lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(C)OC(=O)N1CCCC(C(=O)N2CC[C@@]3(C)c4cccc(O)c4C[C@@H]2C3(C)C)C1
InChIKey: BJDQQOUROBDUBI-JTANGXJZSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: Homogeneous Time-Resolved Fluorescence (HTRF): In vitro inhibition of 11beta -HSD1 by test compounds was determined with HTRF (Homogeneous Time-Resolved Fluorescence) technology (cisbio international, France) detecting cortisol generated from cortisterone by human liver microsomes. Briefly, compounds were incubated for 1 hour at 37 C. in Tris buffer (20 mM tris, 5 mM EDTA, pH 6.0) containing NADPH (200 uM) and cortisone (80 nM). Cortisol generated in the reaction is then detected with a competitive immunoassay, involving two HTRF conjugates: cortisol linked to XL665 and anti-cortisol antibody labeled with Europium cryptate. The incubation period for detection reaction was typically 2 hours. The amount of cortisol is determined by reading the time-resolved fluorescence of the wells (Ex 320/75 nm; Em 615/8.5 nm and 665/7.5 nm). The ratio of the two emission signals is then calculated (Em665*10000/Em615).
Measurement: IC50 = 693 nM (pChEMBL 6.159999847412109)
Descriptors: MW 442.6, ALogP 4.48, HBA 4, HBD 1, PSA 70.08, QED 0.7
Compound (SMILES): CC(C)(O)[C@H](F)CNC(=O)c1cnc(-n2cnc3cc(C#N)cnc32)cc1Nc1cnn(C(F)F)c1
InChIKey: HBYZIUOGLDUDFZ-QGZVFWFLSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 5.7 nM (pChEMBL 8.239999771118164)
Descriptors: MW 499.46, ALogP 2.86, HBA 10, HBD 3, PSA 146.57, QED 0.34
Compound (SMILES): O=C(Nc1cc2[nH]nc(OCCOC(F)(F)F)c2cn1)N[C@H](CO)c1ccccc1
InChIKey: HUEQYKYIFCVQHT-CQSZACIVSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 425.37, ALogP 2.73, HBA 6, HBD 4, PSA 121.39, QED 0.41
Compound (SMILES): Cc1nsc(-c2nnc3n2CCN(C(=O)c2ccc(F)cc2F)[C@@H]3C)n1
InChIKey: OZEMPZBRQPJERB-MRVPVSSYSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Functional Assay: Chinese Hamster Ovary recombinant cells expressing the human NK-3 receptor and a construct that encodes the photoprotein apoaequorin were used for this assay. The antagonist activity of compounds of the invention is measured following pre-incubation (3 minutes) of the compound (at various concentrations) with the cells, followed by addition of the reference agonist (NKA) at a final concentration equivalent to the EC80 (3 nM) and recording of emitted light (FDSS 6000 Hamamatsu) over the subsequent 90-second period. The intensity of the emitted light is integrated using the reader software. Compound antagonist activity is measured based on the concentration-dependent inhibition of the luminescence response to the addition of Neurokinin A.
