lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc(Nc2cc(C(F)F)ccn2)nc(-c2ccc(C(C)(O)C3CCC(C(=O)OC(C)OC(=O)OC(C)C)CC3)nc2)c1
InChIKey: IXPNAXBXAPKVEX-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 pM 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 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45x 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.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 612.67, ALogP 6.99, HBA 10, HBD 2, PSA 132.76, QED 0.18
Compound (SMILES): Cc1cc(C)c(C)c(S(=O)(=O)Nc2ccc(N3CC[C@H](C(=O)O)C3)c3ccccc23)c1C
InChIKey: BVYWIQHJXAEJOD-IBGZPJMESA-N
Target: Kelch-like ECH-associated protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14145
Assay: Binding affinity to recombinant human KEAP1 Kelch domain (321 to 609 residues) expressed in Escherichia coli BL21 (DE3) assessed as thermal stabilization by sypro orange dye based thermal shift assay
Measurement: EC50 = 13000 nM (pChEMBL 4.889999866485596)
Descriptors: MW 452.58, ALogP 4.79, HBA 4, HBD 2, PSA 86.71, QED 0.58
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)[C@@H](C)NC4=O)c2nc1O[C@H](C)C(F)(F)F
InChIKey: YQOPMWZYUDDIAP-PSASIEDQSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
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 = 32.5 nM (pChEMBL 7.489999771118164)
Descriptors: MW 390.37, ALogP 4.07, HBA 4, HBD 2, PSA 79.9, QED 0.71
Compound (SMILES): Cc1noc(C)c1-c1cc(-c2cccnc2-c2ccccc2)c2nc(C3CC3)[nH]c2c1
InChIKey: ZTTDZAWIKNDGPB-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Amplified Luminescent Proximity Homogeneous Assay (ALPHA): Binding of the bromodomain BRD4-I to an acetylated histone H4 peptide was measured using a bead-based Amplified Luminescent Proximity Homogeneous Assay (ALPHA). The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-I was expressed and purified from Escherichia coli as an N-terminal His6-tagged protein. Nickel-Chelate ALPHA acceptor beads (Perkin Elmer) were used to specifically bind BRD4-1 and ALPHA streptavidin donor beads (Perkin Elmer) were used because they specifically recognized the biotinylated H4 peptide. Binding of BRD4-1 to the peptide resulted in proximity of the donor and acceptor beads which leads to an increase in ALPHA signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in ALPHA signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO, 200 nM H4 peptide and 15 nM of BRD4-1 protein. After an assay reaction time of 60 minutes at 25° C., binding was measured with 20 ug/ml streptavidin donor beads and 20 ug/ml nickle-chelate acceptor beads. ALPHA signal was detected on an Envision plate reader (Ex: 320 nm; Em: 570 nm; Ex time: 180 ms). Data were normalized based on a positive (2 uM I-BET) and negative (DMSO) controls and IC50 values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 406.49, ALogP 6.44, HBA 4, HBD 1, PSA 67.6, QED 0.37
Compound (SMILES): CCCCCCCCC(=O)O[C@H]1CC/C(C)=C/CC/C(C)=C/C[C@]2(C(C)C)CC[C@@]1(C)O2
InChIKey: RZMCRLYIPXLJDL-VABOSOLWSA-N
Target: Signal transducer and activator of transcription 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P40763
Assay: Inhibition of IFNgamma-induced STAT3 activation in human HeLa cells expressing STAT3 (unknown origin) preincubated for 15 mins followed by IFN-gamma addition measured after 6 hrs by luciferase reporter gene assay
Measurement: IC50 = 38660 nM (pChEMBL 4.409999847412109)
Descriptors: MW 446.72, ALogP 8.47, HBA 3, HBD 0, PSA 35.53, QED 0.2
Compound (SMILES): C[C@@H]1CN(c2sc(C(F)(F)F)nc2-c2nc3ccccc3n2C)CCN1C(=O)Cn1cccn1
