lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#CC1CN(C(=O)c2ccc3cc(Oc4ccc(C(F)(F)F)cn4)ccc3n2)C1
InChIKey: ZLJXSOXSCVXYDD-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 2530 nM (pChEMBL 5.599999904632568)
Descriptors: MW 398.34, ALogP 4.04, HBA 5, HBD 0, PSA 79.11, QED 0.67
Compound (SMILES): COc1nccc2c1OCC2NC(=O)Cc1ccc(Cl)cc1Cl
InChIKey: NOZJBYNSHDBTFJ-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: In Vitro Assay: Cell line generation was performed in general according to established molecular cloning protocols. Specifically, RNA was extracted from human whole blood using the Qiagen RNeasy kit (Qiagen, CH) according to the manufacturer's instructions. Subsequently cDNA was made (Superscript II, Invitrogen AG, CH) and the human P2X7 gene (genbank ref. BC011913) was amplified with the following primers: ATCGCGGCCGCTCAGTAAGGACTCTTGAAGCCACT and CGCCGCTAGCACCACCATGCCGGCCTGCTGCAGCTGCA. The amplified sequence was subsequently ligated into a pcDNA3.1 (+) NotI, NheI digested plasmid. Human embryonic kidney (HEK) cells (ATCC CRL-1573, Manassas, Va., USA) were transfected with the pcDNA3.1 (+). hP2X7 plasmid using lipofectamine 2000 (Invitrogen AG, CH) according to the manufacturer's instructions. Following a 24 h exposure to DNA, cells were trypsinized and re-seeded at low density in the presence of 250 ug Geneticin. Geneticin resistant cells were then selected during two consecutive rounds of cloning by serial limiting dilution with visual inspection.
Measurement: IC50 = 248 nM (pChEMBL 6.610000133514404)
Descriptors: MW 353.21, ALogP 3.19, HBA 4, HBD 1, PSA 60.45, QED 0.92
Compound (SMILES): O=C(NCCC(=O)N1CCCC1)c1ccc2cc(Oc3ccc(C(F)(F)F)nc3)ccc2n1
InChIKey: QQIHSIXWCPZEOH-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 458.44, ALogP 4.18, HBA 5, HBD 1, PSA 84.42, QED 0.6
Compound (SMILES): CCN(c1c(C)nn(C)c1C)S(=O)(=O)c1c(Cl)cc(-c2ccnc(N3CCNCC3)c2)cc1Cl
InChIKey: CJBFCFNKTFJGHD-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 523.49, ALogP 4.03, HBA 7, HBD 1, PSA 83.36, QED 0.53
Compound (SMILES): COc1cc2c(N)nc(N3CCN(C(=O)C[C@@H]4CCN4)C[C@@H]3c3ccccc3)nc2c(F)c1OC
InChIKey: QZYAVMCERQILPE-FUHWJXTLSA-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 = 610 nM (pChEMBL 6.210000038146973)
Descriptors: MW 480.54, ALogP 2.51, HBA 8, HBD 2, PSA 105.84, QED 0.55
Compound (SMILES): CC[C@@H](CCO)N1C(=O)[C@@](C)(CC(=O)O)C[C@H](c2cccc(Cl)c2)[C@H]1c1ccc(Cl)cc1
InChIKey: BMAXZSKHVQAEBO-KWLZBDKBSA-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 = 10 nM (pChEMBL 8)
Descriptors: MW 478.42, ALogP 5.69, HBA 3, HBD 2, PSA 77.84, QED 0.51
Compound (SMILES): Nc1nc(C(=O)N2Cc3ccccc3C2)c2ccccc2n1
InChIKey: YQRDZESTWPAISV-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Recombinant Hsp90 [His-Tev-huHsp90-alpha (9-236)] was immobilized on a Biacore CM5 chip at 25 degrees C and a flow rate of 30 uL/min using amine coupling at pH 4.50 according to Biacore standard protocol. Hsp90 was applied at a concentration of 20 ug/mL with 50uM 17-DMAG.Data sets were processed and analyzed using the software Biacore 4000 Evaluation. Solvent corrected and double-referenced association and dissociation phase data were fitted to a simple 1:1 interaction model with mass transport limitations
Measurement: Kd = 2152.5 nM (pChEMBL 5.670000076293945)
Descriptors: MW 290.33, ALogP 2.37, HBA 4, HBD 1, PSA 72.11, QED 0.75
Compound (SMILES): O=C(/C=C/c1cccnc1)Nc1ccc(S(=O)(=O)N2CCC(N3CCOCC3)CC2)cc1
InChIKey: CLBDATVAZOOTHJ-FPYGCLRLSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 uM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 456.57, ALogP 2.22, HBA 6, HBD 1, PSA 91.84, QED 0.67
Compound (SMILES): c1nc(-c2n[nH]c3ccc(-c4ccsc4)cc23)cc(N2CCOCC2)n1
InChIKey: SLYUZOLCGREEFV-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 36.4 nM (pChEMBL 7.440000057220459)
Descriptors: MW 363.45, ALogP 3.59, HBA 6, HBD 1, PSA 66.93, QED 0.6
Compound (SMILES): COc1cc(OC)cc(N(CC#Cc2cc(CO)ccn2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: JVHRYVZNKJPNLU-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 3.388 nM (pChEMBL 8.470000267028809)
