lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C(=C/c1n[nH]c2cc(-c3ccncc3)ccc12)\c1ccccc1
InChIKey: MMAITFLGUFEIIV-RMKNXTFCSA-N
Target: CDK4/Cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P11802
Assay: Inhibition of human CDK4/cyclin D3 expressed in Escherichia coli using retinoblastoma (386 to 928 residues) as substrate after 20 mins by gamma32P-ATP based scintillation counting analysis
Measurement: Ki = 780 nM (pChEMBL 6.110000133514404)
Descriptors: MW 297.36, ALogP 4.8, HBA 2, HBD 1, PSA 41.57, QED 0.59
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(C(N)=O)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: ZBMSILUYNLJEMN-UHFFFAOYSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42338
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 439.48, ALogP 2.48, HBA 8, HBD 2, PSA 134.71, QED 0.44
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4cc(O)cc(F)c4)c4c(N)ncnc43)n(-c3ccccc3F)c(=O)c12
InChIKey: LAYNQKZCFROFGL-UHFFFAOYSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42338
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 510.5, ALogP 4.72, HBA 8, HBD 2, PSA 111.85, QED 0.36
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4cn[nH]c4)c4c(N)ncnc43)n(-c3ccccc3Cl)c(=O)c12
InChIKey: WUMGKAVWTFXBSH-UHFFFAOYSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42338
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 482.94, ALogP 4.11, HBA 8, HBD 2, PSA 120.3, QED 0.39
Compound (SMILES): CC(=O)Nc1nc2cc(-c3nn(Cc4cc5cccc(C)c5c(=O)n4-c4ccccc4)c4ncnc(N)c34)ccc2o1
InChIKey: AGZWRHMGHQGYHA-UHFFFAOYSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42338
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 556.59, ALogP 4.84, HBA 10, HBD 2, PSA 146.75, QED 0.31
Compound (SMILES): Cc1cccc2cc([C@H](C)Nc3cc(N)nc(Cl)n3)n(-c3ccccc3)c(=O)c12
InChIKey: FUGXOIKZLQQLKY-AWEZNQCLSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42338
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 405.89, ALogP 4.5, HBA 6, HBD 2, PSA 85.83, QED 0.49
Compound (SMILES): CCOC(=O)C1CN(C(c2ccccc2)c2ccccc2)CCN1C(=O)CC(c1ccccc1)c1ccccc1
InChIKey: KKZWTCQQRNGVOY-UHFFFAOYSA-N
Target: Voltage-dependent P/Q-type calcium channel alpha1A/alpha2delta/beta1b (PROTEIN COMPLEX), Homo sapiens
UniProt: P54289
Assay: Antagonist activity at alpha1A/alpha2delta/beta1b P/Q-type calcium channel (unknown origin) expressed in HEK293 cells assessed as 0.1 Hz current frequency dependent channel blocking for 2 to 3 mins in presence of Ba2+ by whole-cell patch clamp assay
Measurement: IC50 = 5028 nM (pChEMBL 5.300000190734863)
Descriptors: MW 532.68, ALogP 6.07, HBA 4, HBD 0, PSA 49.85, QED 0.25
Compound (SMILES): Cc1cccc2cc(C(C)Nc3ncnc4[nH]cnc34)n(C3CC3)c(=O)c12
InChIKey: XKHASMAALGBCSO-UHFFFAOYSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42338
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 360.42, ALogP 3.48, HBA 6, HBD 2, PSA 88.49, QED 0.58
Compound (SMILES): COc1ccccc1S(=O)(=O)Nc1ccc(-c2nc3cc(NS(=O)(=O)c4ccccc4OC)ccc3o2)cc1
InChIKey: GQPVWDHUHXFDMI-UHFFFAOYSA-N
Target: GroEL/GroES (PROTEIN COMPLEX), Escherichia coli
UniProt: Q548M1
Assay: Inhibition of Escherichia coli GroEL/ES-mediated refolding of the denatured rhodanese
Measurement: IC50 = 33000 nM (pChEMBL 4.480000019073486)
Descriptors: MW 565.63, ALogP 5.11, HBA 8, HBD 2, PSA 136.83, QED 0.25
