lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(c3)-c3cc5ccccc5n3CC(F)CO4)cc12
InChIKey: MMBHJDIVXHFWGP-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 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 641.7, ALogP 7.01, HBA 6, HBD 1, PSA 93.78, QED 0.22
Compound (SMILES): CC(C)n1nc(-c2ccc3onc(N)c3c2)c2c(N)ncnc21
InChIKey: WDKNUDNHMRIRCL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Inhibition of recombinant human GST-tagged mTOR (1360 to 2549 residues) catalytic domain assessed as inhibition of GFP labeled 4EBP1 phosphorylation measured after 1 hr by LanthaScreen assay
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 309.33, ALogP 2.38, HBA 8, HBD 2, PSA 121.67, QED 0.58
Compound (SMILES): C[C@@H](NC(=O)c1nn(-c2ccc(Cl)cc2)c2c1CN(C(=O)c1nc[nH]n1)C/C2=C\c1ccc(Cl)cc1)c1ccccn1
InChIKey: YXYWTRJMRSCHJZ-SQVRNEIYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Inverse agonist activity at human CB1 receptor transfected in HEK293 cells assessed as increase of forskolin-induced cAMP production incubated for 30 mins followed by d2 labeled cAMP addition measured after 60 mins by HTRF assay
Measurement: EC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 599.48, ALogP 5.38, HBA 7, HBD 2, PSA 121.69, QED 0.27
Compound (SMILES): CC1(C)OC[C@H](CNC(=O)c2ccc(NC(=O)N3Cc4ccccc4C3)cc2)O1
InChIKey: WTXMXEQQNFCGJR-IBGZPJMESA-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 = 22.4 nM (pChEMBL 7.650000095367432)
Descriptors: MW 395.46, ALogP 3.12, HBA 4, HBD 2, PSA 79.9, QED 0.83
Compound (SMILES): NC1=NC2(CCO1)c1cc(NC(=O)c3ccc(F)cn3)ccc1Oc1c2cc(C2=CCCOC2)nc1F
InChIKey: IOQMOYXNVRUFHS-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Fluorescence Resonance Energy Transfer (FRET) Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The beta secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 9.3 nM (pChEMBL 8.029999732971191)
Descriptors: MW 505.48, ALogP 3.9, HBA 8, HBD 2, PSA 120.95, QED 0.52
Compound (SMILES): Cc1cc(C#N)cc(C)c1Oc1cc(Cl)nc(NC2CCN(Cc3ccncc3)CC2)c1
InChIKey: WYZVEMYGCLUVLS-UHFFFAOYSA-N
Target: Human immunodeficiency virus type 1 reverse transcriptase (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: Q72547
Assay: Inhibition of recombinant wild type HIV1 p66/p51 using poly(rA) template/oligo(dT)16 primer after 40 mins by PicoGreen-based spectrofluorometric method
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 447.97, ALogP 5.49, HBA 6, HBD 1, PSA 74.07, QED 0.5
Compound (SMILES): Cc1ccc(C2(CCN(C)C)CC(c3ccccc3)=NN2C(=O)Nc2ccc(F)cc2)cc1
InChIKey: HTVBTRMJTNKQJV-UHFFFAOYSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Inhibition of recombinant T-type calcium channel Cav3.2 (unknown origin) expressed in HEK293 cells assessed as inhibition of CaCl2-induced calcium influx preincubated for 3 mins prior CaCl2 addition by Fluo-4-AM dye-based FLIPR assay
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 444.55, ALogP 5.62, HBA 3, HBD 1, PSA 47.94, QED 0.54
Compound (SMILES): COc1ccc(-n2[se]c3ccccc3c2=O)c(C)c1
InChIKey: CWFMODSINTWLPV-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Non-covalent/mixed type inhibition of recombinant human MetAP2 using Met-AMC as substrate preincubated for 30 mins followed by substrate addition by spectrofluorimetric method
Measurement: IC50 = 1350 nM (pChEMBL 5.869999885559082)
Descriptors: MW 318.23
Compound (SMILES): CN1CCC(CNC(=O)c2ccc(NC(=O)N3Cc4ccncc4C3)cc2)C1
InChIKey: LWRPDLIPXWMHIH-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 = 37.7 nM (pChEMBL 7.420000076293945)
Descriptors: MW 379.46, ALogP 2.31, HBA 4, HBD 2, PSA 77.57, QED 0.85
Compound (SMILES): COCCNC(=O)c1ccc2cc(Oc3ccc(C(F)(F)F)nc3)ccc2n1
InChIKey: OEMFCMMKRLPPJB-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 = 3010 nM (pChEMBL 5.519999980926514)
Descriptors: MW 391.35, ALogP 3.82, HBA 5, HBD 1, PSA 73.34, QED 0.65
Compound (SMILES): CC(C)Oc1ccc(/N=c2\[nH]c(=O)n(C[C@H](C)C(=O)O)c(=O)n2Cc2ccc(Cl)cc2)cc1F
InChIKey: IWHFRKDECPOECP-AWEZNQCLSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683)) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37 C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 uM Fluo-3-AM solution (pH 7.5) containing 20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 uL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.).
