lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1noc(C)c1Cn1cc(NC(=O)c2ccc3c(c2)OCO3)cn1
InChIKey: PDPLBSHKAXBPBW-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 340.34, ALogP 2.52, HBA 7, HBD 1, PSA 91.41, QED 0.78
Compound (SMILES): COc1cc(C(=O)N2CCC(=O)Nc3ccccc32)cc(-c2ccccc2)c1
InChIKey: KVWYSEGVHJLHNS-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Inhibition of human His-tagged BRD4 BD1 (49 to 170 residues) using H-SGRGK(Ac)GGK(Ac)GLGK-(Ac)GGAK(Ac)RHRK(Biotin)-OH as substrate preincubated for 30 mins followed by substrate addition measured after 30 mins by TR-FRET assay
Measurement: IC50 = 2740 nM (pChEMBL 5.559999942779541)
Descriptors: MW 372.42, ALogP 4.35, HBA 3, HBD 1, PSA 58.64, QED 0.74
Compound (SMILES): COc1cc(N2CCN(S(C)(=O)=O)CC2)ccc1Nc1ncc(Cl)c(Nc2ccc(N3CCN(C(C)=O)CC3)cc2OC)n1
InChIKey: RMGHGBAKDGPFGP-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 645.19, ALogP 3.38, HBA 11, HBD 2, PSA 132.47, QED 0.36
Compound (SMILES): COc1cc(N(C)CCCO)ccc1Nc1ncc2c(n1)N(C1CCCC1)CCC(=O)N2C
InChIKey: PPPDATIUXAGJGR-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase PLK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53350
Assay: In Vitro Plk1Binding Assay: In vitro Plk1 binding assay: Ambit Kd values in nanomolar. Kd values generated by Ambit binding assay over a concentration range of the compound.
Measurement: Kd = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 454.58, ALogP 3.16, HBA 8, HBD 2, PSA 94.06, QED 0.63
Compound (SMILES): O=C(Nc1cccc(C(F)(F)F)c1)NC(c1nc2ccccc2c(=O)n1-c1ccc(F)cc1)C1CC1
InChIKey: QURHJZNZUKVMFY-UHFFFAOYSA-N
Target: Sonic hedgehog protein (SINGLE PROTEIN), Mus musculus
UniProt: Q62226
Assay: Gli-Luc Assay: Compounds may be tested in the "Gli-Luc" assay below, using the cell line 10T(s12), wherein the cells contain a Hedgehog-responsive reporter construct utilizing Luciferase as the reporter gene. In this way, Hedgehog pathway signaling activity can be measured via the Gli-Luc response.10t1/2(s12) cells are plated in a 96-well micro-titer plate (MTP) at 20,000 cells/well in full medium [DMEM with 10% FBS]. Then plates are placed in the incubator for incubation overnight (O/N), at 37° C. and 5% CO2. After 24 h, the medium is replaced with Luciferase-assay medium (DMEM with 0.5% FBS). Compounds are thawed and diluted in assay medium at 3:1000 (about 300-fold) resulting in a starting concentration of about 30 uM.Subsequently, 150 ul of each 30 uM sample is added to the first wells (in triplicate). The MTP samples are then diluted at 3-fold dilutions to a total of seven wells, ultimately resulting in a regiment of seven dilutions in triplicate, for each compound.