Compound (SMILES): C(=C/c1n[nH]c2cc(-c3ccncc3)ccc12)\c1ccccc1
InChIKey: MMAITFLGUFEIIV-RMKNXTFCSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P11802
Assay: Inhibition of human GST-tagged CDK4/cyclin D1 expressed in Escherichia coli using retinoblastoma (386 to 928 residues) as substrate after 20 mins by gamma32P-ATP based scintillation counting analysis
Measurement: Ki = 780 nM (pChEMBL 6.110000133514404)
Descriptors: MW 297.36, ALogP 4.8, HBA 2, HBD 1, PSA 41.57, QED 0.59
Compound (SMILES): CCCc1nn(C)c2c(=O)[nH]c(-c3cc(N(C)C4CCN(c5ncc(C(=O)NO)cn5)CC4)ccc3OCC)nc12
InChIKey: MXAAVNPVSLHVKO-UHFFFAOYSA-N
Target: Histone deacetylase 3/Nuclear receptor corepressor 2 (HDAC3/NCoR2) (PROTEIN COMPLEX), Homo sapiens
UniProt: O15379
Assay: Inhibition of full length recombinant human C-terminal His-tagged HDAC3/N-terminal GST-tagged NCOR2 (395 to 489 residues) expressed in baculovirus infected Sf9 cells using fluorogenic HDAC substrate after 30 mins by fluorescence analysis
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 561.65, ALogP 2.69, HBA 11, HBD 3, PSA 154.39, QED 0.21
Compound (SMILES): CCOP(=O)(O)c1ccc2cc1CN(C)C(=O)[C@H](Nc1ccc3c(N)nccc3c1)c1ccc(c(C)c1)[C@@H](C)COC(=O)N2
InChIKey: RCTSRSYRSFZKIR-AFJIDDCJSA-N
Target: Coagulation factor VII/tissue factor (PROTEIN COMPLEX), Homo sapiens
UniProt: P08709
Assay: Inhibition of full-length human TF/recombinant human factor 7a assessed as decrease in conversion of factor 10 to factor 10a by measuring S2765 hydrolysis after 15 mins
Measurement: Ki = 0.28 nM (pChEMBL 9.550000190734863)
Descriptors: MW 617.64, ALogP 5.45, HBA 8, HBD 4, PSA 156.11, QED 0.21
Compound (SMILES): COc1ccccc1S(=O)(=O)Nc1ccc(-c2nc3cc(NS(=O)(=O)c4ccccc4OC)ccc3o2)cc1
InChIKey: GQPVWDHUHXFDMI-UHFFFAOYSA-N
Target: HSP60/HSP10 (PROTEIN COMPLEX), Homo sapiens
UniProt: P10809
Assay: Inhibition of human mitochondrial HSP60/10-mediated refolding of the denatured malate dehydrogenase
Measurement: IC50 = 87000 nM (pChEMBL 4.059999942779541)
Descriptors: MW 565.63, ALogP 5.11, HBA 8, HBD 2, PSA 136.83, QED 0.25
Compound (SMILES): CCOC(=O)C1CN(C(c2ccccc2)c2ccccc2)CCN1C(=O)CC(c1ccccc1)c1ccccc1
InChIKey: KKZWTCQQRNGVOY-UHFFFAOYSA-N
Target: Voltage-dependent N-type calcium channel alpha1B/alpha2b/beta1b (PROTEIN COMPLEX), Rattus norvegicus
UniProt: Q02294
Assay: Antagonist activity at rat brain alpha1B/alpha2b/beta1b N-type calcium channel expressed in HEK293 cells assessed as 0.1 Hz current frequency dependent channel blocking for 2 to 3 mins in presence of Ba2+ by whole-cell patch clamp assay
Measurement: IC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 532.68, ALogP 6.07, HBA 4, HBD 0, PSA 49.85, QED 0.25
Compound (SMILES): CCOC(=O)C1CN(C(c2ccccc2)c2ccccc2)CCN1C(=O)CC(c1ccccc1)c1ccccc1
InChIKey: KKZWTCQQRNGVOY-UHFFFAOYSA-N
Target: Voltage-dependent P/Q-type calcium channel alpha1A/alpha2delta/beta1b (PROTEIN COMPLEX), Homo sapiens
UniProt: O00555
Assay: Antagonist activity at alpha1A/alpha2delta/beta1b P/Q-type calcium channel (unknown origin) expressed in HEK293 cells assessed as 0.1 Hz current frequency dependent channel blocking for 2 to 3 mins in presence of Ba2+ by whole-cell patch clamp assay
Measurement: IC50 = 5028 nM (pChEMBL 5.300000190734863)
Descriptors: MW 532.68, ALogP 6.07, HBA 4, HBD 0, PSA 49.85, QED 0.25
Compound (SMILES): CCS(=O)(Cc1cc(F)cc(Nc2ncc(F)c(-c3ccc(F)cc3OC)n2)c1)=NC#N