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 490.92, ALogP 2.92, HBA 7, HBD 2, PSA 118.68, QED 0.5
Compound (SMILES): N#CCCNC(=O)c1cc(NC(=O)c2cc(-c3ncccc3F)c(Cl)cc2Cl)n(-c2ccccc2)n1
InChIKey: RWZRGLYDSCFRQU-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 3.25 nM (pChEMBL 8.489999771118164)
Descriptors: MW 523.36, ALogP 5.28, HBA 6, HBD 2, PSA 112.7, QED 0.32
Compound (SMILES): COc1cc(Nc2n[nH]c3cccnc23)ccc1Cl
InChIKey: RXBIBCQFMWIYGB-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Receptor Activity Assay: The disclosed compounds and compositions can be evaluated for their ability to act as a potentiator of metabotropic glutamate receptor activity, in particular mGluR4 activity, by any suitable known methodology known in the art. For example, Chinese Hamster Ovary (CHO) cells transfected with human mGluR4 or HEK cells co-transfected with rat mGluR4 and the G-protein regulated Inwardly Rectifying Potassium channel (GIRK) were plated in clear bottom assay plates for assay in a Hamamatsu FDSS Fluorometric Plate Reader. The cells were loaded with either a Ca2+-sensitive fluorescent dye or the thallium responsive dye and the plates were washed and placed into a suitable kinetic plate reader. For human mGluR4 assays, a fluorescence baseline was established for 3-5 seconds, the disclosed compounds were then added to the cells, and the response in cells was measured.
Measurement: EC50 = 1110 nM (pChEMBL 5.960000038146973)
Descriptors: MW 274.71, ALogP 3.36, HBA 4, HBD 2, PSA 62.83, QED 0.77
Compound (SMILES): CCCC1C(=O)NC(Cc2ccccc2)C(=O)NCCCc2ccccc2OCCNC(C(C)CC)C(=O)N1C
InChIKey: RUNHUQPXJOCWIE-UHFFFAOYSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 550.74, ALogP 3.49, HBA 5, HBD 3, PSA 99.77, QED 0.51
Compound (SMILES): COc1cncc(-c2nc3ccc(F)cn3c2C2CC2)c1
InChIKey: JQZNZPQADFOCLV-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibition Assay: V79 cell lines stably expressing the either the human CYP11B2 or the human CYP11B1 enzyme were generated using a standard transfection protocol. V79 cells were transfected with plasmids pTriEx3-Hygro-hCyp11B2 or pTriEx3-Hygro-hCyp11B1 using Lipofectamine2000 reagent. V79 cells that stably express the human CYP11B2 or human CYP11B1 enzyme were selected for and maintained in DMEM supplemented with 10% FBS and 400 μg/mL hygromycin for ¿2 weeks. Single cell clones were generated by infinite dilution in DMEM supplemented with 10% FBS and 400 μg/mL hygromycin until single colonies were obtained. Clones V79-hCYP11B2-CLE9 and V79-hCYP11B1-CL8C7, were determined to produce the most aldosterone and cortisol, respectively, and were selected for inhibitor screening. For testing of inhibitors, cells were harvested at 80% confluency with 0.5% Trypsan-EDTA, washed once in PBS, and reconstituted in DMEM+0.1% BSA media at a cell concentration of 600,000 cells/mL for the CYP11B2 assay and 280,000 cells/mL for the CYP11B1 assay. 25 μL of cells were added to a 384 well tissue culture treated plate and mixed with 0.3 μL of inhibitor or DMSO (1% final DMSO concentration) for 1 hour at 37° C., 5% CO2. After pre-incubation with inhibitor, the reaction was initiated by adding 5 μL of substrate (final concentration of 125 nM 11-deoxycorticosterone for the CYP11B2 assay or 250 nM 11-deoxycortisol for the CYP11B1 assay). The reaction was carried out for 3 hours at 37° C., 5% CO2 and was stopped by harvesting the supernatants. The amount of product in the supernatant (aldosterone for CYP11B2 assay and cortisol for the CYP11B1 assay) was measured using HTRF-based assay kit (Aldosterone HTRF-CisBio#64ALDPEB, Cortisol HTRF-CisBio #63IDC002-CORT). IC50s for the inhibitor were determined by plotting the amount of product formed against the concentration of inhibitor using sigmoidal dose-response curve (variable slope) fit in GraphPad.