Descriptors: MW 506.57, ALogP 4.12, HBA 9, HBD 1, PSA 98.42, QED 0.33
Compound (SMILES): Cc1cccc2cc(Cn3nc(C4=CCOCC4)c4c(N)ncnc43)n(-c3ccccc3)c(=O)c12
InChIKey: KJDQWYKQMWVIEC-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 464.53, ALogP 3.87, HBA 8, HBD 1, PSA 100.85, QED 0.43
Compound (SMILES): Clc1ccccc1Nc1ncc(Br)c(Nc2n[nH]c3ccccc23)n1
InChIKey: BUFAGNSLHUASFH-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 >= 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 415.68, ALogP 5.26, HBA 5, HBD 3, PSA 78.52, QED 0.42
Compound (SMILES): CN(c1cc2oc(-c3ccc(F)cc3)c(C(=O)NC3CC3)c2cc1-c1ccc2c(n1)-c1cc3c(F)cccc3n1CO2)S(C)(=O)=O
InChIKey: PNMFTJXXPZJVMV-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: ELISA-Based Assay: Measurement of inhibition by compounds was performed using the HCV replicon system. Several different replicons encoding different HCV genotypes or mutations were used. In addition, potency measurements were made using different formats of the replicon assay, including different ways of measurements and different plating formats. See Jan M. Vrolijk et al., A replicons-based bioassay for the measurement of interferons in patients with chronic hepatitis C, 110 J. VIROLOGICAL METHODS 201 (2003); Steven S. Carroll et al., Inhibition of Hepatitis C Virus RNA Replication by 2'-Modified Nucleoside Analogs, 278(14) J. BIOLOGICAL CHEMISTRY 11979 (2003). However, the underlying principles are common to all of these determinations, and are outlined below.Stable neomycin phosphotransferase encoding replicons-harboring cell lines were used, so all cell lines were maintained under G418 selection prior to the assay. Potency was determined using a cell ELISA assay with an antibody to the replicons encoded NS3/4a protease. See Caterina Trozzi et al., In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor, 77(6) J. Virol. 3669 (2003).
Measurement: IC50 = 7.6 nM (pChEMBL 8.119999885559082)
Descriptors: MW 640.67, ALogP 6.7, HBA 7, HBD 1, PSA 106.67, QED 0.22
Compound (SMILES): c1ccc(-c2cc(-c3ccccc3)c3c(-c4ccccc4)n[nH]c3n2)cc1
InChIKey: JBDFLODGAIHOBF-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Positive allosteric modulation of mGlu5 receptor (unknown origin) assessed as increase in L-glutamate-induced calcium release after 60 mins by cell based FLIPR assay
Measurement: EC50 = 330 nM (pChEMBL 6.480000019073486)
Descriptors: MW 347.42, ALogP 5.96, HBA 2, HBD 1, PSA 41.57, QED 0.44
Compound (SMILES): CC(C)CN(c1ccccc1Cl)S(=O)(=O)c1ccc(-c2ccc(S(N)(=O)=O)cc2)cc1
InChIKey: KAJKOJXUVAJMIF-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In vitro RORc Ligand Binding Assay: On day of the assay, 100 μL of 0.05% CHAPS (in deionized H2O) was added to all wells of the GFB Unifilter plate and allowed soak for 1 h. A wash buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, and 5 mM MgCl2 was prepared to wash the filter plate. To prepare an assay buffer, BSA was added to the wash buffer to reach 0.01% and DTT was added to reach 1 mM. For IC50 mode, 10 mM compound stocks were serially diluted in DMSO with DMSO to give 20× required final concentration in DMSO (15 μL compound + 30 μL DMSO). The 20× compound stocks were diluted in DMSO with Assay Buffer 4-fold to reach 5× the final test concentration in 25% DMSO (10 μL compound + 30 μL Assay Buffer). Solutions were mixed by aspiration several times with a pipette set on 50 μL volume. For the assay, 10 μL of 5× compound stock solutions in 25% DMSO were added to the assay plate in duplicate. Assay plates were 96-well polypropylene V-bottom plates. 10 μL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 μL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 μL of 5 μM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 μL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 μL of 1.5 ug/mL RORc receptor was added to wells (or 40 μL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25 °C. Final concentrations were as follows: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 μM 25-hydroxycholesterol was also present.
