifuged at 1000 rpm for 1 min. Assay buffer was used as background control, and incubated at room temperature for 3 hours. The reaction was stopped with 20 μL of 2.5 mg/mL Streptavidin SPA beads (final conc. 50 μg/well) in buffer using the Multidrop Combi and centrifuged for 1 min. The plate was loaded into the Trilux-Microbeta counter and a delayed read of 10 hours was made. The radioactive signal was measured at 1 min/well. Sinefungin and the internal standard compound were used as tool compounds to determine assay performance.
Measurement: IC50 = 18.2 nM (pChEMBL 7.739999771118164)
Descriptors: MW 304.74, ALogP 2.13, HBA 6, HBD 2, PSA 85.67, QED 0.77
Compound (SMILES): CC(=O)OCC1(C)C(OC(C)=O)CCC2(C)C1CC(OC(C)=O)C1(C)Oc3cc(-c4cccnc4)oc(=O)c3C(O)C21
InChIKey: PMMQOFWSZRQWEV-UHFFFAOYSA-N
Target: Sterol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75908
Assay: Inhibitory Effect on ACAT2 Activity: 1. Purpose:The analogues of Pyripyropene A structure were tested for their inhibitory effect on ACAT2 activity at intact cellular level by a method in which a fluorescent-labeled sterol is used for determining ACAT2 activity.2. Principle:An inhibition curve was plotted based on that different concentrations of compounds would cause the fluorescence intensity to change by inhibiting the synthesis of the ester containing NBD22-fluorescent labeled sterol, thereby IC50 was calculated.3. Process:HepG2 cells were cultured in a 96-well plate at a starting density of 1.5×104 cells per well for 24 hours. After the cholesterol mixture was well mixed, the cells were cultured for another 24 hours. Then NBD22-fluorescent labeled sterol at a final concentration of 0.5 μg/ml, and compounds at a final concentration gradient of 0, 0.008, 0.04, 0.2, 1 and 5 μM were added. Three wells were set for each concentration. After incubating for 6 hours, the fluorescence intensity was measured using a fluorescence analyzer (E488, A535). The fluorescence intensity values were plotted against different concentrations of the compounds and IC50 was obtained.
Measurement: IC50 = 179 nM (pChEMBL 6.75)
Descriptors: MW 583.63, ALogP 3.76, HBA 11, HBD 1, PSA 151.46, QED 0.4
Compound (SMILES): O=C(O)c1ccncc1Nc1nn(CC2CC2)c2ccc(Cl)cc12
InChIKey: CEQIRGOSROIKND-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: Enzymatic Assay: The enzymatic assay of Jarid1A activity is based upon Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The ability of test compounds to inhibit the activity of Jarid1A was determined in 384-well plate format under the following reaction conditions: 1 nM Jarid1A, 300 nM H3K4me3-biotin labeled peptide (Anaspec cat #64357), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-mono- or di-methylated histone H3 lysine 4 (H3K4me1-2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 25 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K4me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO was added to each well of plate, followed by the addition of 2 μl of 3 nM Jarid1A to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 50 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K4me1-2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 342.79, ALogP 3.94, HBA 5, HBD 2, PSA 80.04, QED 0.73
Compound (SMILES): O=C(Nc1ccc(-c2ccccc2F)nc1)c1ccc(OC(F)(F)F)c(NC(=O)C2(N3CCN(C4CC4)CC3)CC2)c1
InChIKey: LZIDJDMKKTZXMT-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: HCT116 TOPFlash Assay: In preparation of the assay, the two cell lines were plated 24 hours before at 10000 cells per well of a 384 micro titre plate (MTP) in 30 μL growth medium. Selective inhibitory activity for small molecules on the mutated Wnt pathway was determined after parallel incubation of both (TOP and FOP) HCT116 reporter cell lines with a compound dilution series from 50 μM to 15 nM in steps of 3.16-fold dilutions in CAFTY buffer (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 5 mM NaHCO3, pH 7.4) containing 2 mM Ca2+ and 0.01% BSA. The compounds were thereby serially prediluted in 100% DMSO and thereafter in addition 50 fold into the CAFTY compound dilution buffer (described above). From this dilution 10 μL were added to the cells in 30 μL growth medium and incubated for 36 hours at 37° C. and 5% CO2. Thereafter luciferase assay buffer (1:1 mixture of luciferase substrate buffer (20 mM Tricine, 2.67 mM MgSO4, 0.1 mM EDTA, 4 mM DTT, 270 μM Coenzyme A, 470 μM Luciferin, 530 μM ATP, ph adjusted to pH 7.8 with a sufficient volume of 5M NaOH) and Triton buffer (30 mL Triton X-100, 115 mL glycerol, 308 mg Dithiothreitol, 4.45 g Na2HPO4.2H2O, 3.03 g TRIS HCl, ad 1I H2O, pH 7.8) was added as equal volume to the compound solution on the cells to determine luciferase expression as a measure of Wnt signaling activity in a luminometer.n order to determine the inhibitory activity of compounds for the WT Wnt signaling pathway, the Super TopFlash vector were cotransfected with pcDNA3 into HEK293 cells.