Measurement: IC50 < 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 496.46, ALogP 5.82, HBA 4, HBD 2, PSA 76.02, QED 0.34
Compound (SMILES): CCC[C@H](c1ccc(C(=O)NCCC(=O)O)cc1)C(c1ccc(Cl)cc1)c1c(C)sc2c(C#N)cc(Cl)cc12
InChIKey: IJVMJWMXQKUNRV-MIJJZIGMSA-N
Target: Glucagon receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P47871
Assay: Binding Assay: A stable CHO (Chinese hamster ovary) cell line expressing cloned human glucagon receptor was maintained as described (Chicchi, et. al. J Biol Chem 272, 7765-9 (1997); Cascieri, et. al. J Biol Chem 274, 8694-7 (1999)). To determine antagonistic binding affinity of compounds, 0.001-0.003 mg of cell membranes from these cells were pre-incubated with 0.100 mg WGA-coated PVT SPA beads (Amersham) for 20 minutes at room temperature in 25 μL of a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM EDTA, 0.1% BSA and 3% glycerol in Costar 384 well plates with clear bottoms (#3706). Next, 25 μL of 125I-Glucagon (New England Nuclear, MA) (1×10−14 mol per well) and either 1 μL solutions of test compounds or 0.001 mM unlabeled glucagon or DMSO were added and mixed. After 4-12 hours incubation at room temperature, the radioactivity bound to the cell membranes was determined in a radioactive emission detection counter (Wallac-Microbeta).
Measurement: IC50 = 7.8 nM (pChEMBL 8.109999656677246)
Descriptors: MW 579.55, ALogP 8.31, HBA 4, HBD 2, PSA 90.19, QED 0.2
Compound (SMILES): O=C(O)c1ccc(-c2c([C@@H]3C[C@H]3c3ccc4ccccc4n3)nc3c(N4CCOCC4)cc(Cl)nn23)cc1F
InChIKey: KIFBSRZFEYQFPU-WOJBJXKFSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6Ã¿His sequence following the start Met to allow metal affinity purification of the recombinant protein. Cells were harvested and the phosphodiesterase protein was purified by metal chelate chromatography on Ni-sepharose 6FF.The affinity of the compounds of Formula (I) for phophodiesterases (PDE) was measured by a scintillation proximity assay (SPA). PDE Yttrium Silicate SPA beads allow PDE activity to be measured by direct binding of the primary phosphate groups of non-cyclic AMP or GMP to the beads via a complex iron chelation mechanism. The amount of bound tritiated product ([3H]-AMP) is measured by liquid scintillation counting.The compounds were dissolved and diluted in 100% DMSO in polystyrene plates to a concentration of 100-fold the final concentration in the assay. Human PDE10A enzyme solution (10 uL) was added.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 543.99, ALogP 5.54, HBA 7, HBD 1, PSA 92.85, QED 0.31
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN2CSC[C@H]2CO1
InChIKey: RFGYFZFUMBQSCW-GICMACPYSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 493.42, ALogP 4.84, HBA 8, HBD 1, PSA 68.74, QED 0.52
Compound (SMILES): COc1cc(OC)cc(N(CCNC2CCN(S(N)(=O)=O)CC2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: RDIKEQDHARQJCT-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 123.03 nM (pChEMBL 6.909999847412109)
Descriptors: MW 566.69, ALogP 2.44, HBA 10, HBD 2, PSA 140.73, QED 0.3
Compound (SMILES): O=C(NCc1ccc(S(=O)(=O)c2ccc(N3CCOCC3)nc2)cn1)c1cnc2[nH]ncc2c1
InChIKey: OAJCZGUQCAZJJT-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT Enzymatic Reaction. The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mixture was incubated for 15 min at rt, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 pM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 min at rt, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 479.52, ALogP 1.35, HBA 9, HBD 2, PSA 143.06, QED 0.42