InChIKey: NCSLFCCQXGPWFZ-CQSZACIVSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Antagonist activity at human recombinant CXCR3 in human venous blood assessed as receptor internalization measured for 30 mins in presence of CXCL10 by flow cytometric analysis
Measurement: IC50 = 3560 nM (pChEMBL 5.449999809265137)
Descriptors: MW 489.53, ALogP 3.65, HBA 8, HBD 0, PSA 72.08, QED 0.44
Compound (SMILES): O=C(C1CCCC1)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(O)c1)C2)CC1CCCCC1
InChIKey: RESVOIMIAVKTCX-OOSCYNTBSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Cavia porcellus
UniProt: P41144
Assay: Displacement of [3H]-diprenorphine from guinea pig kappa opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 438.66, ALogP 5.7, HBA 3, HBD 1, PSA 43.78, QED 0.59
Compound (SMILES): CCCc1nn(C)c2c(=O)[nH]c(-c3cc(S(=O)(=O)N4CCN(Cc5ccc(/C=C/C(=O)NO)cc5)CC4)ccc3OCC)nc12
InChIKey: SBNXVUVSBSHGHC-SDNWHVSQSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Inhibition of full length recombinant human C-terminal His-tagged HDAC2 expressed in baculovirus infected Sf9 cells using fluorogenic HDAC substrate after 30 mins by fluorescence analysis
Measurement: IC50 = 6970 nM (pChEMBL 5.159999847412109)
Descriptors: MW 635.75, ALogP 2.7, HBA 10, HBD 3, PSA 162.75, QED 0.13
Compound (SMILES): Cl.Clc1ccc(C(=CCCN2CCc3ccccc3C2)c2ccc(Cl)cc2)cc1
InChIKey: SMTSKYFAMLVTLI-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2C (SINGLE PROTEIN), Homo sapiens
UniProt: P28335
Assay: Displacement of [3H]-mesulergine from human 5-HT2C receptor by liquid scintillation counting method
Measurement: Ki = 2028 nM (pChEMBL 5.690000057220459)
Descriptors: MW 408.37, ALogP 6.87, HBA 1, HBD 0, PSA 3.24, QED 0.45
Compound (SMILES): CC(=O)c1ccc(OCc2ccc(Oc3cc(-c4nn[nH]n4)ccn3)cc2)c(C)c1O
InChIKey: AJLRUYZRQDKJFI-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13255
Assay: Agonist activity at recombinant human mGlu1 expressed in hamster AV12 cells expressing EAAT1 assessed as increase in intracellular calcium level measured after 24 hrs by FLIPR assay
Measurement: EC50 > 25000 nM (pChEMBL 4.6020002365112305)
Descriptors: MW 417.43, ALogP 3.85, HBA 8, HBD 2, PSA 123.11, QED 0.43
Compound (SMILES): COc1cc2c(cc1F)N(Cc1ccccc1)C[C@@H](C)N(C(=O)Nc1cccnc1)[C@H]2C
InChIKey: QLYUDMMUUNLMPP-MSOLQXFVSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1I (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P0X4
Assay: Channel blocking activity at recombinant human CaV 3.3 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 434.52, ALogP 5.23, HBA 4, HBD 1, PSA 57.7, QED 0.61
Compound (SMILES): Nc1ccc(-c2cccs2)cc1NC(=O)c1ccc2nc(N3CCNCC3)cnc2c1
InChIKey: HKYMRBDGDNAIPA-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC Enzyme Assay: Compounds for testing were diluted in DMSO to 50 fold the final concentration and a ten point three fold dilution series was made. The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 uM tris(2-carboxyethyl)phosphine) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer and pre-incubated with the compounds for 24 hours prior to addition of the substrate. The substrate tripeptide substrate 3 (synthesized in house) for each enzyme was equal to the Km as determined by a substrate titration curve. The enzyme and substrate concentrations used are given in Table 2. The substrates were diluted in assay buffer at 6x their final concentration with 0.3 uM sequencing grade trypsin (Sigma). The substrate/trypsin mix was added to the enzyme/compound mix, the plate was shaken for 60 seconds.
