lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CS(=O)(=O)Nc1cccc(CSc2nc(N)c(C#N)c(-c3ccc(F)c(F)c3)c2C#N)c1
InChIKey: GJYWCDABXNMGIQ-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Luciferase Assay: The stock cultures are grown, at 37° C. and under 5% CO2, in DMEM/F12 medium containing 10% FCS (fetal calf serum) and in each case split 1:10 after 2-3 days. Test cultures are seeded in 384-well plates with 2000 cells per well and grown at 37° C. for approx. 48 hours. The medium is then replaced with a physiological sodium chloride solution (130 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, 20 mM HEPES, 1 mM magnesium chloride hexahydrate, 5 mM sodium bicarbonate, pH 7.4). The substances to be tested, which are dissolved in DMSO, are pipetted into the test cultures (maximum final concentration of DMSO in the test mixture: 0.5%) in a dilution series of from 5x10-11 M to 3x10-6 M (final concentration). 10 minutes later, forskolin is added to the A1 cells and all the cultures are subsequently incubated at 37° C. for four hours. After that, 35 ul of a solution which is composed of 50% lysis reagent (30 mM disodium hydrogenphosphate, 10% glycerol, 3% TritonX100, 25 mM TrisHCl, 2 mM dithiotreitol (DTT), pH 7.8) and 50% luciferase substrate solution (2.5 mM ATP, 0.5 mM luciferin, 0.1 mM coenzyme A, 10 mM tricine, 1.35 mM magnesium sulfate, 15 mM DTT, pH 7.8) are added to the test cultures, which are shaken for approx. 1 minute and the luciferase activity is measured using a camera system.
Measurement: EC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 471.51, ALogP 4.02, HBA 7, HBD 2, PSA 132.66, QED 0.52
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(-c3cncc(Cl)c3)cc1C21N=C(N)N(C(C)C)C1=O
InChIKey: IEYSGHASPXBCSY-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.434): Inhibitory activity of compounds was assessed by a fluorescence quench assay of BACE activity using commercially available substrate HiLyte Fluor 488-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-(QXLT 520)-OH (AnaSpec, San Jose, Calif.) and truncated human beta-secretase (residues 1-458, His6-tagged at the C-terminus) expressed in insect cells D. melanogaster S2 using a baculovirus expression system (Mallender et al., Characterization of recombinant, soluble beta-secretase from an insect cell expression system., Mol Pharmacol 59:619-26, 2001). The assay was performed at room temperature in 96-well white opaque Optiplates aque Optiplates (PerkinElmer, Waltham, Mass.) in a total volume of 200 ul of the incubation mixture containing 50 mM sodium acetate buffer, pH 4.5, 0.4 uM FRET substrate, 2.4 nM enzyme, 5% DMSO, and 0.05% Brij-35. The tested compounds were serially diluted in DMSO and pre-incubated with the substrate. The reaction was started by addition of enzyme.
Measurement: IC50 = 8.9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 452.99, ALogP 4.29, HBA 5, HBD 1, PSA 80.81, QED 0.75
Compound (SMILES): Cc1cnccc1NC(=O)c1cc(-c2ccc(-n3cccn3)cc2)cnc1N
InChIKey: TYXXLNSGWOQLJI-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 : The kinase assay is performed as 384-well Flashplate assay assay (for e.g. Topcount measurement. 0.6 nM TANK binding kinase (TBK1), 800 nM biotinylated MELK-derived peptide (Biotin-Ah-Ah-AKPKGNKDYHLQTCCGSLAYRRR) SEQ ID NO: 2 and 10 uM ATP (spiked with 0.25 uCi 33P-ATP/well) are incubated in a total volume of 50 ul (10 mM MOPS, 10 mM Mg-acetat, 0.1 mM EGTA, 1 mM DTT, 0.02% Brij35, 0.1% BSA, pH 7.5) with or without test compound for 120 Min at 30° C. The reaction is stopped with 25 ul 200 mM EDTA. After 30 Min at room temperature the liquid is removed and each well washed thrice with 100 ul 0.9% sodium chloride solution.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 370.42, ALogP 3.47, HBA 6, HBD 2, PSA 98.72, QED 0.57