Compound (SMILES): CC(C)(O)c1cn(-c2ccc(-c3cc(F)c(CO)c(S(C)(=O)=O)c3)cc2F)c(C(C)(C)c2c(F)cccc2Cl)n1
InChIKey: ZXGHHBLRYXFVLD-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha/oxysterols receptor LXR-beta (PROTEIN COMPLEX), Homo sapiens
UniProt: P19793
Assay: Displacement of [3H]-24,25-epoxycholesterol from human LXRbeta/RXRalpha expressed in baculovirus infected Sf9 cells by scintillation proximity analysis
Measurement: Ki = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 593.07, ALogP 6.06, HBA 6, HBD 2, PSA 92.42, QED 0.27
Compound (SMILES): Cc1cc(C2CCC2)c(-c2nc3c([nH]2)CCN(C)CC3)cc1C(=O)N1CC(c2ccc(C#N)cc2)C1
InChIKey: CLYVHERPOJMOOT-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 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 479.63, ALogP 4.79, HBA 4, HBD 1, PSA 76.02, QED 0.58
Compound (SMILES): Nc1ccccc1NC(=O)CCCCCNC(=O)c1ccccc1C(=O)c1ccccc1
InChIKey: YGDNLQLWEQPVNP-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 2720 nM (pChEMBL 5.570000171661377)
Descriptors: MW 429.52, ALogP 4.43, HBA 4, HBD 3, PSA 101.29, QED 0.25
Compound (SMILES): CCn1cc(-c2oc3cc(N(C)S(C)(=O)=O)c(-c4ccc5c(n4)-c4cc6c(F)cccc6n4CC5)cc3c2C(=O)NC)cn1
InChIKey: YTRLDDUGKGAGCL-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 9.129 nM (pChEMBL 8.039999961853027)
Descriptors: MW 612.69, ALogP 5.45, HBA 8, HBD 1, PSA 115.26, QED 0.27
Compound (SMILES): C[C@H](n1cnnc1-c1nc(NC(=O)c2cc(-n3cnc(C4CC4)c3)c(N3CCC(C#N)CC3)cn2)cs1)C(F)(F)F
InChIKey: VPHMCGNTEDIZJX-HNNXBMFYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: Time-Resolved Fluorescence Resonance energy transfer (TR-FRET) Immunoassay: The assay measures the phosphorylation level of a biotinylated peptide substrate by the ASK1 kinase using HTRF detection. This is a competitive, time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay, based on HTRF KinEASE-STK manual from Cisbio. Test compound, 1 uM STK3 peptide substrate, 4 nM of ASK1 kinase were incubated with 10 mM MOP buffer, pH. 7.0 containing 10 mM Mg-acetate, 0.025% NP-40, 1 mM DTT, 0.05% BSA and 1.5% glycerol for 30 minutes then 100 uM ATP was added to start the kinase reaction and incubated for 3 hr. Peptide antibody labeled with 1xEu3+ Cryptate buffer containing 10 mM EDTA and 125 nM Streptavidin XL665 were added to stop the reaction and phosphorylated peptide substrate was detected using Envision 2103 Multilabeled reader from PerkinElmer. The fluorescence was measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm was calculated for each well.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 582.62, ALogP 4.98, HBA 11, HBD 1, PSA 130.44, QED 0.32
Compound (SMILES): CC(C)(C)OC(=O)N1C[C@@H](Oc2ccc(-c3ccccc3)cc2)C[C@H]1C(=O)N[C@@H](Cc1ccc(OCc2ccccc2)cc1)C(=O)NS(=O)(=O)c1ccc(Cl)c([N+](=O)[O-])c1
InChIKey: GYERYWYSSBUXHI-LUYPYDKDSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Inhibition of 5-FAM-QEDIIRNIARHLAQVGDSMDRSIPPG binding to Mcl-1 (unknown origin) preincubated for 30 mins followed by FAM-labeled peptide addition measured after 20 mins by fluorescence polarization assay
Measurement: Ki = 780 nM (pChEMBL 6.110000133514404)
Descriptors: MW 855.37, ALogP 7.48, HBA 10, HBD 2, PSA 183.48, QED 0.08
Compound (SMILES): Nc1nnc(-c2ccc3[nH]cc(-c4cncc(NC5CCCC5)n4)c3c2)o1