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 430.54, ALogP 3.6, HBA 7, HBD 3, PSA 96.17, QED 0.43
Compound (SMILES): NC(=O)N1CCCc2cc(-c3cncc(C4CCC(=O)N4)c3)cnc21
InChIKey: GZCRPFSQECFJRB-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Aldosterone Synthase Inhibition Assay: The aldosterone synthase inhibition assay employs cynomolgus adrenal gland mitochondria as the source of aldosterone synthase (CYP11B2). Mitochondria are prepared from frozen cynomolgus monkey adrenal glands according to Method A described in by J. D. McGarry et al. (Biochem. J., 1983, 214, 21-28), with a final resuspension in the AT buffer described in R. Yamaguchi et al. (Cell Death and Differentiation, 2007, 14, 616-624), frozen as aliquots in liquid nitrogen and stored at -80° C. until use. Assays are performed in 96-well format in a final volume of 60 uL/well, containing 100 mM potassium phosphate, pH 7.4, 1% (v/v) DMSO, and additionally, 2 uM of corticosterone and 6 mg of mitochondrial protein. Reactions are started by the addition of NADPH to 1 mM and allowed to proceed for 60-90 minutes at 37° C. Reactions are terminated by the addition of 60 uL of acetonitrile. One hundred microliters are then transferred to a glass filter plate and centrifuged at 570xg.
Measurement: IC50 = 159.1 nM (pChEMBL 6.800000190734863)
Descriptors: MW 337.38, ALogP 1.93, HBA 4, HBD 2, PSA 101.21, QED 0.87
Compound (SMILES): Cc1nsc(-c2nnc3n2CCN(C(=O)c2ccc(Cl)cc2)C3C)n1
InChIKey: ZMIOQWILBZBCHW-UHFFFAOYSA-N
Target: Substance-P receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P25103
Assay: Binding Assay: The affinity of compounds of the invention for the NK-1 receptor was evaluated in CHO recombinant cells which express the human NK-1 receptor. Membrane suspensions were prepared from these cells. The following radioligand: [3H] substance P (PerkinElmer Cat#NET111520) was used in this assay. Binding assays were performed in a 50 mM Tris/5 mM MnCl2/150 mM NaCl/0.1% BSA at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 5 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Substance P) at increasing concentrations (diluted in assay buffer) and 2 nM [3H] substance P. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in 0.5% PEI for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki = 32000 nM (pChEMBL 4.5)
Descriptors: MW 374.86, ALogP 2.98, HBA 7, HBD 0, PSA 76.8, QED 0.69
Compound (SMILES): O=C(O)CCc1cc2cc(-c3cc(OC(F)(F)F)ccc3Cl)ccc2o1
InChIKey: WVFSVTANMRLGDI-UHFFFAOYSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Agonist activity at human GPR40 measured after 60 mins by IP1-HTRF assay
Measurement: EC50 > 9950 nM (pChEMBL 5.001999855041504)
Descriptors: MW 384.74, ALogP 5.67, HBA 3, HBD 1, PSA 59.67, QED 0.62
Compound (SMILES): CC(C)CC(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(O)c1)C2)Cc1c(F)cccc1F
InChIKey: OHRHNWLZCCCOQR-KOUNCHBCSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 456.58, ALogP 5.46, HBA 3, HBD 1, PSA 43.78, QED 0.57
Compound (SMILES): COc1cc(OC)cc(N(c2ccc3ncc(-c4cnn(C)c4)nc3c2)C(C)C(=O)NC(C)C)c1
InChIKey: PEJTUNMIBFQQFF-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 474.57, ALogP 4.1, HBA 8, HBD 1, PSA 94.4, QED 0.41
Compound (SMILES): COc1ccccc1OCC(=O)NCC(O)CN1CCc2ccccc2C1
InChIKey: NULKXNMSJKHHEP-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay: Molecular Biology: Full-length human PRMT5 (NM_006109.3) transcript variant 1 clone was amplified from a fetal brain cDNA library, incorporating flanking 5′ sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:4) fused directly to Ala 2 of PRMT5. Full-length human MEP50 (NM_024102) clone was amplified from a human testis cDNA library incorporating a 5′ sequence encoding a 6-histidine tag (MHHHHHH) (SEQ ID NO.:5) fused directly to Arg 2 of MEP50. The amplified genes were sublconed into pENTR/D/TEV (Life Technologies) and subsequently transferred by Gateway attL×attR recombination to pDEST8 baculvirus expression vector (Life Technologies).Protein Expression. Recombinant baculovirus and Baculovirus-Infected Insect Cells (BIIC) were generated according to Bac-to-Bac kit instructions (Life Technologies) and Wasilko, 2006, respectively. Protein over-expression was accomplished by infecting exponentially growing Spodoptera frugiperda (SF9) cell culture at 1.2×106 cell/ml with a 5000 fold dilution of BIIC stock. Infections were carried out at 27° C. for 72 hours, harvested by centrifugation, and stored at −80° C. for purification.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 370.45, ALogP 1.61, HBA 5, HBD 2, PSA 71.03, QED 0.74
Compound (SMILES): CC(C)c1cccc(C(C)C)c1NC(=O)CC(=O)C(Cc1ccccc1)Cc1ccc(OC(=O)CCCCCCN2CCCCCC2=O)cc1
InChIKey: YXOPOKKYMJEBRR-UHFFFAOYSA-N
Target: Sterol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P35610
Assay: Inhibition Assay: Methods for assessing the selectively of ACAT1 inhibitors are known in the art and can be based upon any conventional assay including, but not limited to the determination of the half maximal (50%) inhibitory concentration (IC) of a substance (i.e., 50% IC, or IC50), the binding affinity of the inhibitor (i.e., Ki), and/or the half maximal effective concentration (EC50) of the inhibitor for ACAT1 as compared to ACAT2. See, e.g., Lada, et al. (2004) J. Lipid Res. 45:378-386 and U.S. Pat. No. 5,968,749. ACAT1 and ACAT2 proteins that can be employed in such assays are well-known in the art and set forth, e.g., in GENBANK Accession Nos. NP-000010 (human ACAT1) and NP-005882 (human ACAT2). See also U.S. Pat. No. 5,834,283.