Compound (SMILES): COc1ccc(CC(=O)O)cc1-c1ccc(Cl)c2c1CN(C(=O)CCN1CCc3ccccc31)CC2
InChIKey: GHQSNJQDDVJBPU-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 = 61.5 nM (pChEMBL 7.210000038146973)
Descriptors: MW 505.01, ALogP 4.98, HBA 4, HBD 1, PSA 70.08, QED 0.49
Compound (SMILES): COn1c(NC(c2ccc(Cl)cc2)C(C)C)nc2c(c1=O)CN(C(=O)c1ccc3c(c1)OCCO3)CC2
InChIKey: MXFUVWBKHNWQHI-UHFFFAOYSA-N
Target: Prolactin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P49683
Assay: Inhibition Assay: Receptor-expressing HEK 293 cell membrane fraction was analyzed by modifying the methods of C. J. Langmead et al. (see Langmead C J, Szekeres P G; Chambers J K, Ratcliffe S J, Jones D N C, Hirst W D, et al. Characterization of the binding of [125I]-human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor. Br J Pharmacol 2000; 131: 683-688.). To each well of a 96-well plate, 150 μl of assay buffer (20 mM HEPES, 10 mM EDTA, 1 μl/ml protease inhibiting agent cocktail, pH 7.4), 20 μl of cell membrane fraction, 10 μl of test compound and [3H]-PrRP (final concentration 1 nM) were added and incubated at room temperature for 90 minutes. After completion of the reaction, using a cell harvester, the cell-membrane fraction sample product was filtered under aspiration by a glass fiber filter plate (Unifilter; GF/B) previously treated with 0.5% polyethylene imine. The filter was washed with a 50 mM Tris hydrochloric acid buffer (pH 7.4) three times.
Measurement: Ki = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 525.01, ALogP 3.73, HBA 8, HBD 1, PSA 94.92, QED 0.52
Compound (SMILES): NC(=O)C1CN(C(=O)c2ccc(-c3cc(NC4CC4)n4ncc(/C=C5\NC(=O)NC5=O)c4n3)s2)CCN1
InChIKey: OGGWSIVTFDIJRH-AUWJEWJLSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: 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; 5 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
Measurement: IC50 = 1724.5 nM (pChEMBL 5.760000228881836)
Descriptors: MW 521.56, ALogP 0.11, HBA 10, HBD 5, PSA 175.85, QED 0.22
Compound (SMILES): CC(=O)N[C@@H](C)c1ccc(OC2CCN(c3cc4c(cc3F)OCCO4)C2)cc1
InChIKey: WKNWSOQPSAGUBM-PIVQAISJSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 200 μM f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
Measurement: IC50 = 226 nM (pChEMBL 6.650000095367432)
Descriptors: MW 400.45, ALogP 3.45, HBA 5, HBD 1, PSA 60.03, QED 0.83
Compound (SMILES): COc1cc(F)cc(-c2ccc(-n3c(=O)ccc4cc(S(=O)(=O)Nc5cc(C)ncn5)ccc43)c(OC)c2)c1
InChIKey: QJHSWINHOKIIBL-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 = 219 nM (pChEMBL 6.659999847412109)
Descriptors: MW 546.58, ALogP 4.71, HBA 8, HBD 1, PSA 112.41, QED 0.31
Compound (SMILES): COCCn1ccc(Nc2ncc3c(n2)-c2c(nn(C)c2-c2ccc(-c4cnn(CCO)c4)cc2)CC3)n1
InChIKey: UVJDZKBPVPTIRA-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: DELFIA Assay: The kinase activity is measured by DELFIA assay (dissociation-enhanced lanthanide fluorescence immunoassay, Perkin Elmer). The cytoplasmic kinase domain of human IGF-1R (amino acids 964-1370) is expressed as a fusion protein with a glutathione-S-transferase tag (IGF-1R-GST) in High Five.TM. Cells (Invitrogen). Enzyme activity is measured in the presence of substances and a control substance. Poly-glutamate-tyrosine peptide (pEY, Sigma Aldrich) and biotinylated pEY (bio-pEY) are used as reaction substrates.10 uL of substance in 25% DMSO are mixed with 30 uL of IGF-1R-GST solution (67 mM HEPES pH 7.4, 15 ug/mL pEY, 1.7 ug/mL bio-pEY, 13.3 mM MgCl2, 3.3 mM dithiothreitol, 0.0033% Brij 35, 2 ng IGF-1R-GST) in 96-well plates. The reactions are started with 10 uL of a 750 uM ATP solution. After 40 min at RT the reactions are stopped with 50 .mu.L of stop solution (250 mM EDTA, 20 mM HEPES pH 7.4). 90 uL from each reaction are transferred.