Measurement: IC50 = 116 nM (pChEMBL 6.940000057220459)
Descriptors: MW 479.02, ALogP 4.51, HBA 4, HBD 1, PSA 97.54, QED 0.54
Compound (SMILES): O=C(NCCCCCCNC(=O)N1Cc2ccccc2C1)c1cccc(Cl)c1
InChIKey: HDNLMVPAPLUTGY-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 6.98 nM (pChEMBL 8.15999984741211)
Descriptors: MW 399.92, ALogP 4.36, HBA 2, HBD 2, PSA 61.44, QED 0.65
Compound (SMILES): Cc1nc(-c2ccccc2)sc1C(=O)Nc1cn2nc(-c3cn(C)nc3-c3ccc(F)cc3)ccc2n1
InChIKey: SUCWPIOKZYMTRT-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 34.1 nM (pChEMBL 7.46999979019165)
Descriptors: MW 509.57, ALogP 5.62, HBA 8, HBD 1, PSA 90, QED 0.33
Compound (SMILES): O=C(CCCCCCCNCc1ccc(-c2ccc3ncnc(Nc4ccc(OCc5cccc(F)c5)c(Cl)c4)c3c2)o1)NO
InChIKey: PNEDVLBBOKUUCE-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of N-terminal GST-fused human EGFR (His672 to Ala1210 residues) expressed in baculovirus expression system using biotinylated PTP1B at Tyr66 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 632.14, ALogP 7.94, HBA 8, HBD 4, PSA 121.54, QED 0.05
Compound (SMILES): CO[C@H]1CN(c2ncc(C(=O)Nc3ccc(OC(F)(F)Cl)cc3)cc2-c2cncc(F)c2)C[C@@H]1N
InChIKey: PVNLJYOWGMEBRH-PMACEKPBSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Radio ABL1 (64-515) Assay: For determination of ABL kinase activity, the radiometric filter-binding assay was used. The assay was performed by mixing 10 uL of the compound pre-diluted with 10 uL of ATP (20 uM ATP with 0.1 uCi [γ-33P]-ATP) with the phospho-acceptor peptide poly[Ala6Glu2LysHBr5Tyr1]=polyAEKY) in 20 mM Tris/HCl pH 7.5, 1 mM DTT, 10 mM MgCl2, 0.01 mM Na3VO4, 50 mM NaCl. 10 uL of enzyme (ranging between 5 nM to 20 nM) was added to initiate the reaction. Pre-incubation of enzyme with compounds (when stated) was performed by exposing the enzyme to compounds prior to addition of the substrate mixture (ATP and/or peptide substrate). After 15 mM at room temperature, the reaction was stopped by the addition of 50 uL 125 mM EDTA, and the peptide-bound 33P separated on filter-plates (PVDF or MAIP; Millipore, Volketswil, Switzerland) prepared according to the manufacturer's instructions. Filter-plates were washed 3x with 0.5% H3PO4, followed by addition of 30 uL scintillation cocktail (Microscint, Perkin Elmer) per well and then analysed in a TopCount NXT scintillation counter (Perkin Elmer).