Measurement: IC50 = 3.6 nM (pChEMBL 8.4399995803833)
Descriptors: MW 680.93, ALogP 9.19, HBA 5, HBD 1, PSA 92.78, QED 0.06
Compound (SMILES): O=C(COc1cccc2cccnc12)NC[C@H](O)CN1Cc2ccccc2C1
InChIKey: DOONEOBBMQKESC-IBGZPJMESA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay: General Materials.S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine, KCl, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), and Tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) were purchased from Sigma-Aldrich at the highest level of purity possible. 3H-SAM was purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol. 384-well streptavidin Flashplates were purchased from PerkinElmer.Substrates.Peptide representative of human histone H4 residues 1-15 was synthesized with a C-terminal linker-affinity tag motif and a C-terminal amide cap by 21st Century Biochemicals. The peptide was high high-performance liquid chromatography (HPLC) purified to greater than 95% purity and confirmed by liquid chromatography mass spectrometry (LC-MS). The sequence was Ac-SGRGKGGKGLGKGGA[K-Biot]-amide (SEQ ID NO.:3).Molecular Biology:Full-length human PRMT5 (NM_006109.3) transcript variant 1 clone was amplified from a fetal brain cDNA library, incorporating flanking 5′ sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:4) fused directly to Ala 2 of PRMT5. Full-length human MEP50 (NM_024102) clone was amplified from a human testis cDNA library incorporating a 5′ sequence encoding a 6-histidine tag (MHHHHHH) (SEQ ID NO.:5) fused directly to Arg 2 of MEP50. The amplified genes were sublconed into pENTR/D/TEV (Life Technologies) and subsequently transferred by Gateway attL×attR recombination to pDEST8 baculvirus expression vector (Life Technologies).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 377.44, ALogP 2.11, HBA 5, HBD 2, PSA 74.69, QED 0.66
Compound (SMILES): C=C[C@@H]1C[C@]1(NC(=O)[C@@H]1C[C@@H]2CN1C(=O)[C@H](C(C)(C)C)NC(=O)O[C@@H]1CCC[C@H]1CCCCCc1nc3ccc(OC)cc3nc1O2)C(=O)NS(=O)(=O)C1CC1
InChIKey: PQEXJBQAOYIYHO-IYPPLZFMSA-N
Target: NS3 (SINGLE PROTEIN), Hepatitis C virus
UniProt: A3EZJ3
Assay: Inhibition of HCV subtype 1b NS3 protease D168A mutant pre-incubated for 30 mins before TRF peptide substrate addition and measured after 1 hr by time resolved fluorescence assay
Measurement: Ki = 0.86 nM (pChEMBL 9.069999694824219)
Descriptors: MW 794.97, ALogP 4.08, HBA 11, HBD 3, PSA 195.22, QED 0.34
Compound (SMILES): N#Cc1nc(NC[C@H](O)c2ccccc2)nc(N2CCNCC2)n1
InChIKey: TYRKGQFUSLPRSY-ZDUSSCGKSA-N
Target: Procathepsin L (SINGLE PROTEIN), Homo sapiens
UniProt: P07711
Assay: Inhibition of human cathepsin L using Z-Phe-Arg-AMC as substrate after 30 mins by spectromicrofluorimetric method
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 325.38, ALogP 0.3, HBA 8, HBD 3, PSA 109.99, QED 0.72
Compound (SMILES): Cc1ccccc1COc1ccc(S(=O)(=O)N2CCC[C@](C)(O)[C@@H]2C(=O)NO)cc1
InChIKey: FYNYGUYHKZWCAU-FPOVZHCZSA-N
Target: Disintegrin and metalloproteinase domain-containing protein 17 (SINGLE PROTEIN), Homo sapiens
UniProt: P78536
Assay: Inhibition of TACE in human mononuclear cells assessed as inhibition of LPS-induced TNF-alpha production measured after 4 hrs in presence of LPS by ELISA
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 434.51, ALogP 1.98, HBA 6, HBD 3, PSA 116.17, QED 0.47
Compound (SMILES): On1nnc2cc(CCc3noc(=S)[nH]3)ccc21