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 283.31, ALogP 3.42, HBA 4, HBD 0, PSA 39.42, QED 0.74
Compound (SMILES): O=C(c1cnccn1)N1CCN(C(=O)c2cc(Cc3n[nH]c(=O)c4c3NCCC4)ccc2F)CC1
InChIKey: SVNDWCWXWVHJTA-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 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 477.5, ALogP 1.25, HBA 7, HBD 2, PSA 124.18, QED 0.58
Compound (SMILES): COc1cccc2oc(-c3cnc4ccc(OCCCS(C)(=N)=O)nn34)cc12
InChIKey: DLZQHAOUJWYOTJ-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: MKNK1 Kinase Assay: For the assay 50 mL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM magnesium chloride, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22 °C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μL assay volume is 10 μM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 45 min at 22 °C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.05 μg/ml. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5). The resulting mixture was incubated for 1 h at 22 °C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET reader, e.g. a Rubystar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 400.46, ALogP 3.6, HBA 8, HBD 1, PSA 102.71, QED 0.48
Compound (SMILES): CC(C)C[C@H](NC(=O)c1ccc(C2CC2)c(OCC2CCCO2)n1)C(N)=O
InChIKey: MAEFXTYFMVZJBB-JRZJBTRGSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 375.47, ALogP 2.15, HBA 5, HBD 2, PSA 103.54, QED 0.69
Compound (SMILES): Cc1ccc(C(=O)N2CC(c3ccc(C#N)cc3)C2)cc1-c1[nH]c(CO)nc1C#N
InChIKey: PDYGVRQAMDQWBJ-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 397.44, ALogP 2.86, HBA 5, HBD 2, PSA 116.8, QED 0.7
Compound (SMILES): CN1Cc2c(C3=C[C@@H]4COC(=O)N4CC3)[nH]c3nccc(c23)-c2cc(F)c(F)cc21
InChIKey: FGZHKQSWEGJXQZ-GFCCVEGCSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay : CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were diluted in 100% DMSO then diluted with 1:10 in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. Equal volume of the compound dilutions was added to a final reaction mixture containing LANCE detection buffer (PerkinElmer CR97-100), 100 nM ULight MBP (PerkinElmer TRF0109M), 1000 μM ATP, and CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 1 hour before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer. The reaction was incubated for 1 hour and the signal was read in Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 408.41, ALogP 4.07, HBA 4, HBD 1, PSA 61.46, QED 0.66
Compound (SMILES): Cc1cc(/C=N/NC(=O)[C@@H]2C[C@H]2c2ccc(Cl)cn2)cc2cccnc12
InChIKey: IXRFXQITJITZJD-LKAUBKCLSA-N
Target: Transient receptor potential cation channel subfamily M member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ8
Assay: Inhibition of human TRPM5 transfected in HEK293 cells assessed as inhibition of ATP-mediated calcium mobilization by Calcium3 dye-based FLIPR assay
Measurement: IC50 = 5400 nM (pChEMBL 5.269999980926514)
Descriptors: MW 364.84, ALogP 3.85, HBA 4, HBD 1, PSA 67.24, QED 0.56
Compound (SMILES): CC1(COC(=O)N2CC[C@@H](C#N)[C@H](n3cc(C(N)=O)c(Nc4ccc(F)cc4)n3)C2)COC1
InChIKey: VTZHHLSKLICAJW-KBXCAEBGSA-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 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 456.48, ALogP 2.42, HBA 8, HBD 2, PSA 135.5, QED 0.68
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NCC4CC4)cc(Nc4cccc(Cl)c4)nc23)N1
InChIKey: BFYIETOTLUQMJQ-UUASQNMZSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 = 508.7 nM (pChEMBL 6.289999961853027)
Descriptors: MW 423.86, ALogP 3.13, HBA 7, HBD 4, PSA 112.45, QED 0.36
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NCC5CCOCC5)c(CS(F)(F)(F)(F)F)c4)c(Oc4c[nH]c5nccc-5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: INLMIYOARQASDC-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