Measurement: IC50 = 0.38 nM (pChEMBL 9.420000076293945)
Descriptors: MW 511.59, ALogP 3.09, HBA 11, HBD 2, PSA 120.73, QED 0.31
Compound (SMILES): N#C[C@@H]1CCN(C(=O)N2CC[C@](c3ccc(C(OCc4c(F)cccc4F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)C1
InChIKey: ULWFIWXDJILZFM-WRONEBCDSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 719.63, ALogP 6.98, HBA 5, HBD 0, PSA 90.71, QED 0.19
Compound (SMILES): COc1ncc(-c2ccc3nccc(-c4ccc(CN(C)CCO)cc4)c3c2)cc1NS(=O)(=O)c1ccc(F)cc1
InChIKey: FSMNNDQUPANPBB-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition of PI3Kalpha (unknown origin) using PIP2 as substrate after 1 hr in presence of ATP by kinase-glo luminescence assay
Measurement: IC50 = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 572.66, ALogP 5.34, HBA 7, HBD 2, PSA 104.65, QED 0.23
Compound (SMILES): N#Cc1nc(C(=O)NCC(C(=O)O)C2CC2)c(O)c2ccc(Oc3ccc(F)cc3)cc12
InChIKey: ZFKJTWWTFDTGHS-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 = 950 nM (pChEMBL 6.019999980926514)
Descriptors: MW 435.41, ALogP 3.58, HBA 6, HBD 3, PSA 132.54, QED 0.52
Compound (SMILES): Cc1nc2cccc(-c3cc4c(n3C)C(CO)NC4=O)c2nc1NC(C)(C)C
InChIKey: GFNSSGCOEONQCH-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 = 10.3 nM (pChEMBL 7.989999771118164)
Descriptors: MW 379.46, ALogP 2.93, HBA 6, HBD 3, PSA 92.07, QED 0.65
Compound (SMILES): C=C[C@]1(C)C[C@]1(NC(=O)[C@@H]1C[C@@]2(CN1C(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H]1CCCCN1C(C)C)C1CCCCC1)C(C)(C)C)C(C)(C)C21CCC1)C(=O)NS(=O)(=O)N(C)CC1CC1
InChIKey: UYGSFRWIOMLYCW-KKVQSYGZSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Protease Assay: The inhibitory activity of certain compounds of Table A against HCV NS3-4A serine protease is determined in a homogenous assay using the full-length NS3-4A protein (genotype 1a, strain HCV-1) and a commercially available internally-quenched fluorogenic peptide substrate as described by Taliani, M., et al. 1996 Anal. Biochem. 240:60-67, which is incorporated by reference in its entirety.
Measurement: IC50 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 884.24, ALogP 4.8, HBA 8, HBD 4, PSA 177.33, QED 0.17
Compound (SMILES): Cc1cc(NC(=O)N2CCN(c3ncc(-c4nc(CO)no4)cc3Cl)CC2)ccc1C(F)(F)F
InChIKey: UDCUPMRVPHGUGD-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-Based Assay: Two days prior to performing this assay, cells are seeded on poly-D-lysine-coated 96-well clear-bottom black plates (commercially available from Becton-Dickinson) at 75,000 cells/well in growth media containing 5 uM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1x Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1.6 mM CaCl2 and 20 mM HEPES, pH 7.4 (wash buffer), and loaded using 0.1 mL of wash buffer containing Fluo-4 (3 uM final concentration, commercially available from Molecular Probes). After 1 h, the cells are washed twice with 0.2 mL wash buffer and resuspended in 0.05 mL 1x Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 3.5 mM CaCl2 and 10 mM Citrate, pH 7.4 (assay buffer). Plates are then transferred to a FLIPR for assay.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 496.88, ALogP 3.96, HBA 7, HBD 2, PSA 107.62, QED 0.56
Compound (SMILES): CN(C)Cc1ccc(CSCCN2C(=O)c3ccc([N+](=O)[O-])cc3C2=O)o1
InChIKey: AJFBCSHQVYMJOM-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Inhibition of electric eel AChE using acetylthiocholine as substrate preincubated for 8 mins followed by substrate addition by Ellman's method
Measurement: IC50 = 46000 nM (pChEMBL 4.340000152587891)
Descriptors: MW 389.43, ALogP 2.78, HBA 7, HBD 0, PSA 96.9, QED 0.3
Compound (SMILES): CC(C)(C)c1cc(NC(=O)c2ccc3cc4n(c3c2)CCCNC4=O)sn1
InChIKey: OLJQPXLXHUFJFS-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15349