InChIKey: LARWFEHEXZGWQL-UHFFFAOYSA-N
Target: Catenin beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P35222
Assay: AlphaScreen Assay: Experiments were performed in white opaque 384-well plates from PerkinElmer (Waltham, Mass.), and the samples were read on a Synergy 2 plate reader (Biotek, Winooski, Vt.) with a sensitivity setting of 200 using AlphaScreen protocol with excitation at 680 nm and emission at 570 nm. All dilutions were made in 1× assay buffer containing 25 mM HEPES (pH 7.4), 100 mMNaCl and 0.1% BSA to minimize nonspecific interactions. For inhibitor competition experiment, 5 nM of C-terminal biotinylated Tcf4 45-mer, 20 nM of N-terminal His6-tagged β-catenin and inhibitors at different concentration were incubated in 20 μL of assay buffer for 2 h. Donor and acceptor beads were added to a final concentration of 10 μg/mL in 25 μL of assay buffer. The mixture was incubated at room temperature for 1 h. For each inhibitor competition assay, the negative controls (equivalent to 0% inhibition) refer to 5.0 nM of biotinylated Tcf4 45-mer, 20 nM of β-catenin, 10 g/mL donor and acceptor beads, but no tested peptide inhibitor was presented. The positive controls (equivalent to 100% inhibition) refer to only 5.0 nM of biotinylated Tcf4 45-mer and 10 g/mL donor and acceptor beads in a final volume of 25 μL. The IC50 value was determined by nonlinear least-square analysis of GraphPad Prism 5.0. Experiments were performed in triplicate and carried out in the presence of 1% DMSO.
Measurement: Ki = 7240 nM (pChEMBL 5.139999866485596)
Descriptors: MW 263.28, ALogP 1.5, HBA 7, HBD 2, PSA 92.76, QED 0.55
Compound (SMILES): CC(C)(C)c1cc(NC(=O)Nc2ccc(Oc3ccnc(Nc4ccc5[nH]ccc5c4)n3)c3ccccc23)n(-c2ccc(O)cc2)n1
InChIKey: VDYAAIDBWJMZBD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 142 nM (pChEMBL 6.849999904632568)
Descriptors: MW 624.71, ALogP 8.48, HBA 8, HBD 5, PSA 142.01, QED 0.12
Compound (SMILES): CC(C)C[C@H](O)C(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(O)c1)C2)CC1CCCCC1
InChIKey: OHMKATQKHCVVAN-SFIUMABJSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 456.67, ALogP 4.92, HBA 4, HBD 2, PSA 64.01, QED 0.55
Compound (SMILES): Cc1cccc(COc2ccccc2C(=O)N(CCCN)Cc2cccs2)c1.Cl
InChIKey: UDXGBANGPYONOK-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily M member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z2W7
Assay: Antagonist activity at human recombinant TRPM8 expressed in HEK293 cells assessed as inhibition of icilin-induced calcium increase by Fluo-4 AM based assay
Measurement: IC50 = 448 nM (pChEMBL 6.349999904632568)
Descriptors: MW 394.54, ALogP 4.63, HBA 4, HBD 1, PSA 55.56, QED 0.58
Compound (SMILES): C[C@H]1Cc2c(nnn2-c2ncccc2F)CN1C(=O)c1cccc(Cl)c1Cl
InChIKey: VWBWBQPTYDWJSA-JTQLQIEISA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 18.5 nM (pChEMBL 7.730000019073486)
Descriptors: MW 406.25, ALogP 3.7, HBA 5, HBD 0, PSA 63.91, QED 0.65
Compound (SMILES): O=C(O)COC[C@H]1CC[C@@H](COC(=O)N(c2ccccc2)c2ccccc2)CC1
InChIKey: GDJMTGQHKNDISN-KDURUIRLSA-N
Target: Prostaglandin D2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q13258
Assay: Agonist activity at recombinant human DP1 receptor expressed in CHO-K1 cells assessed as increase in intracellular cAMP level after 1 hr incubation by HTRF method
Measurement: EC50 = 1710 nM (pChEMBL 5.769999980926514)