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 = 1.654 nM (pChEMBL 8.779999732971191)
Descriptors: MW 963.54, ALogP 11.5, HBA 9, HBD 3, PSA 128.89, QED 0.09
Compound (SMILES): O=C(Nc1ccc2[nH]nc(-c3ccccc3)c2c1)c1ccccn1
InChIKey: SLCNGYWKEVVZSF-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 314.35, ALogP 3.88, HBA 3, HBD 2, PSA 70.67, QED 0.6
Compound (SMILES): CC(C)Oc1cc(-c2ncn(/C=C\C(=O)OCc3ccccn3)n2)cc(C(F)(F)F)c1
InChIKey: FOCYRGHXVRQHQV-VURMDHGXSA-N
Target: Exportin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14980
Assay: Inhibition of Crm1 in human U2OS cells expressing GFP-fused HIV1 Rev assessed as decrease in nuclear export of GFP-fused HIV1 Rev after 1 hr by Hoechst 33258 staining-based assay
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 432.4, ALogP 4.36, HBA 7, HBD 0, PSA 79.13, QED 0.41
Compound (SMILES): Oc1ccc(-c2cn3cc(Cl)ccc3n2)cc1
InChIKey: MMJUHKRVPBKDDX-UHFFFAOYSA-N
Target: Dual specificity protein kinase CLK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P49759
Assay: Inhibition of CLK1 (unknown origin)
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 244.68, ALogP 3.36, HBA 3, HBD 1, PSA 37.53, QED 0.71
Compound (SMILES): COCc1ccc(C(C)=O)c2sc(C(=O)Nc3ccc4c(C(=O)N5CC(F)C5)cccc4n3)c(C)c12
InChIKey: RAEVZOTWOOUHLU-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Activity Assay: A human acute lymphoblastic lymphoma T cell line MOLT-4 (which can be purchased under ATCC No. CRL-1582 from ATCC) was cultured in an RPMI1640 medium containing 10% fetal bovine serum to obtain 5108 MOLT-4 cells. The cells were recovered by centrifugation and suspended in 10 ml of buffer solution A (25 mM Tris-HCl, 5 mM 2-mercaptoethanol, 2 mM benzamidine, 2 mM EDTA, and 0.1 mM 4-(2-aminoethyl)benzenesulfonyl hydrochloride, pH 7.5). The cells were homogenized using a Polytron homogenizer and centrifuged (4° C., 25,000 g, 10 minutes). Then, the supernatant was further ultracentrifuged (4° C., 100,000 g, 60 minutes). The obtained supernatant was filtered through a 0.2um filter to obtain a soluble fraction.HiTrap Q HP column (5 ml2, GE Healthcare Japan Corp.) equilibrated with buffer solution A was charged with the obtained soluble fraction. Phosphodiesterase was eluted with 300 ml of buffer solution A containing a linear gradient solution of 0 to 0.8 M NaCl to recover sixty 5-ml fractions. Each fraction was tested for cAMP-metabolizing phosphodiesterase activity. In each fraction, a fraction eluted as an active peak centered around 250 mM NaCl was collected from fractions that had cAMP metabolic activity and did not lose the metabolic activity by 10uM rolipram (PDE4 selective inhibitor) and 10uM milrinone (PDE3 selective inhibitor). In order to confirm whether or not PDE10A mRNA was expressed in the MOLT-4 cells, total RNA was prepared from the MOLT-4 cells according to a standard method and analyzed by RT-PCR using PDE10A gene-specific primers (PDE10A sense primer: 5'-TGCTCCATGGTGGAAGTGGA-3' (SEQ ID NO: 1) and PDE10A antisense primer: 5'-CAACTGGAAGCATGCGGTCA-3' (SEQ ID NO: 2)). As a result, PDE10A mRNA was detected from the total RNA of the MOLT-4 cells. On the other hand, total RNA was prepared from Jurkat cells, one type of human T cell, and similarly subjected to RT-PCR. As a result, PDE10A mRNA was rarely detected. An active peak centered around 250 mM NaCl was not observed in fractions obtained by the treatment of Jurkat cells in the same way as above. Eur J Biochem. 1999, 266 (3), 1118-2 has reported that PDE10A from the rat striatum and testis is eluted as an active peak centered around 250 mM NaCl.
