lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(=O)c1ccc(C(C)=O)c2c(C)c(C(=O)Nc3cc(CO)c4ccccc4n3)sc12
InChIKey: KBWBSHBRZHDULJ-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Activity Assay: A human acute lymphoblastic lymphoma T cell line MOLT-4 (which can be purchased under ATCC No. CRL-1582 from ATCC) was cultured in an RPMI1640 medium containing 10% fetal bovine serum to obtain 5108 MOLT-4 cells. The cells were recovered by centrifugation and suspended in 10 ml of buffer solution A (25 mM Tris-HCl, 5 mM 2-mercaptoethanol, 2 mM benzamidine, 2 mM EDTA, and 0.1 mM 4-(2-aminoethyl)benzenesulfonyl hydrochloride, pH 7.5). The cells were homogenized using a Polytron homogenizer and centrifuged (4° C., 25,000 g, 10 minutes). Then, the supernatant was further ultracentrifuged (4° C., 100,000 g, 60 minutes). The obtained supernatant was filtered through a 0.2um filter to obtain a soluble fraction.HiTrap Q HP column (5 ml2, GE Healthcare Japan Corp.) equilibrated with buffer solution A was charged with the obtained soluble fraction. Phosphodiesterase was eluted with 300 ml of buffer solution A containing a linear gradient solution of 0 to 0.8 M NaCl to recover sixty 5-ml fractions. Each fraction was tested for cAMP-metabolizing phosphodiesterase activity. In each fraction, a fraction eluted as an active peak centered around 250 mM NaCl was collected from fractions that had cAMP metabolic activity and did not lose the metabolic activity by 10uM rolipram (PDE4 selective inhibitor) and 10uM milrinone (PDE3 selective inhibitor). In order to confirm whether or not PDE10A mRNA was expressed in the MOLT-4 cells, total RNA was prepared from the MOLT-4 cells according to a standard method and analyzed by RT-PCR using PDE10A gene-specific primers (PDE10A sense primer: 5'-TGCTCCATGGTGGAAGTGGA-3' (SEQ ID NO: 1) and PDE10A antisense primer: 5'-CAACTGGAAGCATGCGGTCA-3' (SEQ ID NO: 2)). As a result, PDE10A mRNA was detected from the total RNA of the MOLT-4 cells. On the other hand, total RNA was prepared from Jurkat cells, one type of human T cell, and similarly subjected to RT-PCR. As a result, PDE10A mRNA was rarely detected. An active peak centered around 250 mM NaCl was not observed in fractions obtained by the treatment of Jurkat cells in the same way as above. Eur J Biochem. 1999, 266 (3), 1118-2 has reported that PDE10A from the rat striatum and testis is eluted as an active peak centered around 250 mM NaCl.
Measurement: IC50 = 0.89 nM (pChEMBL 9.050000190734863)
Descriptors: MW 432.5, ALogP 4.91, HBA 6, HBD 2, PSA 96.36, QED 0.44
Compound (SMILES): CC1(C)CN(c2cc(Nc3nccc(C(F)(F)F)n3)cc(-c3ccccc3)c2)CCO1
InChIKey: VIHQVVBUGWTXSM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 μM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 293.3 nM (pChEMBL 6.53000020980835)
Descriptors: MW 428.46, ALogP 5.52, HBA 5, HBD 1, PSA 50.28, QED 0.59
Compound (SMILES): CC1(C)CCCCN1c1cnc2cccc(-c3cc4c([nH]3)CCNC4=O)c2n1
InChIKey: IXFNGWMOFJHECL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): 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 384-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 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 375.48, ALogP 3.68, HBA 4, HBD 2, PSA 73.91, QED 0.72
Compound (SMILES): CCOc1ccc(CC(=O)O)cc1-c1ccc(F)c2c1CN(C(=O)CC(C)(C)c1ccc(F)cc1)CC2
InChIKey: UHVRDUDRIMHOFW-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 ul. First, 25 ul 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 ul Binding-Buffer, 50 ul 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 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/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 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 507.58, ALogP 5.91, HBA 3, HBD 1, PSA 66.84, QED 0.41