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 507.9, ALogP 3.87, HBA 7, HBD 2, PSA 102.6, QED 0.47
Compound (SMILES): CSCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(N)=O)NC(=O)CN1CCN(CCNC(=O)CN2CCN(CC(=O)O)CCN(CC(=O)O)CCN(CC(=O)O)CC2)CC1)C(C)C)C(N)=O
InChIKey: VSBYFTODSOFZKG-DNCOJZTMSA-N
Target: Gastrin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30550
Assay: Displacement of [125I]-BBN from GRPR in human PC3 cells after 1 hr by compuGamma counting or microplate scintillation counting method
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 1495.78
Compound (SMILES): CN(c1ccc(Oc2ccccc2)cc1)S(=O)(=O)c1ccccc1Cl
InChIKey: VNNYUTBLBOCLFG-UHFFFAOYSA-N
Target: Progesterone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06401
Assay: Antagonist activity at progesterone receptor in human T47D cells incubated for 24 hrs in presence of P4 by alkaline phosphatase assay
Measurement: IC50 = 390 nM (pChEMBL 6.409999847412109)
Descriptors: MW 373.86, ALogP 4.96, HBA 3, HBD 0, PSA 46.61, QED 0.63
Compound (SMILES): Cc1cccc2c(=O)[nH]c(-c3ccc(N4CCN(C)CC4)nc3)cc12
InChIKey: MENBDHBIZBAFHG-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Chemiluminescent PARP Assay: Tankyrase activity was assayed using a 96-well format HT Universal Chemiluminescent PARP Assay Kit (Trevigen, Inc, cat. no. 4676-096-K) according to the manufacturer's instructions. In short, tankyrase/PARP activity is quantified by the incorporation of biotinylated nicotinamide adenine dinucleotide (biotin-NAD+) onto the immobilised pseudo substrate, Histone. The extent of poly(Biotin-ADP)ribosylation (PARylation) in the presence of increasing dose of inhibitor is then quantified by binding of streptavidin conjugated horse radish peroxidase (strep-HRP) followed by chemiluminescent detection. Prior to assay initiation, inhibitor stocks were prepared in aqueous DMSO (10% (v/v)) from 5 millimolar (mM) stock in 100% DMSO (Sigma Aldrich, cat. no. 265855) as 10x concentrations. For the primary assay (i.e., single dose at 1 micromolar (uM) final concentration) this corresponded to 10 uM in 10% DMSO.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 334.42, ALogP 2.65, HBA 4, HBD 1, PSA 52.23, QED 0.78
Compound (SMILES): CO[C@H]1[C@H](C)C[C@@]2(Cc3ccc(C#N)cc3[C@]23N=C(N)N(C[C@H]2COCCO2)C3=O)C[C@@H]1C
InChIKey: CYSJYHKFWGTAAK-OIIFEQBSSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Fluorescence Quench Assay: The inhibitory activity of compounds was assessed by a fluorescence quench assay of BACE1 activity using commercially available substrate HiLyte Fluor 488-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-(QXLT 520)-OH (SEQ ID NO:1) AnaSpec, San Jose, Calif.) and truncated human beta-secretase, BACE1 (amino acids 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen). The substrate was dissolved at 1 mg/ml in DMSO.The assay was performed in the presence of OptiMEM (supernatant collected over 24 h and cleared from cellular debris by centrifugation) containing the ectodomain of BACE1, 25 ul water containing the desired 2-fold concentration of test compound and 2% DMSO, 1 uM substrate peptide, 20 mM NaOAc, pH 4.4, and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE1 containing OptiMEM diluted 1:10 in water.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 452.56, ALogP 1.95, HBA 7, HBD 1, PSA 110.17, QED 0.75
Compound (SMILES): CCCCCCCC/N=C1/SC[C@@H]2[C@H](O)[C@H](O)CN12
InChIKey: UZNSOHBBKVVYLF-QHRPLHADSA-N
Target: Lysosomal acid glucosylceramidase (SINGLE PROTEIN), Homo sapiens
UniProt: P04062
Assay: Inhibition of human beta-glucocerebrosidase using 4-methylumbelliferyl beta-D glycopyranoside as substrate at pH 7 by fluorescence assay
Measurement: IC50 = 7300 nM (pChEMBL 5.139999866485596)
Descriptors: MW 286.44, ALogP 1.86, HBA 4, HBD 2, PSA 56.06, QED 0.7