InChIKey: DKGCKUUOPCFNNH-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: CDK9/CycT1 Kinase Assay: Recombinant full-length His-tagged human CDK9 and CycT1, expressed in insect cells and purified by Ni-NTA affinity chromatography, were purchased from Invitrogen (Cat. No PV4131). As substrate for the kinase reaction biotinylated peptide biotin-Ttds-YISPLKSPYKISEG (C-terminus in amid form) was used which can be purchased e.g. form the company JERINI Peptide Technologies (Berlin, Germany) For the assay 50 nl of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 ul of a solution of CDK9/CycT1 in aqueous assay buffer [50 mM Tris/HCl pH 8.0, 10 mM MgCl2, 1.0 mM dithiothreitol, 0.1 mM sodium ortho-vanadate, 0.01% (v/v) Nonidet-P40 (Sigma)] were added and the mixture was incubated for 15 min at 22 C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 461.47, ALogP 4.78, HBA 7, HBD 1, PSA 100.26, QED 0.51
Compound (SMILES): COc1cccc(OC)c1C1CC(C)C(=O)N1Cc1cccc(-c2csc(C)n2)c1
InChIKey: LRUWAFBAMKJJRR-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 422.55, ALogP 5.25, HBA 5, HBD 0, PSA 51.66, QED 0.54
Compound (SMILES): CCS(=O)(=O)N(Cc1ccc(F)c(F)c1)c1sc2ccccc2c1C(C)(C)O
InChIKey: BJDMENFYEXGMTK-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 425.52, ALogP 4.76, HBA 4, HBD 1, PSA 57.61, QED 0.62
Compound (SMILES): CC(C)Oc1ccc([C@H](C)Nc2nccc(N3C(=O)OC[C@@H]3[C@@H](C)O)n2)cc1
InChIKey: OJWQIUREUZEYHW-JJRVBVJISA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 358 nM (pChEMBL 6.449999809265137)
Descriptors: MW 386.45, ALogP 3.14, HBA 7, HBD 2, PSA 96.81, QED 0.75
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2[C@H](Cn3cc(-c4ccc(F)cn4)cn3)OC[C@@H]2C)c1
InChIKey: HROJDHGISWMAPH-AOMKIAJQSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Antagonistic Activity Assay: The antagonistic activities of the test compounds on human orexin-1 receptor (hOX1R) and orexin-2 receptor (hOX2R) were measured by modifying from the method described in literature (Toshikatsu Okumura et al., Biochemical and Biophysical Research Communications 280, 976-981, 2001). Chinese hamster ovary (CHO) cells forcibly expressing the hOX1R and hOX2R were seeded into a 96 well Black clear bottom plate (Nunc) at 20,000 cells per well, which were cultured in Ham's F-12 medium containing 0.1 mM MEM non-essential amino acids, 0.5 mg/ml G418, 10% fetal bovine serum (all by Invitrogen) for 16 hours under the conditions of 37° C., 5% CO2. After removing the medium, 100 uL of 0.5 uM Fluo-4AM ester (Dojin) in an assay buffer (25 mM HEPES (Dojin), Hank's balanced salt solution (Invitrogen), 0.1% bovine serum albumin, 2.5 mM probenecid, 200 ug/ml Amaranth (all by Sigma-Aldrich), pH 7.4) was added and the cells were incubated for 60 minutes at 37° C., 5% CO2. After removing the medium, 100 uL of 0.5 uM Fluo-4AM ester (Dojin) in an assay buffer (25 mM HEPES (Dojin), Hank's balanced salt solution (Invitrogen), 0.1% bovine serum albumin, 2.5 mM probenecid, 200 ug/ml Amaranth (all by Sigma-Aldrich), pH 7.4) was added and the cells were incubated for 60 minutes at 37° C., 5% CO2. After removing the assay buffer containing fluo-3AM ester, the test compound was dissolved in dimethyl sulfoxide to be 10 mM and diluted with the assay buffer, 150 uL of which was added and incubated for 30 minutes.