Measurement: IC50 = 0.11 nM (pChEMBL 9.960000038146973)
Descriptors: MW 505.57, ALogP 5.15, HBA 6, HBD 1, PSA 88.6, QED 0.37
Compound (SMILES): COCc1nc2c(C(=O)Nc3cccc(Cl)c3C)cc(NC(=O)c3cc(Br)ccc3Cl)cc2[nH]1
InChIKey: OXQULDJZTAEXNJ-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition Assay: mPGES-1 microsome fractions were prepared from CHO-K1 cells transiently transfected with plasmid encoding the human mPGES-1cDNA. Microsomes were diluted with potassium phosphate buffer containing reduced glutathione (pH7.4), and DMSO containing test compound or DMSO alone was added (such that DMSO final concentration would be 1% in each) and incubated at 4° C. for 20 minutes. Then, the enzymatic reactions were initiated by the addition of PGH2 substrate (final concentration 1 μM) and incubated at 4° C. for 60 seconds. The reaction was terminated by the addition of a citrate solution (final citrate concentration 50 mM) containing ferric chloride (final concentration 1 mg/mL). PGE2 production in the enzyme reaction aliquot was measured using HTRF kit (Cisbio International, catalogue #62P2APEC).
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 562.25, ALogP 6.59, HBA 4, HBD 3, PSA 96.11, QED 0.25
Compound (SMILES): O=C(Nc1cc(C2CCCCC2)no1)c1ccc2cc3n(c2c1)CCCNC3=O
InChIKey: YABZWVBXMRLPIV-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 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 392.46, ALogP 4.06, HBA 5, HBD 2, PSA 89.16, QED 0.7
Compound (SMILES): O=C(OCC12CCN(CC1)CC2)c1c[nH]c2cc3c(cc12)OCCO3
InChIKey: BWFXWPBHQAEGKO-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05941
Assay: Radioligand Binding Assay: Protocol 1: The assay was conducted following the literature reference Meyer E. M., et al., Analysis of 3-(4-Hydroxy, 2-Methoxybenzylidene) Anabaseine Selectivity and Activity at Human and Rat Alpha-7 Nicotinic Receptors, J. Pharmacol. Exp. Ther., 287: 918-925, 1998. The assay was conducted using the following materials: receptor source: rat brain, radioligand: [125I] α-Bungarotoxin (2200 Ci/mmol) with a final concentration of 1 nM, non-specific determinant: Methyllycaconitine (MLA) [1 μM], reference compound: Methyllycaconitine (MLA), and positive control: Methyllycaconitine (MLA). Incubation Conditions: Reactions were carried out in 20 mM HEPES (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2 and 1.2 mM MgSO4 at 37° C. for 90 minutes. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined by liquid scintillation counting and compared to control values in order to ascertain any interactions of test compound with the nicotinic, ganglionic binding site.
Measurement: Ki = 81.1 nM (pChEMBL 7.090000152587891)
Descriptors: MW 342.4, ALogP 2.58, HBA 5, HBD 1, PSA 63.79, QED 0.87
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COC(=O)[C@H](C)N)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: WRANVYZNSQCCSO-XVHYEQEESA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Radioligand Dose-Displacement Binding Assay: Radioligand dose-displacement binding assays for t-opioid receptors used 0.3 nM [3H]-diprenorphine (Perkin Elmer, Shelton, Conn.), with 5 mg membrane protein/well in a final volume of 500 ul binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 2 hr at room temperature. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.), presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by performing three filtration washes with 500 ul of ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 ul/well), and plates were counted using a Packard Top-Count.