Measurement: IC50 = 87 nM (pChEMBL 7.059999942779541)
Descriptors: MW 376.39, ALogP 2.6, HBA 7, HBD 0, PSA 76.8, QED 0.69
Compound (SMILES): CN(C)CCOc1ccc([C@H]2CNC(=O)N2c2ccc3[nH]cnc3c2)cc1
InChIKey: UNARJRXATYBLGH-LJQANCHMSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: Spectrophotometic Assay: Spectrophotometic Assay: This novel assay was used to determine the kinetic parameters for most of the QC substrates. QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R.C.J. 1989 J Neurosci Methods 30, 23-28).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 365.44, ALogP 2.77, HBA 4, HBD 2, PSA 73.49, QED 0.7
Compound (SMILES): C[C@@H](OC(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CC1)C(F)(F)F
InChIKey: RPPZVUWFJDNQRB-CYBMUJFWSA-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 482.44, ALogP 3.66, HBA 7, HBD 2, PSA 126.27, QED 0.61
Compound (SMILES): Cc1nn(C)c(C)c1NS(=O)(=O)c1c(Cl)cc(-c2ccco2)cc1Cl
InChIKey: QREJBKFFAKVFKC-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 = 3600 nM (pChEMBL 5.440000057220459)
Descriptors: MW 400.29, ALogP 4.4, HBA 5, HBD 1, PSA 77.13, QED 0.7
Compound (SMILES): COc1n[nH]c2cc(NC(=O)N[C@@H](c3ccccc3)[C@@H](C)O)nc(CO)c12
InChIKey: ZPRWXYTUCYKXHX-QLJPJBMISA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 371.4, ALogP 1.7, HBA 6, HBD 5, PSA 132.39, QED 0.45
Compound (SMILES): Cc1c(Cl)cccc1NC(=O)N1CC(CC#N)(n2cc(C(N)=O)c(Nc3ccccc3)n2)C1
InChIKey: ZSXZHCUDAITJGK-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 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 463.93, ALogP 3.84, HBA 6, HBD 3, PSA 129.07, QED 0.51
Compound (SMILES): O=C(NCC1(CO)COC1)c1cc(NC(=O)c2cc(-c3ccccn3)c(F)cc2Cl)n(-c2ccccc2)n1
InChIKey: DYHMURVKMVAERV-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 = 24.1 nM (pChEMBL 7.619999885559082)
Descriptors: MW 535.96, ALogP 3.72, HBA 7, HBD 3, PSA 118.37, QED 0.32
Compound (SMILES): COC[C@@H](NC(=O)Nc1cc2[nH]nc(NCC3CC(C(C)(C)O)C3)c2cn1)c1ccccc1
InChIKey: SDKRTYBHKGRDES-MKJPBZQASA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 2.6 nM (pChEMBL 8.59000015258789)
Descriptors: MW 452.56, ALogP 3.68, HBA 6, HBD 5, PSA 124.19, QED 0.34
Compound (SMILES): C[C@@H]1CN(c2cc(-c3n[nH]c4ccc(-c5cnn(C)c5)cc34)ncn2)CCO1
InChIKey: TUPNAVGCYXZCRL-CYBMUJFWSA-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 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 375.44, ALogP 2.65, HBA 7, HBD 1, PSA 84.75, QED 0.59
Compound (SMILES): Cn1cc(CNc2cc(OC[C@H]3C[C@@H]3c3ccc(F)cn3)nn(C)c2=O)cn1
InChIKey: CARSURPIDVVSNV-HIFRSBDPSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product #R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215).
Measurement: Ki = 1.261 nM (pChEMBL 8.899999618530273)
Descriptors: MW 384.42, ALogP 1.84, HBA 8, HBD 1, PSA 86.86, QED 0.67
Compound (SMILES): CCCNc1nccc(N2CC[C@@H](Oc3ccc(C(C)C(=O)NC4COC4)cc3)C2)c1F
InChIKey: MXMDYBZFBODMTP-OTOKDRCRSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Malonyl CoA formation by acetyl CoA carboxylases is stoichometrically linked to the consumption of ATP. ACC2 activity is measured in a NADH-linked kinetic method measuring ADP generated during the ACC reaction using a coupled lactate dehydrogenase/pyruvate kinase reaction. Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetylCoenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
Measurement: IC50 = 626 nM (pChEMBL 6.199999809265137)
Descriptors: MW 442.54, ALogP 3.32, HBA 6, HBD 2, PSA 75.72, QED 0.62
Compound (SMILES): Nc1n[nH]c2cc(NC(=O)C[C@H](COC(F)F)c3ccccc3)ncc12
InChIKey: SOTLBWHGNHPLIH-LLVKDONJSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 361.35, ALogP 2.89, HBA 5, HBD 3, PSA 105.92, QED 0.6
Compound (SMILES): CN1[C@@H](C(=O)NCC[C@@H](O)CO)[C@H](c2cccc(Cl)c2F)[C@@]2(C(=O)Nc3cc(F)c(F)cc32)[C@H]1CC(C)(C)C