Measurement: IC50 = 200.6 nM (pChEMBL 6.699999809265137)
Descriptors: MW 447.47, ALogP 2.86, HBA 8, HBD 0, PSA 90.96, QED 0.47
Compound (SMILES): CC(C)CN1CCCC1c1ccc(NC(=O)Nc2ccccc2N2CC3(CCN(CC(C)(C)C)CC3)c3c(F)c(F)cc(O)c32)cc1
InChIKey: POOLLKUOHVAIGP-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 uL containing 15 fmol of P2Y1 receptor (1.7 ug of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 uM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium Chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 ug of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 uL of the aqueous soluttion was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKERSM (Amersham) Image Reader. Dose-response curves (IC50) were fit by non-linear regression (Toolset an in house data processing program) and binding constants (Ki) calculated using the Cheng-Prusoff relationship (Ki=IC50/(1+L/Kd) in which a Kd for 2MeS-ADP to the P2Y1 receptor was determined to be 14 nM.
Measurement: Ki = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 645.84, ALogP 8.64, HBA 5, HBD 3, PSA 71.08, QED 0.24
Compound (SMILES): O=C(NCC1COC1)c1cc(NC(=O)c2cc(-c3ncccc3F)ccc2Cl)n(-c2ccccc2)n1
InChIKey: NFKKQPLTSYTUGG-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 = 79.3 nM (pChEMBL 7.099999904632568)
Descriptors: MW 505.94, ALogP 4.36, HBA 6, HBD 2, PSA 98.14, QED 0.39
Compound (SMILES): CN(C)C(=O)/C(C#N)=C/c1cccc(Oc2cnc3[nH]ccc3n2)c1
InChIKey: WFGUWJCHAKVDEN-MDWZMJQESA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Phosphorylation Assay: Phosphorylation of the appropriate peptide substrate (fluorescently labeled Srctide) by recombinant JAK3 (catalytic domain, aa 781-1124) was measured in the presence and absence of different concentrations of test compounds. Test compound, enzyme, fluorescently labeled substrate, and cofactors (ATP and Mg2+) were combined in a well of a microtiter plate and incubated for 2 hours at 25° C. The composition of the reaction buffer was 100 mM HEPES pH 7.5, 5 mM MgCl2, 0.01% Triton-X 100, 0.1% Bovine Serum Albumin, and 1% DMSO. Enzyme concentration in the final reaction mixture was 0.5 nM while peptide substrate was at 1 uM. ATP was used at 1xKm concentration corresponding to 2 uM. Serial dilutions (3-fold steps) of compounds were prepared in DMSO. At the end of the incubation, the reaction was stopped by the addition of 20 mM EDTA. Substrate and product (i.e. phosphorylated substrate) were separated electrophoretically and both were quantified by fluorescence intensity.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 333.35, ALogP 2.75, HBA 5, HBD 1, PSA 94.9, QED 0.58
Compound (SMILES): CCOC(=O)/N=C(\N)c1ccc(CC2NCCn3c2nc2cc(C4(C(=O)N5CCCC5)CCCC4)ccc23)cc1
InChIKey: ATMALHVUQAHUDQ-UHFFFAOYSA-N
Target: Thrombin (SINGLE PROTEIN), Oryctolagus cuniculus
UniProt: Q28731
Assay: Inhibition of rabbit thrombin-induced platelet aggregation in rabbit platelet-rich plasma preincubated with rabbit liver microsomes for 4 hrs followed by addition to platelet
Measurement: IC50 = 88200 nM (pChEMBL 4.050000190734863)
Descriptors: MW 542.68, ALogP 4.22, HBA 6, HBD 2, PSA 114.84, QED 0.36
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: QOFVNRHULNZMOX-URURHIFTSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 2431.86
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)NC(C)(C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)NC(C)(C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: HEUQHBOLBXTWOQ-XFHYZCPMSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 2300.7
Compound (SMILES): O=[N+]([O-])c1ccc(CN2C[C@H](CO)[C@@H](O)[C@H](O)C2)cc1
InChIKey: PUVBDYZJWIEJKE-RAIGVLPGSA-N
Target: Lysosomal acid glucosylceramidase (SINGLE PROTEIN), Homo sapiens
UniProt: P04062
Assay: Enzyme Assay: To 2.5 μl of supernatant (in 96-well plates) was added 17.5 μl reaction buffer (citrate phosphate buffer, pH 4.5, no Triton X-100), and 50 μl of 4-methyl umbelliferone (4-MU)-labeled substrate, β-glucopyranoside, or a labeled negative controls α-glucopyranoside or α-galacatopyranoside). Plates were incubated at 370 for 1 hour, followed by the addition of 70 μl stop buffer (0.4 M glycine-NaOH, pH 10.6). Activity of GCase was determined by measuring the emission at 460 nm by exciting at 355 nm using a 1 second read time per well (Victor2 multilabel counter-Wallac).