Compound (SMILES): N#Cc1nc(C(=O)NCC(C(=O)O)c2ccccc2)c(O)c2ccc(Oc3ccccc3)cc12
InChIKey: LEMDOGKUYBBUNK-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 1790 nM (pChEMBL 5.75)
Descriptors: MW 453.45, ALogP 4.2, HBA 6, HBD 3, PSA 132.54, QED 0.38
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3C(CNS(C)(=O)=O)O4)cc12
InChIKey: AWEQYVSYQYQUJC-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: ELISA-Based Assay: Measurement of inhibition by compounds was performed using the HCV replicon system. Several different replicons encoding different HCV genotypes or mutations were used. In addition, potency measurements were made using different formats of the replicon assay, including different ways of measurements and different plating formats. See Jan M. Vrolijk et al., A replicons-based bioassay for the measurement of interferons in patients with chronic hepatitis C, 110 J. VIROLOGICAL METHODS 201 (2003); Steven S. Carroll et al., Inhibition of Hepatitis C Virus RNA Replication by 2'-Modified Nucleoside Analogs, 278(14) J. BIOLOGICAL CHEMISTRY 11979 (2003). However, the underlying principles are common to all of these determinations, and are outlined below.Stable neomycin phosphotransferase encoding replicons-harboring cell lines were used, so all cell lines were maintained under G418 selection prior to the assay. Potency was determined using a cell ELISA assay with an antibody to the replicons encoded NS3/4a protease. See Caterina Trozzi et al., In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor, 77(6) J. Virol. 3669 (2003).
Measurement: IC50 = 2.6 nM (pChEMBL 8.59000015258789)
Descriptors: MW 721.76, ALogP 5.26, HBA 9, HBD 2, PSA 152.84, QED 0.22
Compound (SMILES): S=c1[nH]c(C2CCCCC2)c2c3cc[nH]c3ncn12
InChIKey: MZSZFDGQDHRGQK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 430 nM (pChEMBL 6.369999885559082)
Descriptors: MW 272.38, ALogP 3.92, HBA 3, HBD 2, PSA 48.88, QED 0.66
Compound (SMILES): O=C1c2ccc(C3CC3)cc2OCCN1c1cccc(-c2ncnc3[nH]c(-c4ccc(CN5CCOCC5)cc4)cc23)c1CO
InChIKey: PSIWUKBLYARZHY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Mus musculus
UniProt: P35991
Assay: Inhibition of C57BL/6J mouse BTK assessed as decrease in anti-mouse IgM antibody stimulated BCR mediated CD69 upregulation preincubated for 30 mins followed by antibody challenge after 18 to 24 hrs by flow cytometry
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 601.71, ALogP 5.53, HBA 7, HBD 2, PSA 103.81, QED 0.25
Compound (SMILES): Brc1cnc(Nc2ccccc2Cc2ccccc2)nc1Nc1cc(C2CC2)[nH]n1
InChIKey: GPLPSDJXUVUQQY-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 461.37, ALogP 5.92, HBA 5, HBD 3, PSA 78.52, QED 0.32
Compound (SMILES): Cn1nc(C2CCN(C(=O)C(C)(C)O)CC2)nc1-c1cnc(N)c(-c2nnc(C(C)(C)C)o2)n1
InChIKey: ORHNDZXUGUCSGY-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: In Vitro Enzyme Inhibition Assay: The inhibition of PI3K-beta, PI3K-alpha, PI3K-gamma and PI3K-delta was evaluated in a Kinase Glo based enzyme activity assay using human recombinant enzymes. Compounds in 100% DMSO were added to assay plates by acoustic dispensing. PI3K enzyme was added in a Tris buffer (50 mM Tris pH7.4, 0.05% CHAPS, 2.1 mM DTT, and 10 mM magnesium chloride) and allowed to preincubate with compound for 20 minutes prior to addition of substrate solution containing PIP2 and ATP. The enzyme reaction was stopped after 80 minutes by the addition of Kinase Glo detection solution containing Lucferin and Luciferase (from Kinase Glo® Plus Luminecent Kinase Assay kit (Promega #V3772). Plates were left for 30 minutes at room temperature then read on a Pherastar Instrument with a standard Luminescence filter block. The final concentration of DMSO, ATP and PIP2 in the assay were, 1%, 8 uM, and 80 uM respectively.