InChIKey: HFUZHVLXCKATBR-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 1.009 nM (pChEMBL 9)
Descriptors: MW 361.41, ALogP 3.61, HBA 7, HBD 3, PSA 118.54, QED 0.51
Compound (SMILES): O=C(NC1CCC1)c1cccc(-c2n[nH]c3ccc(-c4nc[nH]n4)cc23)c1
InChIKey: LOWJUWIPVBPXKY-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 <= 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 358.41, ALogP 3.3, HBA 4, HBD 3, PSA 99.35, QED 0.52
Compound (SMILES): O=C(c1cc(Cc2n[nH]c(=O)c3c2NCCC3)ccc1F)N1CCC(O)CC1
InChIKey: WRDSIDFKRBNIBF-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 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 386.43, ALogP 1.45, HBA 5, HBD 3, PSA 98.32, QED 0.74
Compound (SMILES): O=C(O)COc1ccc(Cl)cc1C1c2scnc2CCN1C(=O)OCc1ccccc1
InChIKey: QNGMDXQNDSIYSW-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 μl. First, 25 μl of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 μl Binding-Buffer, 50 μl of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 μl CRTH2 membrane fragments, reaching a final concentration of 20 μg/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 μl of Microscint-40 (Packard) was added to each well and the re
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 458.92, ALogP 4.54, HBA 6, HBD 1, PSA 88.96, QED 0.58
Compound (SMILES): COC(=O)N[C@@H](C)CNc1nccc(-c2cn(C(C)C)nc2-c2c(F)ccc(NS(C)(=O)=O)c2F)n1
InChIKey: MKCVFAPLFKXTKT-ZDUSSCGKSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf V600E mutant (unknown origin) using biotinylated Mek substrate incubated for 60 mins by amplified luminescence proximity homogeneous assay
Measurement: IC50 = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 523.57, ALogP 3.39, HBA 9, HBD 3, PSA 140.13, QED 0.39
Compound (SMILES): c1ccc(OCCNCC2CSC3(CCCCC3)S2)cc1
InChIKey: IPRUKNODGOFJGL-UHFFFAOYSA-N
Target: Alpha-1A adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35348
Assay: Displacement of [3H]prazosin from recombinant human alpha1a adrenergic receptor expressed in CHO cell membranes after 30 mins by TopCount liquid scintillation counting method
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 323.53, ALogP 4.16, HBA 4, HBD 1, PSA 21.26, QED 0.79
Compound (SMILES): O=C(CN1CCN(c2ccnc3cc(Cl)ccc23)CC1)NCc1ccccn1
InChIKey: WWZVEBSYTZIRFV-UHFFFAOYSA-N
Target: DNA gyrase subunit B (SINGLE PROTEIN), Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
UniProt: A0QNE0
Assay: Inhibition of Mycobacterium smegmatis 155 6His-tagged DNA gyrase B catalytic domain ATPase activity expressed in Escherichia coli BL21 (DE3) pLysS cells assessed as inorganic phosphate release after 120 mins in presence of ATP by malachite green dye based assay
Measurement: IC50 = 12200 nM (pChEMBL 4.909999847412109)
Descriptors: MW 395.89, ALogP 2.72, HBA 5, HBD 1, PSA 61.36, QED 0.72
Compound (SMILES): Nc1ncc(C2CCOCC2)nc1-c1ccc(C(=O)N[C@@H]2CCCN(S(=O)(=O)c3ccccc3)C2)c(F)c1
InChIKey: HHUMMTOMWKWWQT-HXUWFJFHSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Kinase Assay (New): Compound potency against activated ERK2 is 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 is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 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 is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is 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, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions are 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 are determined using a four-parameter fit.