Measurement: Ki = 18630 nM (pChEMBL 4.730000019073486)
Descriptors: MW 282.3, ALogP -0.26, HBA 6, HBD 3, PSA 107.07, QED 0.52
Compound (SMILES): Cc1nc(C)c(-c2csc(Nc3ccc(C(=O)Nc4ccc(Cl)cc4)cn3)n2)s1
InChIKey: SPEDBOZUFYBCIJ-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Antagonist activity at human recombinant TRPV4 expressed in CHO-K1 cells assessed as blockade of 1000 nM 4alphaPDD-induced calcium mobilization by FLIPR assay
Measurement: IC50 = 9.5 nM (pChEMBL 8.020000457763672)
Descriptors: MW 441.97, ALogP 5.93, HBA 7, HBD 2, PSA 79.8, QED 0.4
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1cnn(Cc2ccc(-c3ccncc3)cc2)c1
InChIKey: KCGGXHJBWUPJKT-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 412.5, ALogP 3.52, HBA 6, HBD 2, PSA 98.72, QED 0.5
Compound (SMILES): Cn1cc(-c2cc(C(N)=O)nc3cc(CN4CCOC(CC(F)(F)F)C4)ccc23)cn1
InChIKey: ATFSSOIYERKWIZ-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader 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 various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 433.43, ALogP 2.89, HBA 6, HBD 1, PSA 86.27, QED 0.67
Compound (SMILES): CC(=O)N[C@@H](C)c1ccc(OC2CCN(c3ccc(OCC(F)F)cc3)C2)cc1
InChIKey: RLJUAUANLFNJQS-ZDGMYTEDSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: 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, acetyl Coenzyme A (both 200 μM 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 = 360 nM (pChEMBL 6.440000057220459)
Descriptors: MW 404.46, ALogP 4.19, HBA 4, HBD 1, PSA 50.8, QED 0.72
Compound (SMILES): CN(C)c1nc(NCc2ccc(NC(=O)C3CCN(Cc4ccccc4)CC3)cc2)c2ccccc2n1
InChIKey: DBCOLASKGYSTCT-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf33.13 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 1767 nM (pChEMBL 5.75)
Descriptors: MW 494.64, ALogP 5.16, HBA 6, HBD 2, PSA 73.39, QED 0.35
Compound (SMILES): COc1cc(Nc2nc3cc(C4(O)c5ccccc5C(=O)N4c4ccccc4)ccc3[nH]2)cc(OC)c1
InChIKey: JEFOXOJBOIUZJE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 >= 10000 nM (pChEMBL 5)
Descriptors: MW 492.54, ALogP 5.18, HBA 6, HBD 3, PSA 99.71, QED 0.3
Compound (SMILES): Cc1ccc(CCn2c3c(c4ccccc42)C(=O)NCCC3)cc1
InChIKey: WVRIJJWQTMVCQA-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Equus caballus
UniProt: P81908
Assay: Inhibition of horse serum BChE pretreated for 20 mins followed by butyrylthiocholine iodide substrate addition measured for 5 mins by Ellman's method
Measurement: IC50 = 68200 nM (pChEMBL 4.170000076293945)
Descriptors: MW 318.42, ALogP 3.87, HBA 2, HBD 1, PSA 34.03, QED 0.78
Compound (SMILES): O=C(Nc1ccc(N2CCNCC2)cc1)c1ccc(Cl)cc1
InChIKey: IAGQUTLPQWNFTE-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 25.6 nM (pChEMBL 7.590000152587891)
Descriptors: MW 315.8, ALogP 3, HBA 3, HBD 2, PSA 44.37, QED 0.91
Compound (SMILES): COC(=O)Cc1ccc(Oc2cc3cc(-c4ccccn4)[nH]c3cc2C2CCC(=O)N2C)cc1
InChIKey: QKCSVBTZZNFVST-UHFFFAOYSA-N
Target: Hexokinase-4 (SINGLE PROTEIN), Homo sapiens
UniProt: P35557
Assay: Glucokinase-Activating Assay: To test the exemplified compounds, the following assay was employed. Recombinant human liver glucokinase was expressed as a FLAG fusion protein in E. coli, and purified on ANTIFLAG M2 AFFINITY GEL (Sigma). The assay was carried out at 30° C. in a 96-well plate. In the plate was distributed 69 ul each of assay buffer (25 mM Hepes Buffer: pH=7.2, 2 mM MgCl2, 1 mM ATP, 0.5 mM TNAD, 1 mM dithiothreitol), to which was added 1 ul of a DMSO solution of the compound or DMSO as control. Then, 20 ul of pre-ice-cooled enzyme mixture (FLAG-GK, 20 U/ml G6PDH) was distributed thereto, to which was added 10 ul of 25 mM glucose as substrate to initiate the reaction (final glucose concentration=2.5 mM). After starting the reaction, the absorbance at 