Compound (SMILES): N#C[C@@H]1CCN(CCC2CCOCC2)C[C@H]1n1cc(C(N)=O)c(Nc2ccc(F)cc2)n1
InChIKey: FMEGDGWLVGDMLI-LAUBAEHRSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 440.52, ALogP 3.07, HBA 7, HBD 2, PSA 109.2, QED 0.68
Compound (SMILES): Cc1ccc2c(n1)[C@H]([C@@H](C)C(=O)Nc1ccc(Cl)c(-c3nc4ccccc4o3)c1)CC[C@H]2C
InChIKey: RGHIUACNCDFLPP-MDYRTPRTSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of Smo-mediated Hh signalling pathway in mouse Shh Light2 cells by Gli-luciferase reporter gene assay
Measurement: IC50 = 243 nM (pChEMBL 6.610000133514404)
Descriptors: MW 459.98, ALogP 7.11, HBA 4, HBD 1, PSA 68.02, QED 0.35
Compound (SMILES): CCS(=O)(=O)N1CCN(C(=O)c2cc(Cc3n[nH]c(=O)c4c3CCCC4)ccc2F)CC1
InChIKey: FGOAWWMQQYDPCK-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 462.55, ALogP 1.49, HBA 5, HBD 1, PSA 103.44, QED 0.73
Compound (SMILES): CC(C)Oc1ccc(-c2nc(-c3cccc4c3CC[C@H]4NCC(=O)N3CCNCC3)no2)cc1C#N
InChIKey: OILQAQNPQIPOSI-HSZRJFAPSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 > 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 486.58, ALogP 3.07, HBA 8, HBD 2, PSA 116.31, QED 0.52
Compound (SMILES): CCn1nc(-c2ccccc2Cl)nc2c(=O)n(C)c(=O)nc1-2
InChIKey: RXVOZHUSTRGKDU-UHFFFAOYSA-N
Target: Dihydrolipoyl dehydrogenase (SINGLE PROTEIN), Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
UniProt: P9WHH9
Assay: Inhibition Assay: The assay was performed in a manner similar to that described in Bryk et al., Biochemistry (2010) 49:1616-1627 and modified for an online robotics screening system.A solution containing Lpd (100 nM) and 100 mM sodium phosphate buffer (pH 7.0) was prepared. Another solution containing the substrate lipoamide (2 mM), 100 mM sodium phosphate buffer (pH 7.0), EDTA (4 mM), and NADH (200 uM) was also prepared. The detection reagent, DTNB was dissolved in DMSO and diluted to a concentration of 375 uM with a 100 mM sodium phosphate buffer (pH 7.0).4 uL/well of Lpd solution was dispensed into 1536-well clear bottom plates. Next, 50 nL of each test compounds (1 mM dissolved in DMSO) was added into each well. After one hour of incubation, 4 uL/well of lipoamide (LPA) solution was added, so that in each well, the concentrations of Lpd, LPA, and the test compound were 50 nM, 1 mM, and 6.25 uM, respectively.
Measurement: IC50 < 40 nM (pChEMBL 7.3979997634887695)
Descriptors: MW 317.74, ALogP 1.18, HBA 7, HBD 0, PSA 82.67, QED 0.71
Compound (SMILES): Cc1ccnc(C2CN([C@H](C)c3nc4c(cnn4C4CCCC4)c(=O)[nH]3)C2)n1
InChIKey: VFQBJSVQLWNMPH-CYBMUJFWSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition of PDE9A (unknown origin) using [3H]-cGMP as substrate after 30 mins by microbeta counting method
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 379.47, ALogP 2.49, HBA 7, HBD 1, PSA 92.59, QED 0.75
Compound (SMILES): CN(C)C1(c2ccccc2)CCC2(CC1)SCCc1c2[nH]c2ccccc12
InChIKey: JCROKVWSGUVMAM-UHFFFAOYSA-N
Target: Opioid growth factor receptor-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5TC84
Assay: Binding Assay: The cyclohexane derivatives of the general formula I were investigated in a receptor binding assay with 3H-nociceptin/orphanin FQ with membranes from recombinant CHO-ORL1 cells. This test system was conducted in accordance with the method described by Ardati et al. (Mol. Pharmacol., 51, 1997, p. 816-824). The concentration of 3H-nociceptin/orphanin FQ in these experiments was 0.5 nM. The binding assays were carried out with in each case 20 μg of membrane protein per 200 μl batch in 50 mM hepes, pH 7.4, 10 mM MgCl2 and 1 mM EDTA. The binding to the ORL1 receptor was determined using in each case 1 mg WGA-SPA beads (Amersham-Pharmacia, Freiburg) by incubation of the batch for one hour at room temperature and subsequent measurement in a Trilux scintillation counter (Wallac, Finland).