InChIKey: NCVZNOICJNGHGC-AXSIKVPYSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Fluorescence-polarization Binding Assay: The binding affinity of the MDM2 inhibitors was determined using an optimized, sensitive and quantitative fluorescence polarization-based (FP-based) binding assay using a recombinant human His-tagged MDM2 protein (residues 1-118) and a fluorescently tagged p53-based peptide. The design of the fluorescence probe was based upon a previously reported high-affinity p53-based peptidomimetic compound (5-FAM-βAla-βAla-Phe-Met-Aib-pTyr-(6-Cl-LTrp)-Glu-Ac3c-Leu-Asn-NH2 (SEQ ID NO: 1)) (García-Echeverría et al., J. Med. Chem. 43: 3205-3208 (2000)). This tagged peptide is called PMDM6-F. MDM2 protein was serially double diluted in a Dynex 96-well, black, round-bottom plate, and the PMDM6-F peptide was added at 1 nM concentration. The assay was performed in the buffer: 100 mM potassium phosphate, pH 7.5; 100 μg/mL bovine gamma globulin; 0.02% sodium azide, 0.01% Triton X-100) and the polarization values were measured after 3 h of incubation using an ULTRA READER (Tecan U.S. Inc).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 568.04, ALogP 3.71, HBA 5, HBD 4, PSA 101.9, QED 0.41
Compound (SMILES): COC(=O)C[C@@H](C)n1c(=O)[nH]/c(=N\c2ccc(OC(C)C)c(F)c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: FJXMNXYGVWTZBZ-OAHLLOKOSA-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 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 504.95, ALogP 3.32, HBA 8, HBD 1, PSA 107.68, QED 0.47
Compound (SMILES): CC[C@@H](N[C@H](C)CC(N)=O)c1ccc(OC)c(C(=O)c2ccccc2)c1F
InChIKey: MSQHYHHPFFDMFR-CZUORRHYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: FRET-Based Assay: The HCV NS3 protease functions have been extensively studied and are considered as potential targets for antiviral therapy: see for example the many references listed in the introductory section of this application. Therefore, the activity of the compounds of the invention as anti-HCV agents was assessed using a full length HCV NS3 protease.The protease activity of the full length NS3/4a was measured using a FRET-based assay utilizing a peptide substrate derived from the NS4A/B cleavage site (Anaspec) and labelled at one end with a quencher (QXL520) and at the other with a fluorophore (5-FAMsp). NS3/4a (produced in-house by literature methods) was incubated with test compounds and peptide substrate in 50 mM Tris pH8, 20 mM DTT, 1% CHAPS, 10% glycerol and 5% DMSO. The reaction was followed by monitoring the change in fluorescence on a Molecular Devices Gemini plate reader for 30 minutes at room temperature.
Measurement: IC50 = 490 nM (pChEMBL 6.309999942779541)
Descriptors: MW 372.44, ALogP 3.37, HBA 4, HBD 2, PSA 81.42, QED 0.66
Compound (SMILES): FC(F)(F)c1ccc(Nc2nc(N[C@@H]3CCOC3)nc3c2CCN(c2ncccc2C(F)(F)F)CC3)cc1
InChIKey: ROGIRUYAXVLHPQ-QGZVFWFLSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 538.5, ALogP 5.46, HBA 7, HBD 2, PSA 75.2, QED 0.42
Compound (SMILES): C=C(C)[C@H]1Cc2c(ccc3c2O[C@@H]2COc4cc(OC)c(OC)cc4[C@@H]2[C@]32CC3O[C@]3(O)[C@@H](O)O2)O1
InChIKey: VKSPBPTXXGCAIS-SASZYTRVSA-N
Target: Pancreatic triacylglycerol lipase (SINGLE PROTEIN), Sus scrofa
UniProt: P00591
Assay: Inhibition Assay: Particularly, pancreatic lipase inhibiting activity was measured by the conventional method known to those in the art (Kim, J. H.; Kim, H. J.; Park, H. W.; Youn, S. H.; Choi, D. Y.; Shin, C. S. FEMS Microbiol. Lett. 2007, 276, 93.). To prepare the enzyme buffer, 30 mL (10 units) of pig pancreatic lipase solution (Sigma, St. Louis, Mo.) and 1 mM EDTA (pH 6.8) were mixed with 10 mM MOPS (morpholinepropanesulphonic acid), which was added to 850 mL of tris buffer (100 mM Tris-HCl and 5 mM CaCl2, pH 7.0). Then, 100 mL of the rotenoisin A or B of the present invention was mixed with the prepared enzyme buffer at the experimental concentration. As the positive control, 880 mL of Orlistat (Roche, Basel, Switzerland) was mixed with the enzyme buffer. 20 mL of substrate solution containing 20 mM p-nitrophenylbutyrate dissolved in dimethyl formaide was added to the enzyme buffer, followed by culture for 15 minutes at 37° C. that was the temperature adequate for enzyme reaction.