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 469.55, ALogP 1.68, HBA 11, HBD 2, PSA 161.97, QED 0.57
Compound (SMILES): N#CCc1cnc(N)c(-c2ccc(C(=O)N[C@H](CO)c3ccccc3)c(F)c2)c1
InChIKey: VHQKGXVJGHIURG-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 = 202 nM (pChEMBL 6.699999809265137)
Descriptors: MW 390.42, ALogP 3, HBA 5, HBD 3, PSA 112.03, QED 0.6
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)cc(N4CCN(C(=O)C5CC5(F)F)CC4)nc23)N1
InChIKey: XLDPATMKVYWXOK-AUWJEWJLSA-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 = 41017 nM (pChEMBL 4.389999866485596)
Descriptors: MW 472.46, ALogP 0.79, HBA 8, HBD 3, PSA 123.97, QED 0.43
Compound (SMILES): COc1cc(C(=O)N(C)C)ccc1Nc1cc2cc(-c3cnn(CC(C)(C)O)c3)ccc2cn1
InChIKey: WFXYXLAUKHZGKK-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Kinase Assay: The enzyme reaction (total volume 10 μl) was carried out in black 384-well low volume plates containing full length MPS1 (12.5 nM or 3 nM), fluorescent labelled peptide [known as H236, which has the sequence: 5FAM-DHTGFLTEYVATR-CONH2](5 μM), ATP (10 μM), either DMSO (1% v/v) or the test compound (in the range 0.25 nM-100 μM in 1% DMSO) and assay buffer (50 mM HEPES (pH 7.0), 0.02% NaN3, 0.01% BSA, 0.1 mM Orthovandate, 10 μM MgCl2, 1 μM DTT, Roche protease inhibitor). The reaction was carried out for 60 min at room temperature and stopped by the addition of buffer (10 μl) containing 20 mM EDTA, 0.05% (v/v) Brij-35, in 0.1M HEPES-buffered saline (Free acid, Sigma, UK). The plate was read on a Caliper EZ reader II (Caliper Life Sciences).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 459.55, ALogP 4.32, HBA 7, HBD 2, PSA 92.51, QED 0.43
Compound (SMILES): O=C(O)CCNC(=O)c1ncc2cc(Oc3cccnc3)ccc2c1O
InChIKey: PXXCPPPHJZXXGB-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 2310 nM (pChEMBL 5.639999866485596)
Descriptors: MW 353.33, ALogP 2.33, HBA 6, HBD 3, PSA 121.64, QED 0.62
Compound (SMILES): O=C(Nc1ccccc1)N1CCN(Cc2cccc(OS(=O)(=O)c3ccccc3)c2)CC1
InChIKey: FACBXOHFYSYKNJ-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: Biological Assay: Assay Method 1: T84 frozen pellets (contents of 1-4x15 cm culture dishes) were homogenized in 300 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixture was prepared from 50 uL of the cell homogenate, 10 uL of the test compound, and 40 uL of anandamide [1-3H-ethanolamine] (3H-AEA; Perkin-Elmer, 10.3 Ci/mmol), which was added last, for a final tracer concentration of 200 nM. The reaction mixture was incubated at rt for 1 hour (h). During the incubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loaded with 25 uL of activated charcoal (Multiscreen column loader, catalog number MACL09625, Millipore) and washed once with 100 uL of MeOH. Also during the incubation, 96-well DYNEX MicroLite plates (catalog number NL510410) were loaded with 100 uL of MicroScint40 (catalog number 6013641, Packard Bioscience, Meriden, Conn., USA).