Measurement: IC50 = 29.6 nM (pChEMBL 7.53000020980835)
Descriptors: MW 583.59, ALogP 5.29, HBA 6, HBD 2, PSA 86.8, QED 0.35
Compound (SMILES): Cc1c2ccccc2n2c(C(=O)NC[C@@H]3CCCN3C(C)C)cn(-c3cnn(C)c3)c(=O)c12
InChIKey: TVILFXSKIKHUPN-SFHVURJKSA-N
Target: Glucagon-like peptide 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43220
Assay: GLP-1 Assay with GLP1 Endogenous Agonist: To assess the effects of test compounds on GLP-1 or glucagon potency and efficacy, a high-throughput calcium mobilization assay was used essentially as previously described (Morris L C, Days E L, Turney M, Mi D, Lindsley C W, Weaver C D, Niswender K D, A Duplexed High-Throughput Screen to Identify Allosteric Modulators of the Glucagon-Like Peptide 1 and Glucagon Receptors. J. Biomol. Screen. 2014 Feb. 13, 19(6):847-858). Briefly, human GLP-1R or Glucagon Receptor 9-3-H cells over-expressing a promiscuous G-protein (Millipore, Billerica, Mass.) were plated at 15,000 cells/well in black-walled 384-well plates (Greiner Bio-one, Monroe, N.C.) in Dulbecco's Modified Eagles Medium (DMEM) with 10% FCS, 4.0 mM L-glutamine, non-essential amino acids (NEAAs), and 10.0 mM HEPES without antibiotics. After overnight attachment, cells were washed twice with assay buffer (HBSS supplemented with 20 mM HEPES and 1.0 mM probenecid) using an ELx405CW cell washer (Bio-Tek, Winooski, Vt.) and then loaded with the calcium sensitive dye fluo-4 AM (Invitrogen, Grand Island, N.Y.) at a final concentration of 2.0 μM in assay buffer. After a 45-minute incubation at room temperature, the dye was removed by washing, leaving 20 μL of assay buffer. Next, the cell plate was introduced alongside a 384-well compound plate containing 0.1% final DMSO control wells and 11-point concentration response curves of putative GLP-1 PAMs created using a non-pipet based liquid transfer instrument, ECH0555 (Labcyte, Sunnyvale, Calif.). After a 20 μL compound addition to the appropriate wells, kinetic fluorescent measurements were collected for 2 minutes using an FDSS6000 (Hamamatsu, Bridgewater, N.J.) with 488 nm excitation and 480/540 emission filters. After 2 minutes, 10 μL of an EC20 concentration of GLP-1 peptide 7-36 amide (Phoenix Pharmaceuticals, Burlingame, Calif.), Glucagon (Phoenix Pharmaceuticals, Burlingame, Calif.) Exendin-4 (Tocris Bioscience, Bristol, UK), or Liraglutide (Victoza, Novo-Nordisk, Denmark) was added and fluorescence was monitored for an additional 2 minutes to observe potentiation of the calcium flux signal. Control wells for vehicle, EC20 peptide, and Emax peptide resided in columns #1 and 24 and rows H and I to account for any plate variations. Recombinant peptides were reconstituted into assay buffer supplemented with 0.1% fatty acid free BSA (Sigma #A6003) and diluted into borosilicate glass tubes to maximize peptide recovery (Goebel-Stengel M, Stengel A, Tache Y, Reeve J R Jr., The importance of using the optimal plasticware and glassware in studies involving peptides. Anal. Biochem. 2011 Jul. 1, 414(1):38-46).
Measurement: EC50 = 6700 nM (pChEMBL 5.170000076293945)
Descriptors: MW 446.56, ALogP 2.89, HBA 7, HBD 1, PSA 76.57, QED 0.51
Compound (SMILES): CC(=O)N[C@@H](C)CO[C@H]1C[C@H](COc2ccc3nc(N(C)CC#N)sc3n2)C1
InChIKey: GBXQSVGEDXZZGR-QEJZJMRPSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 892 nM (pChEMBL 6.050000190734863)
Descriptors: MW 403.51, ALogP 2.35, HBA 8, HBD 1, PSA 100.37, QED 0.64