Assay: Kinase Glo Plus Assay: Human RSK2 protein, purchased from Invitrogen, is used to measure kinase activity utilizing Kinase Glo Plus (Promega) a homogeneous assay technology, which uses a luciferin-luciferase based ATP detection reagent to quantify residual ATP. The assay is performed using 0.75 nM His-RSK2, 0.75 .mu.M ATP and 1.0 uM S6 Kinase/RSK Substrate Peptide 1 (Upstate, catalog #12-124), in assay buffer consisting of 25 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 uM Na3VO4, 0.5 mM DTT, and 1% DMSO. Solutions of test compounds at various concentrations are prepared by 1:3 fold serial dilution of a 1 mM solution of compound in DMSO. The DMSO solutions are further diluted with assay buffer to a final concentration of DMSO of 5%. The assay is performed in a 384 well, white, non-binding plate (Corning, catalogue #3574). Solutions of test compounds (10 uL) are transferred to a dry assay plate, followed by addition of 20 uL kinase.
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 382.49, ALogP 3.78, HBA 5, HBD 2, PSA 76.02, QED 0.71
Compound (SMILES): COc1cc(C(=O)N2CCN(C)CC2)ccc1Nc1nc(Cc2c(Cl)cccc2Cl)n2ccnc2c1C(N)=O
InChIKey: HHFQVKTVYJPJEF-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Enzyme Inhibition Assay: ALK kinase assays were conducted with the indicated final concentrations unless otherwise specified. In 384 well black plates (Axygen), 8 ul of compound (2% DMSO) was incubated with 8 ul Lck-peptide substrate (0.5 uM, biotin-Ahx-GAEEEIYAAFFA-COOH) and 8 ul of a mixture of ALK (3 nM, Millipore) and ATP (50 uM) in reaction buffer (50 mM Hepes, pH 7.4; 10 mM MgCl2; 2 mM MnCl2; 0.1 mM sodium orthovanadate; 0.01% BSA and 1 mM DTT (added fresh before assay) for 1 h at room temperature. Reactions were then quenched by the addition of 30 ul quench solution (streptavidin-allophycocyanin and Europium-cryptate PT66 monoclonal antibody in 40 mM Hepes, pH 7.4; 480 mM KF; 66 mM EDTA; 0.01% Tween-20; and 0.1% BSA) at room temperature. Plates were read 1 h after quenching on an Envision Multilaber Reader and IC50 values were calculated using a sigmoidal fit of the concentration/inhibition response curves. These values were converted to apparent Ki values.
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 568.47, ALogP 3.87, HBA 8, HBD 2, PSA 118.09, QED 0.35
Compound (SMILES): O=S(=O)(NCC1CC1)c1ccc(NCC2CCOCC2)cc1C(F)(F)F
InChIKey: XJHHIIQDMWDELO-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Binding Assay: The binding assays were performed by testing the ability of the new compounds to displace the radiolabeled synthetic non-selective cannabinoid agonist [3H]CP55940 (168 Ci/mmol; PerkinElmer) from the human CB1 (hCB1) or human CB2 (hCB2) receptors on membranes derived from stably transfected HEK-293 cells (PerkinElmer). Membranes were diluted in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, pH=7.4). The amount of membrane was determined for each batch of membranes according to protein binding assay. The minimum amount of membrane that gave 50% specific binding was used for the binding assay. In most assays, binding was tested using 8 μg and 4 μg protein of hCB1 and hCB2 membranes, respectively. The tested compounds were dissolved in DMSO and diluted in assay buffer to a final concentration of 0.1% solvent. Total binding of [3H]CP55940 was evaluated with 1.5 nM to hCB1 and with 0.5 nM to hCB2, according to Kd affinity of [3H]CP55940 for the respective membranes. The ability of the tested compounds to displace [3H]CP55940 was evaluated first at a single concentration point of 100 nM for binding toward hCB2 or hCB1. In certain cases, the displacement was tested at compound concentrations ranging from 0.03 nM to 10 μM. Non-specific binding was measured by the addition of 1 μM of unlabelled CP55940 to the tubes. Binding assays were performed in triplicate in a total volume of 500 μl for 60 minutes at 30° C., in a shaking bath. Free and bound radioligands were separated by rapid filtration through GF/C filter plates (PerkinElmer) that had been presoaked with 0.1% Polyethylenimine (Sigma). Filters were shaken for 1 hour in 7 ml scintillation fluid (PerkinElmer) and radioactivity was determined by liquid scintillation counter (Wallac; PerkinElmer).