Measurement: IC50 = 45.9 nM (pChEMBL 7.340000152587891)
Descriptors: MW 539.63, ALogP 3.34, HBA 7, HBD 2, PSA 127.51, QED 0.49
Compound (SMILES): COc1cc(CN(CC(C)C)C(CC(=O)O)c2ccc(Cl)c(F)c2)ccc1OCCN1C(=O)CCC1=O
InChIKey: KDRDXBGSLNQNGZ-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 535.01, ALogP 4.69, HBA 6, HBD 1, PSA 96.38, QED 0.37
Compound (SMILES): CC[C@@H]1CN(c2scnc2-c2nc3ccccc3[nH]2)CCN1C(=O)Cn1cnc2cccnc21
InChIKey: AXOCJQGAWLLMQP-MRXNPFEDSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.015%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 nM, is also is used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 472.58, ALogP 3.56, HBA 8, HBD 1, PSA 95.83, QED 0.42
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(Cc3ccc(F)c(C#N)c3)CC2)C1
InChIKey: QANLPBPAEYOOIU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 495.57, ALogP 3.42, HBA 8, HBD 1, PSA 113.45, QED 0.44
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)nc(NCc4nccs4)nc23)N1
InChIKey: OIVNGMFAALMIET-YHYXMXQVSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 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; 75 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 the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 397.42, ALogP 0.95, HBA 10, HBD 4, PSA 138.23, QED 0.36
Compound (SMILES): C=CC(=O)Nc1cccc(-c2cnc3[nH]cc(-c4cccc(S(=O)(=O)N(C)C(C)(C)C)c4)c3c2)c1
InChIKey: RGEOJTBGCSOIGD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: In Vitro Inhibition Assay: ITK and JAK3 Kinase assay procedures:Enzyme was incubated with substrate peptide in reaction buffer in the presence and absence of test compounds or Staurosporine. All additions were done on ice, followed by the addition of ATP mix. Wells were uniformly mixed using an Eppendorff plate shaker and incubated at 30° C. for 20 min, and stopped by the addition of 5 μL of 3% phosphoric acid. Volume was increased to 100 μL by adding 0.8% phosphoric acid which was then transferred to PC filter mats (Millipore), pre-equilibrated with 70% ethanol and water. Plates were washed thrice with 100 μL 0.8% phosphoric acid and dried for an hour at 60° C. 100 μL scintillation fluid was added into each well and reading taken in Perkin Elmer TOPCOUNT beta counter. The data analysis was performed by averaging the duplicate top count readings for each standard, negative, positive control (enzyme control) and samples and subtracting the average negative control from each reading which results in corrected values.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 488.61, ALogP 5.44, HBA 4, HBD 2, PSA 95.16, QED 0.35
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)c1ccc(N2C[C@H](O)[C@@H](O)C2)c(-c2cncnc2)c1
InChIKey: WKZIKPYEJBZHNQ-PMACEKPBSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Radio ABL1 (64-515) Assay: For determination of ABL kinase activity, the radiometric filter-binding assay was used. The assay was performed by mixing 10 uL of the compound pre-diluted with 10 uL of ATP (20 uM ATP with 0.1 uCi [γ-33P]-ATP) with the phospho-acceptor peptide poly[Ala6Glu2LysHBr5Tyr1]=polyAEKY) in 20 mM Tris/HCl pH 7.5, 1 mM DTT, 10 mM MgCl2, 0.01 mM Na3VO4, 50 mM NaCl. 10 uL of enzyme (ranging between 5 nM to 20 nM) was added to initiate the reaction. Pre-incubation of enzyme with compounds (when stated) was performed by exposing the enzyme to compounds prior to addition of the substrate mixture (ATP and/or peptide substrate). After 15 mM at room temperature, the reaction was stopped by the addition of 50 uL 125 mM EDTA, and the peptide-bound 33P separated on filter-plates (PVDF or MAIP; Millipore, Volketswil, Switzerland) prepared according to the manufacturer's instructions. Filter-plates were washed 3x with 0.5% H3PO4, followed by addition of 30 uL scintillation cocktail (Microscint, Perkin Elmer) per well and then analysed in a TopCount NXT scintillation counter (Perkin Elmer).
Measurement: IC50 < 3 nM (pChEMBL 8.52299976348877)
Descriptors: MW 460.41, ALogP 2.84, HBA 7, HBD 3, PSA 107.81, QED 0.54
Compound (SMILES): Cn1c(=O)n(CC(C)(C)C)c2ccc(C3=CCC(C(=O)NCc4ncco4)CC3)nc21
InChIKey: JMKYLIZUPROHTI-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Fluorescence Laser Imaging Plate Reader (FLIPR) Based Assay: The compounds of the present invention may be tested in a fluorescence laser imaging plate reader (FLIPR) based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr-cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with dose responses of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity. The potentiation response is monitored after a subsequent addition of an EC20 concentration of glutamate (900 nM).