Measurement: IC50 = 28400 nM (pChEMBL 4.550000190734863)
Descriptors: MW 496.51, ALogP 2.54, HBA 9, HBD 2, PSA 108.37, QED 0.49
Compound (SMILES): CCC[C@@H]1c2nnc(-c3nc(C)ns3)n2CCN1C(=O)c1ccc(F)cc1
InChIKey: GEUWDXLPGYKMAG-CQSZACIVSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Functional Assay: Chinese Hamster Ovary recombinant cells expressing the human NK-3 receptor and a construct that encodes the photoprotein apoaequorin were used for this assay. The antagonist activity of compounds of the invention is measured following pre-incubation (3 minutes) of the compound (at various concentrations) with the cells, followed by addition of the reference agonist (NKA) at a final concentration equivalent to the EC80 (3 nM) and recording of emitted light (FDSS 6000 Hamamatsu) over the subsequent 90-second period. The intensity of the emitted light is integrated using the reader software. Compound antagonist activity is measured based on the concentration-dependent inhibition of the luminescence response to the addition of Neurokinin A.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 386.46, ALogP 3.24, HBA 7, HBD 0, PSA 76.8, QED 0.69
Compound (SMILES): Cc1ccc([C@H]2C[C@@H]2COc2cc(NCc3cnn(C)c3)c(=O)n(C)n2)nc1
InChIKey: XFQFOFMRXLQIRL-CVEARBPZSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product #R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215).
Measurement: Ki = 0.072 nM (pChEMBL 10.140000343322754)
Descriptors: MW 380.45, ALogP 2.01, HBA 8, HBD 1, PSA 86.86, QED 0.67
Compound (SMILES): N#CC1(NC(=O)[C@@H]2C[C@@H](S(=O)(=O)c3ccccc3-n3ccnn3)CN2C(=O)C2(c3ncc(I)cc3F)CC2)CC1
InChIKey: PRLYGGLINQVATF-XLIONFOSSA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Enzyme Inhibition Assay: Enzyme activity is measured by observing the increase in fluorescence intensity caused by cleavage of a peptide substrate containing a fluorophore whose emission is quenched in the intact peptide.Assay buffer: 100 mM potassium phosphate pH 6.5, EDTA-Na 5 mM, Triton X-100 0.001%, DTT 5 mM.Enzymes (all at 1 nM): human and mouse Cathepsin S, Cat K, Cat B, Cat L.Substrate (20 μM): Z-Val-Val-Arg-AMC, except for Cat K which uses Z-Leu-Arg-AMC (both from Bachem).Z=Benzyloxycarbonyl.AMC=7-Amino-4-Methyl-Coumarin.DTT=dithiothreitol.Final volume: 100 μL.Excitation 360 nm, Emission 465 nm.Enzyme is added to the substance dilutions in 96-well microtitre plates and the reaction is started with substrate. Fluorescence emission is measured over 20 minutes, during which time a linear increase is observed in the absence of inhibitor.