Measurement: Ki = 996 nM (pChEMBL 6)
Descriptors: MW 392.44, ALogP 3.23, HBA 4, HBD 2, PSA 67.43, QED 0.75
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N2CCC[C@@H]2C)c2n1CCOC2)c1ccccc1C(F)(F)F
InChIKey: SHILZLXEZORMIR-HNAYVOBHSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 463.5, ALogP 4.54, HBA 4, HBD 1, PSA 63.57, QED 0.71
Compound (SMILES): Cc1ncc(C)n2nc(/C=C/c3ccc4cc(F)ccc4n3)nc12
InChIKey: LDYXRCAAXRZLNX-SOFGYWHQSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Inhibition Assay: A PDE10A assay may for example, be performed as follows: The assay is performed in 60 uL samples containing a fixed amount of the relevant PDE enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 225 pCi of 3H-labelled cyclic nucleotide substrate, tritium labeled cAMP to a final concentration of 5 nM and varying amounts of inhibitors. Reactions are initiated by addition of the cyclic nucleotide substrate, and reactions are allowed to proceed for one hr at room temperature before being terminated through mixing with 15 uL 8 mg/mL yttrium silicate SPA beads (Amersham). The beads are allowed to settle for one hr in the dark before the plates are counted in a Wallac 1450 Microbeta counter.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 319.34, ALogP 3.6, HBA 5, HBD 0, PSA 55.97, QED 0.57
Compound (SMILES): CNC(=O)N(C)CCC#Cc1cn2c(-c3ccc(C(=O)NC4CC4)cc3)cnc2c(NCC(C)C)n1
InChIKey: VCVUVVNOSISFMD-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Mps-1 Kinase Assay: For the assay 50 nL of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 ul of a solution of Mps-1 in assay buffer [0.1 mM sodium- ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 ul of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 uM=> final conc. in the 5 ul assay volume is 10 uM) and peptide substrate (1.67 uM=> final conc. in the 5 ul assay volume is 1 uM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 ul assay volume). The reaction was stopped by the addition of 3 ul of a solution of HTRF detection reagents (100 mM Hepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nM anti-phospho(Ser/Thr)-Europium-antibody [#AD0180, PerkinElmer LAS, Rodgau-Jugesheim, Germany]. The resulting mixture was incubated 1 h at 22° C. to allow the binding of the phosphorylated peptide to the anti-phospho(Ser/Thr)-Europium-antibody. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Europium-labelled anti-phospho(Ser/Thr) antibody to the Streptavidin-XLent. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a Viewlux TR-FRET reader (PerkinElmer LAS, Rodgau-Jugesheim, Germany).