Measurement: Ki = 147.99 nM (pChEMBL 6.829999923706055)
Descriptors: MW 482.62, ALogP 2.81, HBA 7, HBD 1, PSA 83.25, QED 0.6
Compound (SMILES): CCn1nc(N2CCCC2)nc1/C=C/c1nc2nc(C)cc(C)n2n1
InChIKey: SORCAMPMGWCYHG-BQYQJAHWSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Scintillation Proximity Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 μl contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. # A-7906), 50 nM cGMP (Sigma, cat. # G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. # TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat # SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 μl of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate solution (stop reagent). After 1 h under shaking, the plate was centrifuged one minute at 170 g to allow beads to settle. Afterwards, radioactive counts were measured on a Perkin Elmer Top-Count Scintillation plate reader.
Measurement: IC50 = 205.12 nM (pChEMBL 6.690000057220459)
Descriptors: MW 338.42, ALogP 2.12, HBA 8, HBD 0, PSA 77.03, QED 0.73
Compound (SMILES): CNCCN1CCC(OCc2ccc(OCC(F)(F)F)cc2)CC1
InChIKey: FPMCBNOZDJXPHM-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96LA8
Assay: Inhibition of human full length PRMT6 (1 to 375 residues) expressed in baculovirus expression system assessed as inhibition of methylation activity preincubated with protein followed by addition of biotin labeled histone H4 (1 to 24) peptide as substrate and [3H]-SAM measured after 3 hrs by scintillation proximity assay
Measurement: IC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 346.39, ALogP 2.83, HBA 4, HBD 1, PSA 33.73, QED 0.78
Compound (SMILES): Cn1nccc1-n1nnc2c1CCN(C(=O)c1cccc(C(F)(F)F)c1Cl)C2
InChIKey: PEUALQSPZSYZBK-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 2108.6 nM (pChEMBL 5.679999828338623)
Descriptors: MW 410.79, ALogP 2.87, HBA 6, HBD 0, PSA 68.84, QED 0.65
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccccc3)n2)CCN(C(=O)Cc2ccc(C(F)(F)F)cc2)CC1
InChIKey: TUSQTYLHQVAXDY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF 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 uL of 1.11x enzyme and 1.11x substrate in 1x 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 room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x 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.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 510.52, ALogP 4.22, HBA 6, HBD 2, PSA 117.04, QED 0.49
Compound (SMILES): CCNC(=O)Nc1ccc(-c2nc(N3CCOC[C@@H]3CC)nc3c2C(=O)N(CC)CC(C)(C)O3)cc1F
InChIKey: IZVQXADISQNPBH-KRWDZBQOSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The test detects the phosphorylation of the substrate GFP-4EBP1 using a terbium-labelled antibody that is specific for the phospho-Thr46 epitope. Binding of the Tb-antibody to the phosphorylated GFP peptide allows the transfer of energy from Tb, excited at 340 nm, to GFP with an increase in the fluorescence emission of the GFP at 520 nm. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 ul. To 1 ul of inhibitor in 100% DMSO are added (final concentrations) 400 nM of GFP-4EBP1 (Invitrogen PV4759), 8 uM of ATP, 200 ng/ml of enzyme (human recombinant aa mTOR 1360-2549, Invitrogen PV4753) in a buffer of HEPES 50 mM pH 7.5, EGTA 1 mM, MnCl2 10 mM, BSA 0.1 mg/ml, glycerol 0.5%, NV-10 0.1 mg/ml, DTT 2 mM. The mixture is incubated for 30 minutes at room temperature. 10 ul of solution are taken up and the reaction is quenched by adding 10 ul of Tb-antibody solution (Invitrogen PV4757)/EDTA.
Measurement: IC50 = 116 nM (pChEMBL 6.940000057220459)
Descriptors: MW 514.6, ALogP 3.67, HBA 7, HBD 2, PSA 108.92, QED 0.61