Measurement: EC50 = 594.1 nM (pChEMBL 6.230000019073486)
Descriptors: MW 423.52, ALogP 3.27, HBA 7, HBD 1, PSA 94.95, QED 0.68
Compound (SMILES): N#Cc1ccc(CNc2n[nH]c3ccc(-c4cn(Cc5ccccc5)nn4)cc23)cc1
InChIKey: CGLZEAOJMVZEMM-UHFFFAOYSA-N
Target: Cell division cycle 7-related protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: O00311
Assay: Inhibition Assay: 11 data point. Cdc7 kinase assays were carried out in 25 mM HEPES, pH 7.5, 1 mM DTT, 10 mM MgCl2, 100 μM Na3VO4, and 0.075 mg/ml Triton X-100 using 12 ng baculovirus-expressed Cdc7/Dbf4 and 2 μM Jerini peptide substrate A-A11 (biotin-C6linker-TPSDSLIYDDGLS) (SEQ ID NO:5). Reactions were initiated with λ-[33P]-ATP (1 μM, 20 mCi/μmol) and quenched after 1 hour with 5 volumes of stop buffer (50 mM EDTA, 2 M NaCl). The reactions were incubated for 30 minutes on streptavidin-coated plates, washed, and quantified using a TopCount scintillation plate reader (Packard). IC50 values are determined via non-linear regression fitting of the data. Ki values are generated assuming ATP-competitive (equilibrium) inhibition and using the experimentally determined apparent ATP Km of Cdc7 (0.7 μM).
Measurement: Ki = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 405.47, ALogP 4.35, HBA 6, HBD 2, PSA 95.21, QED 0.44
Compound (SMILES): CCOC(=O)C1=C(C)N(c2cccc(C(F)(F)F)c2)C(=O)N(C)C1c1ccc(C#N)cc1C(=O)N1CCN2CCCC2C1
InChIKey: NDEVQXWGQNRAHY-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available.The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA. Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 uL, of these compound dilutions were mixed with 10 uL Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 uL, substrate solution in assay buffer were added (250 uM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 595.62, ALogP 4.95, HBA 6, HBD 0, PSA 97.19, QED 0.45
Compound (SMILES): CC(C)n1cc(C(=O)c2ccnc(NC(=O)Cn3cnnn3)c2)c2cnccc21
InChIKey: GBQOCKJMPOOLAW-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: HTRF Assay: Isolated TRK Enzyme assays use the HTRF KinEASE-TK kit (Cisbio Cat#62TK0PEJ) with recombinant His-tagged cytoplasmic domains of each TRK receptor sourced from Invitrogen (see table below). This activity-assay measures the phosphorylation of tyrosine residues within a substrate from the HTRF kit which has been validated by Cisbio for a variety of tyrosine kinases including the TRK receptors.Assay Details: Invitrogen Amino FAC FAC Assay ReactionTarget Cat# acids enzyme ATP TimeTRKA PV3144 aa 441-4 nM 40 uM 35 min (NTRK1) 796 TRKB PV3616 aa 526-1 nM 1.4 uM 40 min (NTRK2) 838 TRKC PV3617 aa 510- 10 nM 15 uM 30 min (NTRK3) 8250.5 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135 disclosed in WO2005/116035 of 150 uM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 uM.