Descriptors: MW 397.47, ALogP 4.87, HBA 4, HBD 1, PSA 76.07, QED 0.69
Compound (SMILES): Cc1ncc(C(CNC(=O)c2cccc(Cl)c2Cl)N2CCOCC2)cn1
InChIKey: DVEMCJONIRIHRK-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO42H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates. 93-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1X); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes. Two sets of drug plates were prepared: A) Mixtures of compound plus agonist were prepared as follows, in order to determine close response: BzATP: 11 point ½ log, diluted in buffer, starting from 1 mM. Testing compounds: 11 point ½ log, diluted in 2% DMSO buffer starting from 10 μM. B) Agonist only mixture was prepared with BzATP at a single concentration in buffer (concentration determined by dose response). Compound mixtures (A) were added to assay plates containing cells and placed at room temperature for 30 minutes, then BzATP (B) was added. Fluorescence was read using the Tetra FLIPR® (Molecular Devices, Inc., Sunnyvale, Calif., USA) and IC50 values were calculated by standard methods to determine antagonist activity.
Measurement: IC50 = 69 nM (pChEMBL 7.159999847412109)
Descriptors: MW 395.29, ALogP 2.9, HBA 5, HBD 1, PSA 67.35, QED 0.84
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3F)ncnc2cc1OC[C@@H]1CN2CCCC[C@@H]2C(=O)N1
InChIKey: ZDTMGSSRQLKYTK-SCLBCKFNSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 520.39, ALogP 4.56, HBA 7, HBD 2, PSA 88.61, QED 0.46
Compound (SMILES): O=C(/C=C/c1ccccc1)Nc1ccc(C(=O)NS(=O)(=O)c2ccc(NCCSc3ccccc3)c([N+](=O)[O-])c2)cc1
InChIKey: BDOWOEXBFLRGBF-WOJGMQOQSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Inhibition of 5-FAM-Bid peptide binding to MCL-1 (unknown origin) by fluorescence polarization assay
Measurement: Ki = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 602.69, ALogP 5.57, HBA 8, HBD 3, PSA 147.51, QED 0.06
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCC(N2CCC(O)CC2)CC1
InChIKey: OUVGXRDDQMDFAQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 273 nM (pChEMBL 6.559999942779541)
Descriptors: MW 440.52, ALogP 2.87, HBA 7, HBD 3, PSA 120.2, QED 0.64
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1C=Cc2c(ncn2-c2cnccn2)C1
InChIKey: SZTATUNQORHJBD-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 4111.5 nM (pChEMBL 5.389999866485596)
Descriptors: MW 405.77, ALogP 3.96, HBA 5, HBD 0, PSA 63.91, QED 0.65
Compound (SMILES): COc1ccc(C2(OC/C=C\Cn3ccnc3CCC(=O)O)c3ccccc3-c3ccccc32)cc1
InChIKey: PIEZLFLTLKUGEC-SREVYHEPSA-N
Target: GABA transporter 3 (SINGLE PROTEIN), Mus musculus
UniProt: P31649
Assay: Inhibition of mouse GAT3 expressed in HEK293 cells assessed as decrease in [3H]GABA uptake preincubated for 25 mins followed by [3H]GABA addition measured after 4 mins by liquid scintillation counter method
Measurement: IC50 = 15848.93 nM (pChEMBL 4.800000190734863)
Descriptors: MW 480.56, ALogP 5.45, HBA 5, HBD 1, PSA 73.58, QED 0.31
Compound (SMILES): N#CC[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: FZXMPCXMYBNDEB-XYPYZODXSA-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 = 32 nM (pChEMBL 7.5)
Descriptors: MW 280.34, ALogP 2.79, HBA 5, HBD 1, PSA 82.66, QED 0.78
Compound (SMILES): O=C(OCC12CCN(CC1)CC2)c1c[nH]c2cc(O)ccc12