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 487.61, ALogP 3.37, HBA 6, HBD 3, PSA 103.66, QED 0.42
Compound (SMILES): NC(=O)c1cc(N[C@H]2CCNC2=O)nc(-c2ccc(Oc3ccc(Cl)cc3F)cc2)n1
InChIKey: HICKPJYJOOIJSI-HNNXBMFYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: In vitro assays were performed in a recombinant cell line expressing cDNA encoding the alpha subunit (Nav1.7, SCN9a, PN1, NE) of human Nav1.7 (Accession No. NM002977). The cell line was provided by investigators at Yale University (Cummins et al, J. Neurosci. 18(23): 9607-9619 (1998)). For dominant selection of the Nav1.7-expressing clones, the expression plasmid co-expressed the neomycin resistance gene. The cell line was constructed in the human embryonic kidney cell line, HEK293, under the influence of the CMV major late promoter, and stable clones were selected using limiting dilution cloning and antibiotic selection using the neomycin analogue, G418. Recombinant beta and gamma subunits were not introduced into this cell line. Additional cell lines expressing recombinant Nav1.7 cloned from other species can also be used, alone or in combination with various beta subunits, gamma subunits or chaperones.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 441.85, ALogP 3.13, HBA 6, HBD 3, PSA 119.23, QED 0.54
Compound (SMILES): C[C@H]1[C@@H](c2cc(C(F)(F)F)cc(C(F)(F)F)c2)OC(=O)N1CC1=C(c2cc(-c3ccc(C(=O)O)cc3C(F)(F)F)ccc2Cl)CCC(C)(C)C1
InChIKey: OBGPYICAGBJCJA-ADKRDUOOSA-N
Target: Cholesteryl ester transfer protein (SINGLE PROTEIN), Homo sapiens
UniProt: P11597
Assay: Cholesteryl Ester Transfer Assay: As a protein source for cholesteryl ester transfer, plasma from healthy persons was used, and as a cholesteryl ester receptor, LDL from healthy persons was used. Samples were treated with each test compound to final concentrations of 16, 80, 400, 2000 and 10000 nM and analyzed in duplicate. For the cholesteryl ester transfer test, 20 μl of plasma, 50 μl of LDL (0.25 mg/ml) and 50 μl of rHDL-agarose (0.25 mg/ml) were added, and a solution containing a test compound was added, followed by reaction at 37° C. Then, centrifugation was performed at 4° C. for 3 minutes to stop the reaction, and 150 μl of the supernatant was taken and transferred to a 96-well plate for radioactivity measurement, and the radioactivity of the plate was measured with a beta-ray detector.
Measurement: IC50 = 7.5 nM (pChEMBL 8.119999885559082)
Descriptors: MW 734.06, ALogP 11.31, HBA 3, HBD 1, PSA 66.84, QED 0.26
Compound (SMILES): CC(=O)C1=C(C)N(c2cccc(C(F)(F)F)c2)C(=O)N(C)C1c1ccc(C#N)cc1C(=O)N(C)Cc1cn[nH]c1
InChIKey: SFFDECBQEKWPBN-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available.The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA. Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 uL, of these compound dilutions were mixed with 10 uL Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 uL, substrate solution in assay buffer were added (250 uM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 550.54, ALogP 5.05, HBA 5, HBD 1, PSA 113.4, QED 0.46
Compound (SMILES): CC(C)OC(=O)/C=C/n1cnc(-c2cccc(Cl)c2)n1
InChIKey: KLXKTTGROYBSDM-VOTSOKGWSA-N
Target: Exportin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14980
Assay: Inhibition of Crm1 in human U2OS cells expressing GFP-fused HIV1 Rev assessed as decrease in nuclear export of GFP-fused HIV1 Rev after 1 hr by Hoechst 33258 staining-based assay
Measurement: IC50 >= 10000 nM (pChEMBL 5)
Descriptors: MW 291.74, ALogP 3.02, HBA 5, HBD 0, PSA 57.01, QED 0.64
Compound (SMILES): COc1ccc(Br)c(CC2CCN(CCc3cc(OC)c(OC)cc3Cl)CC2)c1
InChIKey: YDSPMZSSSJLARO-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Inhibition of human SERT expressed in CHO cell membranes assessed as reduction in [3H]serotonin uptake preincubated for 10 mins followed by [3H]serotonin addition measured after 20 mins by liquid scintillation counting method
Measurement: Ki = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 482.85, ALogP 5.63, HBA 4, HBD 0, PSA 30.93, QED 0.49
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(N3CCC(Oc4ccc(OC(F)(F)F)cc4)CC3)n2)c1
InChIKey: MRNSTJNYJIZTQO-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 760 nM (pChEMBL 6.119999885559082)
Descriptors: MW 488.47, ALogP 3.92, HBA 8, HBD 2, PSA 101.5, QED 0.52
Compound (SMILES): COc1nc(-c2ccccc2Cl)cc2nc(-c3cc(C(C)(C)C)c(Br)o3)[nH]c12
InChIKey: PJHANERVGJSZGP-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: HEK293 cells stably expressing canine TRPM8 were routinely grown as monolayers in Dulbecco's minimum essential medium supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 400 μg/mL G418. Cells were maintained in 5% CO2 at 37° C. At 24 hr prior to assay, cells were seeded in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) at a density of 5,000 cells per well in culture medium and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed, and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then incubated for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR™ or FDSS. Cells were treated with compounds of the formula (I) at varying concentrations and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to each well to achieve a final concentration that produces an approximately 80% maximal response.