Measurement: IC50 = 2.372 nM (pChEMBL 8.619999885559082)
Descriptors: MW 675.48, ALogP 2.05, HBA 9, HBD 1, PSA 150.94, QED 0.37
Compound (SMILES): Cc1nc([C@](C)(O)C#Cc2cc3c(cc2F)OCCn2c-3nc(C(N)=O)c2C2CC2)no1
InChIKey: NVNGWTVBDRHXCY-JOCHJYFZSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki = 3.3 nM (pChEMBL 8.479999542236328)
Descriptors: MW 437.43, ALogP 2.01, HBA 8, HBD 2, PSA 129.29, QED 0.6
Compound (SMILES): Nc1ncc([C@H]2CC[C@H](O)CC2)nc1-c1ccc(C(=O)N[C@H](CNCCF)c2cc(F)cc(Br)c2)c(F)c1
InChIKey: HFYQZBBECYMACW-ZFUCPZCZSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 (20 pM) Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.374 nM (pChEMBL 9.430000305175781)
Descriptors: MW 592.46, ALogP 4.82, HBA 6, HBD 4, PSA 113.16, QED 0.27
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N[C@@H](CO)C(C)C)n2c1COCC2)c1ccccc1
InChIKey: CYUMJJKNLUZYSO-MOPGFXCFSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 4400 nM (pChEMBL 5.360000133514404)
Descriptors: MW 413.52, ALogP 2.65, HBA 5, HBD 3, PSA 92.59, QED 0.62
Compound (SMILES): Cc1nc(NC(=O)N[C@H](C)c2ccccc2)cc2[nH]nc(N3CCCC3C(=O)O)c12
InChIKey: WMHACIVVKKYBHS-ZGTOLYCTSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 74.7 nM (pChEMBL 7.130000114440918)
Descriptors: MW 408.46, ALogP 3.2, HBA 5, HBD 4, PSA 123.24, QED 0.51
Compound (SMILES): C[C@@H]1CN(c2ncnc3[nH]cc(-c4cnn(C)c4)c23)CCO1
InChIKey: CZUHAOMXATZIIC-SNVBAGLBSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 67 nM (pChEMBL 7.170000076293945)
Descriptors: MW 298.35, ALogP 1.58, HBA 6, HBD 1, PSA 71.86, QED 0.78
Compound (SMILES): CC(C)(O)[C@H](F)CNC(=O)c1cnc(-n2ncc3cc(C#N)cnc32)cc1NC1CC1
InChIKey: UXOKRVWJKRLBSR-QGZVFWFLSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 423.45, ALogP 2.1, HBA 8, HBD 3, PSA 128.75, QED 0.53
Compound (SMILES): Cc1cc([C@](C)(O)C#Cc2ccc3c(c2)-c2nc(C(N)=O)c(C(=O)N[C@@H]4CCOC4)n2CCO3)no1
InChIKey: HDAYWGVVZKYWFN-PUAOIOHZSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 491.5, ALogP 1.12, HBA 9, HBD 3, PSA 154.73, QED 0.46
Compound (SMILES): Cc1cc(N=S(C)(C)=O)cc2ncnc(Nc3ccc(Cl)cc3O[C@H](C)C(=O)NCC(F)(F)F)c12
InChIKey: KTXHWOZYGOHYOE-CYBMUJFWSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: In vitro Kinase Assay: The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled eIF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 529.97, ALogP 5.14, HBA 7, HBD 2, PSA 105.57, QED 0.44
Compound (SMILES): O=C([C@@H]1CCCN(c2ccc3nc(-c4ccc(OC(F)(F)F)cc4)[nH]c3n2)C1)N1CCCC1
InChIKey: MSLLTRZKRVTGDV-MRXNPFEDSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: In Vitro DGAT2 Assay: For determination of IC50 values, the reactions were carried out in 384-well white Polyplates (Perkin Elmer) in a total volume of 20 uL. To 1 uL of compounds dissolved in 100% DMSO and spotted at the bottom of each well, 5 uL of 0.04% bovine serum albumin (BSA) (fatty acid free, Sigma Aldrich) was added and the mixture was incubated at room temperature for 20 minutes. To this mixture, 10 uL of hDGAT2 membrane fraction (0.01 mg/mL) diluted in 100 mM Hepes-NaOH, pH 7.4, 20 mM MgCl2 containing 200 nM methyl arachidonyl fluorophosphonate (Cayman Chemical; dried from ethyl acetate stock solution under argon gas and dissolved in DMSO as 5 mM stock) was added. After this mixture was preincubated at room temperature for 2 hours, DGAT2 reactions were initiated by the addition of 4 uL of substrates containing 30 uM [1-14C]decanoyl-CoA (custom-synthesized by Perkin Elmer, 50 mCi/mmol) and 125 uM 1,2-didecanoyl-sn-glycerol (Avanti Polar Lipids) dissolved in 12.5% acetone. The reaction mixtures were incubated at room temperature for 40 min and the reactions were stopped by addition of 5 uL of 1% H3PO4. After the addition of 45 uL MicroScint-E (Perkin-Elmer), plates were sealed with Top Seal-A covers (Perkin-Elmer) and phase partitioning of substrates and products was achieved using a HT-91100 microplate orbital shaker (Big Bear Automation, Santa Clara, Calif.). Plates were centrifuged at 2,000×g for 1 min in an Allegra 6R Centrifuge (Beckman Coulter) and then were sealed again with fresh covers before reading in a 1450 Microbeta Wallac Trilux Scintillation Counter (Perkin Elmer). DGAT2 activity was measured by quantifying the generated product [14C]tridecanoylglycerol in the upper organic phase.