InChIKey: IAFWPIRKIGHVRK-UHFFFAOYSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Cytochrome P450 Inhibition: Protocol 2: Stock solution for NCEs (test articles) and standard inhibitors were prepared at a concentration of 20 mM in DMSO. Stock solutions were diluted with DMSO to get substocks which were further diluted 10-fold in 80:20 MeCN:water to obtain intermediate substocks. Working stocks were prepared by diluting the intermediate substocks 50-fold in buffer.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 300.36, ALogP 2.52, HBA 4, HBD 2, PSA 65.56, QED 0.85
Compound (SMILES): Cc1ccncc1-n1c([C@H](C)Nc2nc(N)nc(N)c2C#N)nc2cccc(Cl)c2c1=O
InChIKey: UAFKEIXHLUCWPA-NSHDSACASA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of human full length PI3K p110delta/p85alpha using phosphatidylinositol 3,4,5-trisphosphate as substrate measured after 30 mins by TR-FRET assay
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 447.89, ALogP 2.74, HBA 10, HBD 3, PSA 161.42, QED 0.43
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(-c4ccc5nccn5c4)c(F)c3)CC2)C1
InChIKey: LGVQWGBMXQBALU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In vitro JAK Kinase Assay: The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.)
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 600.66, ALogP 4.5, HBA 9, HBD 1, PSA 124.03, QED 0.3
Compound (SMILES): N#Cc1nc2ccc(C(O)(c3ccc(C(F)(F)F)nc3)c3cnc[nH]3)cc2c(C#N)c1Oc1ccccc1
InChIKey: SHUIPTRYEZOHNC-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: RORγt Reporter Assay: The reporter assay was performed by transiently transfecting HEK293T cells with 5 μg of pBIND-RORγt LBD or pBIND-RORγt LBD-AF2 and 5 μg pGL4.31 (Promega Cat no. C935A) using Fugene 6 (Invitrogen Cat no. E2691) at a 1:6 ratio of DNA: Fugene 6 in a T-75 flask in which cells were at least 80% confluent. Twenty four hours after bulk transfection, cells were plated into 96-well plates at 50,000 cells/well in phenol-red free DMEM containing 5% Lipid Reduced FCS and Pen/Strep. Six hours after plating, cells were treated with compounds for 24 hours. Media was removed and cells were lysed with 50 μL 1x Glo Lysis Buffer (Promega). Dual Glo Luciferase Reagent (50 μL/well) was then added and firefly luciferase luminescence was read on an Envision after a ten minute incubation. Finally, Stop and Glo reagent (50 μL/well) was added and renilla luciferase luminescence was read on an Envision after a ten minute incubation.
Measurement: IC50 = 6000 nM (pChEMBL 5.21999979019165)
Descriptors: MW 512.45, ALogP 5.19, HBA 7, HBD 2, PSA 131.5, QED 0.33
Compound (SMILES): Cc1cccc(C(=O)N2C[C@@H](C)OC[C@H]2Cc2cccc(-c3ncccn3)c2)c1-n1nccn1
InChIKey: QDURYEZFCXJVOM-DENIHFKCSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 = 40000 nM (pChEMBL 4.400000095367432)
Descriptors: MW 454.53, ALogP 3.5, HBA 7, HBD 0, PSA 86.03, QED 0.46
Compound (SMILES): Cc1c([C@H]2CN3CCN(C(=O)Cc4ccc(-n5cnnn5)nc4)C[C@H]3CO2)ccc2c1COC2=O
InChIKey: YPUSXXSDSCDEJP-LAUBAEHRSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG by electrophysiology method
Measurement: IC50 = 56500 nM (pChEMBL 4.25)
Descriptors: MW 475.51, ALogP 0.86, HBA 10, HBD 0, PSA 115.57, QED 0.51
Compound (SMILES): Cc1cc2ccc1[C@@H](C)COC(=O)Nc1ccc(C3(C(=O)O)CCC3)c(c1)CN(C)C(=O)[C@@H]2Nc1ccc2c(N)ncc(F)c2c1
InChIKey: PROYMPSAMYSXNO-WENCNXQZSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Inhibition of thrombin (unknown origin) at 25 degC by chromogenic substrate assay
Measurement: Ki > 13300 nM (pChEMBL 4.875999927520752)