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 451.55, ALogP 3.8, HBA 5, HBD 1, PSA 78.95, QED 0.58
Compound (SMILES): CN1CCc2cc(-c3cnc(N)c(-c4nnc(C5CCNCC5)o4)c3)ccc21
InChIKey: KBDRJATVSJLQHO-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Kinase Activity Assay: The kinase reaction buffer consists of 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, and 2 mM DTT. In the assay, 1 nM LRRK2 WT or 250 pM LRRK2 G2019S kinase is incubated with the test compound (typically at 0 to 30 μM) for 30 minutes before the kinase reaction is initiated by addition of 1.3 mM ATP and 0.4 μM fluorescein-LRRKtide. The reaction mixture (20 μl total volume) is incubated for 2 hours at 30 °C. before the reaction is terminated by addition of 10 mM EDTA and 1 nM terbium-labelled anti-phospho-LRRKtide antibody (final volume 20 μl). The mixture is further incubated for 30 minutes at RT. TR-FRET is measured by excitation of the terbium-donor with 340 nm light and subsequent (delay time 100 μs) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a time window of 1000 μs. The measurement is repeated 10 times for fluorescein and 10 times for terbium emission with a 2000 μs time window between repeats. TR-FRET measurements are performed on a Biomek Synergy plate. The TR-FRET signal is calculated as the emission-ratio at 520 nm over 495 nm.
Measurement: IC50 = 380 nM (pChEMBL 6.420000076293945)
Descriptors: MW 376.46, ALogP 2.84, HBA 7, HBD 2, PSA 93.1, QED 0.73
Compound (SMILES): O=C(O)CC1CCC(c2ccc(NC(=O)CCNC(=O)c3ccc(OCC4CC4)cc3)cc2)CC1
InChIKey: YAECHSHJWWIDQR-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Enzyme Inhibition Assay: Human DGAT-1 activity was determined as follows: Assay buffer [20 mM HEPES (pH 7.4), 100 mM MgCl2, 0.04% BSA] containing 500 μM of enzyme substrate (didecanoyl glycerol) and 7.5 μM radiolabeled acyl-CoA substrate ([14C]decanoyl-CoA) was added to each well of a phospholipid FlashPlate (PerkinElmer Life Sciences). A small aliquot of lysate (5 μg/well) or mouse liver microsome (0.1 mg/well) was added to start the reaction, which was allowed to proceed in a 20° C. bath for 1 hour. and maintained overnight at room temperature. Next day, measurement was performed by Wallac 1450 Microbeta Trilux Liquid Scintillation Counter and Luminometer (PerkinElmer Life Science). In the control group, 0.1% DMSO was only added, and for the background value, 0.1% DMSO was only added without the cell fragment having the over-expressed human DGAT1 (5 μg/well) or the liver microsome of mice (0.1 mg/well), which was the same as the control group.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 478.59, ALogP 4.98, HBA 4, HBD 3, PSA 104.73, QED 0.42
Compound (SMILES): Cc1nc(C2CN(c3ncnc4[nH]cc(-c5cccc(C#N)c5)c34)CCO2)no1
InChIKey: SAOVTENLRUIQAM-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 387.4, ALogP 2.77, HBA 8, HBD 1, PSA 116.75, QED 0.57
Compound (SMILES): O=C(c1cc(F)ccc1-c1ncccn1)N1CCCOC1Cn1ccc(-c2ccc(F)cn2)n1
InChIKey: YZRHBGIKWQSFJJ-UHFFFAOYSA-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 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 462.46, ALogP 3.57, HBA 7, HBD 0, PSA 86.03, QED 0.45
Compound (SMILES): C=CCOc1cc(-c2ccc3c(/C=C/c4ccc(OC)c(OC)c4)n[nH]c3c2)ccc1O