Measurement: IC50 = 24.8 nM (pChEMBL 7.610000133514404)
Descriptors: MW 459.47, ALogP 4.36, HBA 5, HBD 1, PSA 74.35, QED 0.63
Compound (SMILES): O=C(Nc1cnc2ccc(N3CCC[C@@H]3c3cc(F)ccc3F)nn12)N1CCS(=O)(=O)CC1
InChIKey: JOYTWSNCHBVCGB-QGZVFWFLSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: ELISA Assay: An enzyme-linked immunosorbant assay (ELISA) was used to assess TrkA kinase activity in the presence of inhibitors. Immulon 4HBX 384-well microtiter plates (Thermo part #8755) were coated with a 0.025 mg/mL solution of poly (Glu, Ala, Tyr; 6:3:1; Sigma P3899). Various concentrations of test compound, 2.5 nM TrkA (Invitrogen Corp., histidine-tagged recombinant human TrkA, cytoplasmic domain), and 500 uM ATP were incubated for 25 minutes at ambient temperature in the coated plates while shaking. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The reaction mixture was removed from the plate by washing with PBS containing 0.1% (v/v) Tween 20. The phosphorylated reaction product was detected using 0.2 ug/mL of a phosphotyrosine specific monoclonal antibody (clone PY20) conjugated to horseradish peroxidase in conjunction with the TMB Peroxidase Substrate System (KPL).
Measurement: IC50 = 884.4 nM (pChEMBL 6.050000190734863)
Descriptors: MW 476.51, ALogP 2.61, HBA 7, HBD 1, PSA 99.91, QED 0.62
Compound (SMILES): COc1cccc([C@@H](C)NC(=O)c2ccc(-c3cn[nH]c3)cc2F)c1
InChIKey: SPMLNRHNYBYMMZ-GFCCVEGCSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 43.3 nM (pChEMBL 7.360000133514404)
Descriptors: MW 339.37, ALogP 3.72, HBA 3, HBD 2, PSA 67.01, QED 0.74
Compound (SMILES): CN(C)CC/C=C/C(=O)Nc1cc2c(Nc3ccc(F)c(Cl)c3)ncnc2cc1OC[C@]12COC[C@H]1C2
InChIKey: LKPIQEOIBNBGGY-RUTRQQBQSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Time-Resolved Fret (TR-FRET) Assay: 1) The compound is dissolved in DMSO to prepare a 10 mM solution, and was diluted to 100 micoM with water. When used for IC50 measurement, series dilutions of 10 fold from 100 micoM are used. Kinase activity was determined with time-resolved Fret (TR-FRET) assay (LanthaScreen® kinase activity assay, from InVitrogen).2) The assay is performed in a Black 384-well plate (from Corning). The kinase and the compound was incubated for 30 mM at room temperature. ATP (1 mM) and fluorescein-poly GT were added, and the reaction was incubated for 15 mM. Detection agent SA-XL665 (from Cisbio Assay) and TK Ab-Cryptate detection antibody (from InVitrogen) were added to stop the reaction.3) The 384-well plate was sealed and incubated for 1 hour. The fluorescence was then measured at 620 nM (Cryptate) and 665 nM (XL655) wavelength.4) Each concentration of compound was done in triplicate, and vehicle (without compound) and a positive control were used.
Measurement: IC50 = 0.54 nM (pChEMBL 9.270000457763672)
Descriptors: MW 526.01, ALogP 5.03, HBA 7, HBD 2, PSA 88.61, QED 0.36