Measurement: IC50 = 33.9 nM (pChEMBL 7.46999979019165)
Descriptors: MW 460.76, ALogP 6.61, HBA 4, HBD 1, PSA 63.94, QED 0.37
Compound (SMILES): Cn1c(C[C@H](N)C(=O)O)cnc1S[C@H]1[C@H]2C(=O)C=C3S[C@H]4C[C@]31C1=C(N4)C(=O)c3[nH]cc4c3C1=[N+]2CC4
InChIKey: DFPHRMRRFSKANC-CAQGJSCNSA-O
Target: Hypoxia-inducible factor 1-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q16665
Assay: Displacement of GST-tagged p300-CH1 domain (323 to 423 residues) from synthetic biotinylated HIF-1alpha C-TAD domain (786 to 826 residues) (unknown origin) measured after overnight incubation by fluorescence analysis
Measurement: IC50 = 2200 nM (pChEMBL 5.659999847412109)
Descriptors: MW 535.63, ALogP 0.57, HBA 9, HBD 4, PSA 146.11, QED 0.4
Compound (SMILES): CC(C)(c1nc(-c2cc(F)cc3[nH]ccc23)nc2c1OC[C@@H]1COCCN21)S(C)(=O)=O
InChIKey: NZTQVSPKHVTMQI-ZDUSSCGKSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: LanthaScreen Kinase Activity Assay: The enzymatic mTOR activity was measured using a LanthaScreen kinase activity assay (Invitrogen). The enzyme was purchased from Invitrogen (PV4754), as well as the GFP-labelled substrate (4EBP1-GFP; PV4759) and the Tb-antip4EBP1(pThr46) antibody (PV4757). The assay was performed in 50 Mm HEPES buffer, pH 7.5, containing 1.5 mM MnCl2, 10 mM MgCl2, 1 mM EGTA, 2.5 mM DTT and 0.01% Tween-20. The concentration of the assay components were the following: 0.24 nM mTOR kinase, 400 nM 4EBP1-GFP, 10 mM ATP and serial dilutions of the compound (inhibitor) to be evaluated. After 1 h incubation at room temperature, 20 mM EDTA was used to stop the reaction and terbium-labelled antibody (4 nM) added to detect phosphorylated product. The antibody associates with the phosphorylated product resulting in an increased TR-FRET value. The TR-FRET value (a dimensionless number) was calculated as the ratio of the acceptor signal (GFP, emission at 520 nm) to the donor signal (terbium, emission at 495 nm).
Measurement: IC50 = 99.3 nM (pChEMBL 7)
Descriptors: MW 446.5, ALogP 2.64, HBA 7, HBD 1, PSA 97.41, QED 0.66
Compound (SMILES): Cc1cc(Nc2cc(C(F)(F)F)ccn2)nc(-c2ccc(NC3CCN(C(=O)c4ccc5ccccc5c4)CC3)nc2)c1
InChIKey: OKDWROIAKDTBCK-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 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 582.63, ALogP 7.48, HBA 6, HBD 2, PSA 83.04, QED 0.22
Compound (SMILES): NC(=O)C12CC3CC(C1)C(NC(=O)c1ccc(F)c(NS(=O)(=O)c4cccc(Cl)c4)c1)C(C3)C2
InChIKey: MAZLHGLTNUFYED-UHFFFAOYSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: HTRF Assay : The inhibiting activity of 11β-HSD1 derived from microsomal fractions was measured using the HTRF assay (62CO2PEB, Cisbio). Different concentrations of compounds were added to 96-well plates, followed by the addition of TE buffer (20 mM Tris buffer and 5 mM EDTA, pH 6.0) containing 200 μM NADPH (N1630, Sigma) and 160 nM cortisone (C2755, Sigma), The reactions were initiated by the addition of human microsomal fractions (M0317, Sigma), and were allowed to incubate for 2 h at 37° C. Europium (Eu3+) cryptate and XL665-conjugated cortisol were then added to each well and incubated for an additional 2 h at room temperature.