InChIKey: BXMUFQGDIWSQBJ-BJMVGYQFSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of unphosphorylated human 50 residues truncated cytoplasmic domain of VEGFR2 E990V mutant expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using poly(E4Y1) as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 428.49, ALogP 5.69, HBA 5, HBD 2, PSA 76.6, QED 0.35
Compound (SMILES): CCc1ccc(Nc2n[nH]c3c2CN(C(C)=O)CC3)cc1
InChIKey: NWIFMQIVWVMZLD-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Displacement of biotinylated ligand from recombinant His-tagged BRD4 bromodomain-1 (unknown origin) measured after 10 mins by TR-FRET assay
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 284.36, ALogP 2.62, HBA 3, HBD 2, PSA 61.02, QED 0.91
Compound (SMILES): COc1cc(-c2ccc(F)c(C)c2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ncccn3)ccc21
InChIKey: AQFOFFUVEFOKQU-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 534.54, ALogP 4.84, HBA 7, HBD 1, PSA 103.18, QED 0.33
Compound (SMILES): CN(C)CC1CCCN(c2cc(Nc3nccc(C(F)(F)F)n3)cc(-c3ccccc3)c2)C1
InChIKey: FRVVGNWBHUUPGV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 μM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 251.1 nM (pChEMBL 6.599999904632568)
Descriptors: MW 455.53, ALogP 5.68, HBA 5, HBD 1, PSA 44.29, QED 0.52
Compound (SMILES): CC(C)Oc1ccc(Nc2ncc(N(S(C)(=O)=O)S(C)(=O)=O)c(=O)n2Cc2ccc(Cl)cc2)cc1F
InChIKey: VZZCIFSQDDQFGD-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 in M MgCl2, 5.0 mM CaCl2, 5.6 D-glucose, 2.5 mM probenecid, 1.0% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence inte
Measurement: IC50 = 536 nM (pChEMBL 6.269999980926514)
Descriptors: MW 559.04, ALogP 3.34, HBA 9, HBD 1, PSA 127.67, QED 0.42
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)nc(N4CCC(N5CCCCC5)CC4)nc23)N1
InChIKey: SMAWZWIKVAQWAO-ATVHPVEESA-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 451.54, ALogP 1.33, HBA 9, HBD 3, PSA 119.79, QED 0.46
Compound (SMILES): Cc1nn(C)c(C)c1N(CC1CC1)S(=O)(=O)c1c(Cl)cc(Br)cc1Cl
InChIKey: BMECBYDYFQOQNT-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
UniProt: Q388H8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 670 nM (pChEMBL 6.170000076293945)
Descriptors: MW 467.22, ALogP 4.71, HBA 4, HBD 0, PSA 55.2, QED 0.64
Compound (SMILES): CC(C)(O)C(=O)c1ccc(-c2ccc(CC[C@](C)(C(=O)NO)S(C)(=O)=O)cc2)cc1
InChIKey: BARRZEJAJSIBKB-JOCHJYFZSA-N
Target: UDP-3-O-acyl-N-acetylglucosamine deacetylase (SINGLE PROTEIN), Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG12228)
UniProt: P47205
Assay: Enzyme Assay: IC50 determination in the LpxC enzyme assay was carried out in a similar manner to that described by Malikzay et al in the 2006 Poster, Screening LpxC (UDP-3-O(R-3-hydroxymyristoyl)-GlcNAc deacetylase) using BioTrove Rapid Fire HTS Mass Spectrometry (aNew Lead Discovery and bInflammation and Infectious Disease, cStructural Chemistry, Schering-Plough Research Institute, Kenilworth, N.J. 07033, (BioTrove, Inc. 12 Gill St., Suite 4000, Woburn, Mass. 01801). Briefly, Pseudomonas aeruginosa LpxC enzyme (0.1 nM) purified from E. coli-overexpressing bacteria was incubated at 25° C. in a final volume of 50 ul containing 0.5 uM UDP-3-O(R-3-hydroxydecanoyl)-N-acetylglucosamine, 1 mg/mL BSA, and 50 mM sodium phosphate buffer, pH 8.0 in the presence and absence of inhibitor compound. At the end of 1 hour, 5 ul of 1N HCl was added to stop the enzyme reaction; the plates were centrifuged, and then processed with the BioTrove Rapidfire HTMS Mass Spectrometry System.