405 nm was measured every 30 seconds for 10 minutes to evaluate the compound based on the initial increase for 5 minutes. FLAG-GK was added so that the increase of absorbance after 5 minutes fell between 0.05 to 0.1 in the presence of 1% DMSO. The OD values of the respective compounds were measured in the respective concentrations, wherein the OD value of DMSO as control is regarded as 100%. From the OD values at the respective concentrations, Emax (%, 2.5 mM Glu) and EC50 (nM, 2.5 mM Glu) were calculated and used as indicators of the GK activation capability of the compounds. According to the above assay, the GK activation capability of the exemplified compounds of the present invention was determined. The following table shows the results. Where different enantiomers of the same compound were tested, two numbers are provided. Where 3 numbers are provided, for example, Ex. #30, data for the racemic form is also shown.
Measurement: EC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 455.51, ALogP 5.03, HBA 5, HBD 1, PSA 84.52, QED 0.41
Compound (SMILES): Cc1cccc(NC(=O)Nc2ccc(C3CN=C4c5ccccc5NC(=O)N4C3)c(C)c2)c1
InChIKey: UXOPRZHXAFAIIL-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mLs per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mLs per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 2.7 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 μl per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mLs per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mLs per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 439.52, ALogP 5.34, HBA 3, HBD 3, PSA 85.83, QED 0.51
Compound (SMILES): CN1C(=O)N(c2cccc(C(F)(F)F)c2)C2=C(C(=O)CC2)C1c1ccc(C#N)cc1C(=O)NCc1nccn1C
InChIKey: JFRQDDQQYKSJSA-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: 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 μL of these compound dilutions were mixed with 10 μl 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 μL substrate solution in assay buffer were added (250 μM 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.
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 548.53, ALogP 4.47, HBA 6, HBD 1, PSA 111.33, QED 0.51
Compound (SMILES): Cc1c(C(=O)Nc2cccc(C(F)(F)F)c2)cc(C2CCN(C(=O)OCc3ccccc3)CC2)n1CCCN1CCOCC1
InChIKey: DGBAWOXGLXNPSZ-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 1: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) In the day before the experimental day, frozen cell were thawed and washed with the culture medium (MEM-α, 10% FBS, 2 mmol/L GlutaMax, 50 unit Penicillin, 50 μg/mL Streptomycin). Then cells were suspended in the culture medium. (3) The 384-well plates which hTRPV4/CHO cells were seeded at densities of 4000 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for overnight was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 20 mmol/L HEPES, 2.5 mmol/L probenecid, pH7.4), and the buffer were remained at 20 μL/well. (5) 10 μl of dye loading buffer (9 μmol/L Fluo 4-AM, 0.09% Pluronic F-127/assay buffer) was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour. (final 3 μmol/L Fluo4-AM). (6) The assay plate was washed with the assay buffer, and the buffers were remained at 20 μL/well. Then the assay plate was incubated for 10 min at room temperature. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the Fluorescence analysis system FLIPR TETRA (Molecular devices). (8) After incubation for 5 min, 20 μL of 4α-PDD solution were applied to each well of assay plate and mixed with the FLIPR TETRA. (final 1 Î¼mol/L 4α-PDD). (9) The fluorescent intensity was measured with FLIPR TETRA system for 10 min from the point of time addition the compound solution, at Ex 470-495 nm, Em 515-575 nm wavelength.