Measurement: Ki = 3.7 nM (pChEMBL 8.430000305175781)
Descriptors: MW 376.57, ALogP 5.68, HBA 2, HBD 1, PSA 19.03, QED 0.62
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(OCCCS(C)(=O)=O)c([N+](=O)[O-])c4)c(Oc4cccc5[nH]ccc45)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: LFVCWNFYAYKXPY-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 0.149 nM (pChEMBL 9.829999923706055)
Descriptors: MW 890.48, ALogP 8.24, HBA 11, HBD 2, PSA 181.25, QED 0.06
Compound (SMILES): COc1cc(N2CCN(C(C)=O)CC2)ccc1Nc1ncc(F)c(Nc2ccc(N3CCNCC3)cc2OC)n1
InChIKey: RQJRTVVSDOXVNN-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 550.64, ALogP 3.2, HBA 10, HBD 3, PSA 107.12, QED 0.39
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NCC5CCOCC5)c(S(=O)(=O)C(F)(F)F)c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: VFHRTBRFCWDRKZ-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki = 0.09 nM (pChEMBL 10.050000190734863)
Descriptors: MW 955.52, ALogP 9.05, HBA 11, HBD 3, PSA 163.03, QED 0.1
Compound (SMILES): CC(C)c1cccc(NC(=O)c2ccc3cc4n(c3c2)CCCNC4=O)c1
InChIKey: DUCUIRXEVXZKPK-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15349
Assay: Kinase Glo Plus Assay: Human RSK2 protein, purchased from Invitrogen, is used to measure kinase activity utilizing Kinase Glo Plus (Promega) a homogeneous assay technology, which uses a luciferin-luciferase based ATP detection reagent to quantify residual ATP. The assay is performed using 0.75 nM His-RSK2, 0.75 .mu.M ATP and 1.0 uM S6 Kinase/RSK Substrate Peptide 1 (Upstate, catalog #12-124), in assay buffer consisting of 25 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 uM Na3VO4, 0.5 mM DTT, and 1% DMSO. Solutions of test compounds at various concentrations are prepared by 1:3 fold serial dilution of a 1 mM solution of compound in DMSO. The DMSO solutions are further diluted with assay buffer to a final concentration of DMSO of 5%. The assay is performed in a 384 well, white, non-binding plate (Corning, catalogue #3574). Solutions of test compounds (10 uL) are transferred to a dry assay plate, followed by addition of 20 uL kinase.
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 361.45, ALogP 4.15, HBA 3, HBD 2, PSA 63.13, QED 0.74
Compound (SMILES): O=C(NCC(F)(F)F)c1cccc(Nc2ncnc(N3CC(Oc4ccc(F)cc4)C3)n2)c1
InChIKey: YGGLYIYKFRPJGG-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 4620 nM (pChEMBL 5.329999923706055)
Descriptors: MW 462.41, ALogP 3.31, HBA 7, HBD 2, PSA 92.27, QED 0.52
Compound (SMILES): C[C@@]1(c2cc(NC(=O)c3ncc(F)cc3F)ccc2F)CS(=O)(=O)[C@]2(CCOC2)C(=N)N1
InChIKey: PGYKXVCGXDDQDR-PMACEKPBSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3x the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 ul) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol.
Measurement: Ki = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 468.46, ALogP 2.12, HBA 6, HBD 3, PSA 121.24, QED 0.63
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(Br)cc1[C@@]21N=C(N)N(CC(C)(C)O)C1=O
InChIKey: JSGJFFJENPMRRB-JHVJFLLYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Assay: For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham).
Measurement: IC50 = 4226 nM (pChEMBL 5.369999885559082)
Descriptors: MW 450.38, ALogP 2.7, HBA 5, HBD 2, PSA 88.15, QED 0.74
Compound (SMILES): C#CC(O)(c1ccc2nc(OC)c(Cc3ccc(C(F)(F)F)cc3)c(Cl)c2c1)C1CN(C(=O)OC(C)(C)C)C1
InChIKey: KGQNXXWLTAQQPJ-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells.Â  Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20.
Measurement: Kd = 3100 nM (pChEMBL 5.510000228881836)
Descriptors: MW 561, ALogP 6.19, HBA 5, HBD 1, PSA 71.89, QED 0.38