Descriptors: MW 625.7, ALogP 6.25, HBA 7, HBD 4, PSA 146.88, QED 0.21
Compound (SMILES): C[C@H]1CCCCN1c1cccc(-c2c[nH]c3ccc(-c4nnc(N)s4)cc23)n1
InChIKey: WNIIXCCZBNUGMW-ZDUSSCGKSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 9.735 nM (pChEMBL 8.010000228881836)
Descriptors: MW 390.52, ALogP 4.71, HBA 6, HBD 2, PSA 83.72, QED 0.53
Compound (SMILES): N#Cc1ccc(OCC(=O)NCC(O)CN2CCc3ccccc3C2)cc1
InChIKey: JOVUEDKSBHZOHS-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay: Molecular Biology: Full-length human PRMT5 (NM_006109.3) transcript variant 1 clone was amplified from a fetal brain cDNA library, incorporating flanking 5′ sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:4) fused directly to Ala 2 of PRMT5. Full-length human MEP50 (NM_024102) clone was amplified from a human testis cDNA library incorporating a 5′ sequence encoding a 6-histidine tag (MHHHHHH) (SEQ ID NO.:5) fused directly to Arg 2 of MEP50. The amplified genes were sublconed into pENTR/D/TEV (Life Technologies) and subsequently transferred by Gateway attL×attR recombination to pDEST8 baculvirus expression vector (Life Technologies).Protein Expression. Recombinant baculovirus and Baculovirus-Infected Insect Cells (BIIC) were generated according to Bac-to-Bac kit instructions (Life Technologies) and Wasilko, 2006, respectively. Protein over-expression was accomplished by infecting exponentially growing Spodoptera frugiperda (SF9) cell culture at 1.2×106 cell/ml with a 5000 fold dilution of BIIC stock. Infections were carried out at 27° C. for 72 hours, harvested by centrifugation, and stored at −80° C. for purification.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 365.43, ALogP 1.47, HBA 5, HBD 2, PSA 85.59, QED 0.78
Compound (SMILES): CCc1ccc(S(=O)(=O)[C@@]2(c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)CCN(C(=O)[C@H]3CC[C@H](C(=O)O)CC3)C2)cc1
InChIKey: LABVKPUHHCCTTJ-DYLHXGEVSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Inverse agonist activity at GAL4-fused RORgammat LBD (267 to 516 residues) (unknown origin) expressed in human Jurkat cells measured after 18 hrs by native IL17 promoter driven steady-glo luciferase assay
Measurement: EC50 = 84 nM (pChEMBL 7.079999923706055)
Descriptors: MW 637.61, ALogP 6.33, HBA 4, HBD 1, PSA 91.75, QED 0.36
Compound (SMILES): CN1CCC(Oc2ccc(-c3cc4c(-c5cccc(N6CCOc7cc(C8CC8)ccc7C6=O)c5CO)ncnc4[nH]3)cc2)CC1
InChIKey: AEVXRMQSNKOQLJ-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 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 615.73, ALogP 6.17, HBA 7, HBD 2, PSA 103.81, QED 0.23
Compound (SMILES): CC(C)COC(=O)N1CCC(N2CC(CC#N)(n3cc(-c4ncnc5[nH]ccc45)cn3)C2)CC1
InChIKey: UYKHLAQFMHOIFI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In vitro JAK Kinase Assay: The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.)
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 462.56, ALogP 3, HBA 8, HBD 1, PSA 115.96, QED 0.6
Compound (SMILES): O=S(=O)(Nc1nccs1)c1ccc2c(c1)OCCN2c1cccc(OC(F)F)c1
InChIKey: YMQJEMQDDDAVNP-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 439.46, ALogP 4.08, HBA 7, HBD 1, PSA 80.76, QED 0.62
Compound (SMILES): FC(F)(F)c1cccc(CCNC2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)c1
InChIKey: IFPBVNDZBIKCOL-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 > 1000 nM (pChEMBL 6)
Descriptors: MW 428.46, ALogP 4.48, HBA 5, HBD 2, PSA 70.9, QED 0.49