Measurement: IC50 = 6510 nM (pChEMBL 5.190000057220459)
Descriptors: MW 390.41, ALogP 1.87, HBA 9, HBD 1, PSA 120.48, QED 0.5
Compound (SMILES): COc1ccc(CCNC(=O)c2ccc(NC(=O)N3Cc4cccnc4C3)cc2)cc1OC
InChIKey: RTRWENWQHAWRRC-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 = 35.4 nM (pChEMBL 7.449999809265137)
Descriptors: MW 446.51, ALogP 3.62, HBA 5, HBD 2, PSA 92.79, QED 0.58
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(-c3ccc4[nH]cc(C)c4c3)cc1C21N=C(C)C(N)=N1
InChIKey: RJIJEEIOHDLRFZ-MJPADRBUSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: TR-FRET Assay: The β-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human β-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc 10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in −80° C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 μg/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 μM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 μl) was added to the plate then 1 μl of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 μl) was then added, mixed and the reaction proceeded for 15 minutes at r.t. The reaction was stopped with the addition of Stop solution (7 μl, NaAcetate, pH 9). The fluorescence of the product was measured on a Victor II plate reader with an excitation wavelength of 340 nm and an emission wavelength of 615 nm. The assay was performed in a Costar 384 well round bottom, low volume, non-binding surface plate (Corning #3676). The final concentration of the enzyme was 2.7 g/ml; the final concentration of substrate was 100 nM (Km of 250 nM). The dimethylsulphoxide control, instead of test compound, defined the 100% activity level and 0% activity was defined by wells lacking enzyme (replaced with reaction buffer). A control inhibitor was also used in dose response assays and had an IC50 of 150 nM.
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 426.56, ALogP 5.26, HBA 4, HBD 2, PSA 75.76, QED 0.59
Compound (SMILES): O=S(=O)(c1ccc(Cl)nc1)N(Cc1ccc(F)c(C(F)(F)F)c1)c1sc2ccccc2c1Cl
InChIKey: UCGAHLBFTDBSDR-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 535.37, ALogP 7.16, HBA 4, HBD 0, PSA 50.27, QED 0.2
Compound (SMILES): O=C(Nc1nc2ccccc2s1)c1cccc2c1CN(c1ccc(-c3cnn(CC4CCCOC4)c3)c(C(=O)O)n1)CC2
InChIKey: RYLYKMUOJBVPPS-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 594.7, ALogP 5.49, HBA 9, HBD 2, PSA 122.47, QED 0.25
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(F)cc3F)CC2)C1
InChIKey: GXXIRGRFCRZVEY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 502.53, ALogP 3.33, HBA 7, HBD 1, PSA 106.73, QED 0.45
Compound (SMILES): CC(C)Nc1cc(Nc2ccc(C#N)cc2)ncc1-c1nnc(NC[C@@H](F)CO)o1
InChIKey: RZCXAFDYADYWET-CQSZACIVSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL 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% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction 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 = 22.9 nM (pChEMBL 7.639999866485596)
Descriptors: MW 411.44, ALogP 3.31, HBA 9, HBD 4, PSA 131.92, QED 0.42
Compound (SMILES): NC(=O)C12CC3CC(C1)C(NC(=O)c1cccc(NS(=O)(=O)c4ccccc4Cl)c1)C(C3)C2
InChIKey: KFMQHJITHKCHGD-UHFFFAOYSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: HTRF Assay : The inhibiting activity of 11β-HSD1 derived from microsomal fractions was measured using the HTRF assay (62CO2PEB, Cisbio). Different concentrations of compounds were added to 96-well plates, followed by the addition of TE buffer (20 mM Tris buffer and 5 mM EDTA, pH 6.0) containing 200 μM NADPH (N1630, Sigma) and 160 nM cortisone (C2755, Sigma), The reactions were initiated by the addition of human microsomal fractions (M0317, Sigma), and were allowed to incubate for 2 h at 37° C. Europium (Eu3+) cryptate and XL665-conjugated cortisol were then added to each well and incubated for an additional 2 h at room temperature.
Measurement: IC50 = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 488.01, ALogP 3.55, HBA 4, HBD 3, PSA 118.36, QED 0.58
Compound (SMILES): COc1cccc(Cn2nc(-c3cccc(Cl)c3)c3c2CCN(C(C)=O)C3)c1
InChIKey: PSJXUKRBYYNTCL-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: Biological 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 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: NaCl 96 mM, KCl 2 mM, CaCl2 1.8 mM, MgCl2 1 mM, HEPES 5 mM (pH adjusted to 7.4 with NaOH). All experiments were performed at room temperature.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 395.89, ALogP 4.17, HBA 4, HBD 0, PSA 47.36, QED 0.67