Measurement: Ki = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 612.69, ALogP 6.31, HBA 6, HBD 1, PSA 76.04, QED 0.31
Compound (SMILES): Cc1cccc(Cl)c1S(=O)(=O)Nc1cccc2cccnc12
InChIKey: VKUZBVCGWSWKSA-UHFFFAOYSA-N
Target: Ferroportin (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP59
Assay: Antagonist activity at C-terminal halo-tag fused human ferroportin expressed in MDCK cells assessed as inhibition of hepcidin-induced ferroportin internalization after overnight incubation by Draq5 staining-based fluorescence assay
Measurement: IC50 = 100000 nM (pChEMBL 4)
Descriptors: MW 332.81, ALogP 4, HBA 3, HBD 1, PSA 59.06, QED 0.79
Compound (SMILES): C[C@H]1CN(c2ccccc2-c2ccccc2)CCN1CCCCCC(=O)NCc1ccccc1
InChIKey: GPICSZOLXGYSHL-VWLOTQADSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: Displacement of [3H]-5-CT from human 5-HT7b receptor expressed in HEK293 cell membranes after 1 hr by microbeta counting analysis
Measurement: Ki = 36.5 nM (pChEMBL 7.440000057220459)
Descriptors: MW 455.65, ALogP 5.74, HBA 3, HBD 1, PSA 35.58, QED 0.4
Compound (SMILES): NS(=O)(=O)OC[C@@H]1C[C@@H](Oc2cc(N3Cc4ccccc4C3)ncn2)C[C@@H]1O
InChIKey: GUKGTVMKUPBWOJ-XHSDSOJGSA-N
Target: NEDD8 activating enzyme (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13564
Assay: Inhibition of recombinant human NAEalpha/N-terminal His-tagged NAEbeta expressed in baculovirus infected insect Sf9 cells using N-terminal FLAG tagged Nedd8 as substrate incubated for 105 mins in presence of N-terminal GST-tagged Ubc12 by FRET assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 406.46, ALogP 0.74, HBA 8, HBD 2, PSA 127.87, QED 0.72
Compound (SMILES): Cc1cc2ccc1[C@@H](C)COC(=O)Nc1ccc([C@@H](CC(F)F)C(=O)O)c(c1)CN(C)C(=O)[C@@H]2Nc1ccc2c(N)ncc(F)c2c1
InChIKey: VORDOLJPJWNPOM-GJABKNSGSA-N
Target: Coagulation factor VII (SINGLE PROTEIN), Homo sapiens
UniProt: P08709
Assay: In Vitro Assay: FVIIa-Xase Ki (37° C.): S2765 (0.5 mM), PCPS (25 μM), calcium chloride (5 mM), full-length human TF (3 nM), human FVIIa (5 pM), and FVIIa inhibitor were incubated for 15 min at 37° C. Reactions were initiated by the addition of human FX (range of concentrations). Preliminary experiments revealed that the plasma purified FX contains a residual amount of human FVIIa which could not be removed by affinity chromatography. The residual FVIIa is sufficient, when combined with PCPS, calcium and TF to catalyze the conversion of FX to Xa, and was increased by the addition of 5 pM human FVIIa. FXa in turn hydrolyses S2765, which was monitored for 60 min at 405 nm.
Measurement: Ki = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 649.67, ALogP 6.57, HBA 7, HBD 4, PSA 146.88, QED 0.18
Compound (SMILES): COc1cc(-c2ccnc3[nH]c4ccc(-c5ccc(N6CCN(C)CC6)cc5)cc4c23)c(OC)cc1Cc1ccccc1
InChIKey: ZLHADXKVVSTNRO-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: ELISA-Based In Vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad).
Measurement: IC50 = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 568.72, ALogP 7.41, HBA 5, HBD 1, PSA 53.62, QED 0.22
Compound (SMILES): CC(=O)N1C2CCC1CN(C(=O)CC1([C@H]3COC[C@@H](C4CC4)N3S(=O)(=O)c3ccc(Cl)cc3)CC1)C2
InChIKey: RDEIVTBXCYQJJZ-BRLSKQCNSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of recombinant human CYP3A4 expressed in baculovirus infected insect cells using fluorescent substrate
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 536.09, ALogP 2.9, HBA 5, HBD 0, PSA 87.23, QED 0.56
Compound (SMILES): COc1ccccc1CC(=O)Nc1cnn(Cc2c(C)noc2C)c1
InChIKey: RAYQCUAXPIUNDR-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 = 1160 nM (pChEMBL 5.940000057220459)
Descriptors: MW 340.38, ALogP 2.73, HBA 6, HBD 1, PSA 82.18, QED 0.75