Measurement: IC50 = 0.278 nM (pChEMBL 9.5600004196167)
Descriptors: MW 433.53, ALogP 2.55, HBA 6, HBD 3, PSA 120.77, QED 0.33
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(F)c(C(=O)c2c[nH]c3ncc(-c4cccc(S(C)(=O)=O)c4)cc23)c1F
InChIKey: LCVHYZOPJTTYEL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 533.58, ALogP 4.29, HBA 6, HBD 2, PSA 126.06, QED 0.33
Compound (SMILES): CN1CCC(c2ccc(Nc3nc4c(NCc5cccc(S(C)(=O)=O)c5)cccn4n3)cc2)CC1
InChIKey: APRIKMQKELYDRU-UHFFFAOYSA-N
Target: Focal adhesion kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05397
Assay: In Vitro Enzyme Assay: The ability of compounds to inhibit the kinase activity of baculovirus-expressed human FAK and JAK kinase using the time-resolved fluorescence (TRF) assay system.
Measurement: IC50 = 9999 nM (pChEMBL 5)
Descriptors: MW 490.63, ALogP 4.3, HBA 8, HBD 2, PSA 91.63, QED 0.4
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(=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: QSMLKXNHLIEUKG-CCHCRRDSSA-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 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 2344.66
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3CCOC[C@@H]3C)nc(-c3ccc(NC(=O)OCC(C)(C)O)cc3)c2C1=O
InChIKey: UXHJVGZARQEKOQ-KRWDZBQOSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The test detects the phosphorylation of the substrate GFP-4EBP1 using a terbium-labelled antibody that is specific for the phospho-Thr46 epitope. Binding of the Tb-antibody to the phosphorylated GFP peptide allows the transfer of energy from Tb, excited at 340 nm, to GFP with an increase in the fluorescence emission of the GFP at 520 nm. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 ul. To 1 ul of inhibitor in 100% DMSO are added (final concentrations) 400 nM of GFP-4EBP1 (Invitrogen PV4759), 8 uM of ATP, 200 ng/ml of enzyme (human recombinant aa mTOR 1360-2549, Invitrogen PV4753) in a buffer of HEPES 50 mM pH 7.5, EGTA 1 mM, MnCl2 10 mM, BSA 0.1 mg/ml, glycerol 0.5%, NV-10 0.1 mg/ml, DTT 2 mM. The mixture is incubated for 30 minutes at room temperature. 10 ul of solution are taken up and the reaction is quenched by adding 10 ul of Tb-antibody solution (Invitrogen PV4757)/EDTA.
Measurement: IC50 = 2448 nM (pChEMBL 5.610000133514404)
Descriptors: MW 527.62, ALogP 3.32, HBA 9, HBD 2, PSA 126.35, QED 0.58
Compound (SMILES): NC(=O)c1cc(C2=CCCC2)c2ccccc2n1
InChIKey: ONFZHRZTMRGDRC-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 = 822 nM (pChEMBL 6.079999923706055)
Descriptors: MW 238.29, ALogP 2.9, HBA 2, HBD 1, PSA 55.98, QED 0.87
Compound (SMILES): CCN(CC)CCOc1ccc2c(c1)C1(CCNCC1)c1[nH]c3ccccc3c1C2=O
InChIKey: GGYVQDJNEABTCR-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 687.68 nM (pChEMBL 6.159999847412109)
Descriptors: MW 417.55, ALogP 4.1, HBA 4, HBD 2, PSA 57.36, QED 0.63
Compound (SMILES): O=C(/C=C/c1cccnc1)Nc1ccc(S(=O)(=O)c2cnc3ccccc3c2)cc1
InChIKey: QAAUCCWOBWBSIC-KPKJPENVSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 uM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine.