Measurement: IC50 = 78.3 nM (pChEMBL 7.110000133514404)
Descriptors: MW 506, ALogP 3.69, HBA 4, HBD 3, PSA 118.36, QED 0.55
Compound (SMILES): Cc1ncoc1-c1nnc(SCCCN2CC[C@@]3(C[C@@H]3c3ccc(F)cc3)C2)n1C
InChIKey: XRDPJIJIWYVCKW-XMSQKQJNSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]-spiperone from human dopamine D3 receptor expressed in CHO-K1 cell membranes after 90 mins by liquid scintillation counting
Measurement: Ki = 28.84 nM (pChEMBL 7.539999961853027)
Descriptors: MW 427.55, ALogP 4.28, HBA 7, HBD 0, PSA 59.98, QED 0.41
Compound (SMILES): CCn1cc(-c2cc3c(-c4cc(C5CC5)ccn4)n[nH]c3cn2)cn1
InChIKey: BDVVVKKOYBBKGI-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 11.5 nM (pChEMBL 7.940000057220459)
Descriptors: MW 330.4, ALogP 3.78, HBA 5, HBD 1, PSA 72.28, QED 0.62
Compound (SMILES): CN1CCN(c2ccc(C(=O)Nc3n[nH]c4cc(OCCOCc5ccc(C(F)(F)F)cc5)ccc34)cc2)CC1
InChIKey: RJHKFEDJXGONBU-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Biochemical Assay: The assay was performed using FLT3 cytoplasmic domain product and purified in house as GST fused protein. The FLT3 protein (1 microM) was pre activated with 800 microM ATP for 1 hour at 28° C in order to obtain a linear kinetic. Kinase buffer was composed of 50 mM HEPES pH 7.9 containing 4 mM MgCl2, 1 mM DTT, 10 microM Na3VO4, and 0.2 mg/mL BSA. The FLT3 kinase assay was run with a final pre activated enzyme concentration of 2 nM, in the presence of 254 microM ATP (residual ATP from KIT pre activation step is negligible), 8 nM 33P-γ-ATP and 55 microM of substrate BioDB n*24 (Aminoacidic sequence: GGKKKVSRSGLYRSPSMPENLNRPR- SEQ ID NO: 1).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 553.59, ALogP 5.18, HBA 6, HBD 2, PSA 82.72, QED 0.28
Compound (SMILES): N#Cc1ccc2[nH]c(NC(=O)c3ccc4cc5n(c4c3)CCCNC5=O)nc2c1
InChIKey: SRYWIXAMNRCZBU-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15349
Assay: Kinase Glo Plus Assay: Human RSK2 protein, purchased from Invitrogen, is used to measure kinase activity utilizing Kinase Glo Plus (Promega) a homogeneous assay technology, which uses a luciferin-luciferase based ATP detection reagent to quantify residual ATP. The assay is performed using 0.75 nM His-RSK2, 0.75 .mu.M ATP and 1.0 uM S6 Kinase/RSK Substrate Peptide 1 (Upstate, catalog #12-124), in assay buffer consisting of 25 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 uM Na3VO4, 0.5 mM DTT, and 1% DMSO. Solutions of test compounds at various concentrations are prepared by 1:3 fold serial dilution of a 1 mM solution of compound in DMSO. The DMSO solutions are further diluted with assay buffer to a final concentration of DMSO of 5%. The assay is performed in a 384 well, white, non-binding plate (Corning, catalogue #3574). Solutions of test compounds (10 uL) are transferred to a dry assay plate, followed by addition of 20 uL kinase.
Measurement: IC50 = 4.9 nM (pChEMBL 8.3100004196167)
Descriptors: MW 384.4, ALogP 2.78, HBA 5, HBD 3, PSA 115.6, QED 0.49
Compound (SMILES): CC[C@H](C)[C@@H]1NCCOc2ccccc2CCCNC(=O)[C@@H](Cc2cscn2)NC(=O)[C@@H](C)N(C)C1=O
InChIKey: NOMMNLQFTSGKMR-WGWURMLHSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 529.71, ALogP 2.16, HBA 7, HBD 3, PSA 112.66, QED 0.56