Measurement: IC50 = 3.7 nM (pChEMBL 8.430000305175781)
Descriptors: MW 415.47, ALogP 4.11, HBA 5, HBD 1, PSA 89.02, QED 0.5
Compound (SMILES): NC1=N[C@@]2(c3ccc(F)cc3F)CO[C@@H](c3ccc4c(c3)CCO4)C[C@H]2CS1
InChIKey: LVYIZOZZLBROIC-ZKYUUJBMSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Cell-Free Assay: A synthetic APP substrate that can be cleaved by beta-secretase having N-terminal biotin and made fluorescent by the covalent attachment of Oregon Green at the Cys residue is used to assay beta-secretase activity in the presence or absence of the inhibitory compounds. The substrate is Biotin-GLTNIKTEEISEISY^EVEFR-C[Oregon Green]KK-OH. The BACE1 enzyme is affinity purified material from conditioned media of CHO-K1 cells that have been transfected with a soluble BACE construct (BACE1deltaTM96His). Compounds are incubated in a log dose response curve from a top concentration of 100 uM with BACE1 enzyme and the biotinylated fluorescent peptide in 384-well black plates (Thermo Scientific #4318). BACE1 is at a final concentration of 0.1 nM with a final concentration of peptide substrate of 150 nM in a reaction volume of 30 uL assay buffer [100 mM sodium acetate, pH 4.5 (brought to pH with acetic acid), and 0.001% Tween-20]. Plates are covered and incubated for 3 hours at 37° C. The reaction is stopped with the addition of 30 uL of 1.5 uM Streptavidin (Pierce, #21125). After a 10 minute incubation at room temperature, plates are read on a PerkinElmer EnVision for fluorescence polarization (Ex485 nm/Em530 nm). The activity of the beta-secretase enzyme is detected by changes in the fluorescence polarization that occur when the substrate is cleaved by the enzyme. Incubation in the presence of compound inhibitor demonstrates specific inhibition of beta-secretase enzymatic cleavage of the synthetic APP substrate.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 402.47, ALogP 3.93, HBA 5, HBD 1, PSA 56.84, QED 0.83
Compound (SMILES): Cc1ccncc1C1=CC[C@H]2[C@@H]3CC[C@H]4CC(=O)NCC[C@]4(C)[C@H]3CC[C@]12C
InChIKey: NSIQPNJAAQCXJJ-RSIBYXMTSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human H295R cells assessed as decrease in testosterone levels after 24 hrs by ELISA
Measurement: IC50 <= 15 nM (pChEMBL 7.823999881744385)
Descriptors: MW 378.56, ALogP 5.15, HBA 2, HBD 1, PSA 41.99, QED 0.73
Compound (SMILES): COc1cc(F)cc(C(=O)Nc2cn3nc(-c4cn(C)nc4-c4ccc(F)cc4)ccc3n2)c1
InChIKey: GNFHJOCKJPRPNE-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 6.8 nM (pChEMBL 8.170000076293945)
Descriptors: MW 460.44, ALogP 4.34, HBA 7, HBD 1, PSA 86.34, QED 0.42
Compound (SMILES): CC1=C(C(=O)Nc2nc3ccccc3s2)C2(CCCC2)N=C(Nc2nc3ccccc3o2)N1
InChIKey: ZRNNDDXZPXXDDT-UHFFFAOYSA-N
Target: Galactokinase (SINGLE PROTEIN), Homo sapiens
UniProt: P51570
Assay: qHTS Assay: Assay details and protocol: The primary assay monitored ATP depletion using Promega's KinaseGlo technology, where ATP levels are measured through luminescence generated from firefly luciferase, a bioluminescent ATP-dependent enzymes. ATP was held at 35 μM, near its reported KM value, and the KM for galactose was determined under the 1536-well assay conditions to be 50-100M (FIG. 1A). As well, the IC50 for a commercially available CD45 inhibitor (N-(9,10-dioxo-9,10-dihydrophenanthren-2-yl)pivalamide) was confirmed (previously found to inhibit GALK (FIG. 1B)). This was used as the positive control for the assay. The assay used 5 nM GALK and a 1 hr incubation time, which gave sufficient signal:background and stability for the HTS.
Measurement: IC50 = 25902.4 nM (pChEMBL 4.590000152587891)
Descriptors: MW 458.55, ALogP 5.03, HBA 8, HBD 3, PSA 104.44, QED 0.4