lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Nc1ncccc1S(=O)(=O)CC[C@@H]1OCC[C@@]2(S(=O)(=O)c3ccc(Cl)cc3)c3c(F)ccc(F)c3OCC12
InChIKey: YCPUSFAJKCUASD-NHHFAVJZSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Electrochemiluminescence (ECL)-based Immunoassay: To measure γ-secretase activity, membranes were incubated at 37° C. for 1 h in 50 μL of buffer containing 20 mM Hepes (pH 7.0) and 2 mM EDTA. At the end of the incubation, Aβ 40 and Aβ 42 were measured using an electrochemiluminescence (ECL)-based immunoassay. Aβ 40 was identified with antibody pairs TAG-G2-10 and biotin-W02, while Aβ 42 was identified with TAG-G2-11 and biotin-4G8. The ECL signal was measured using an ECL-M8 instrument (IGEN International, Inc.) according to the manufacturer's instructions.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 585.05, ALogP 3.93, HBA 8, HBD 1, PSA 125.65, QED 0.46
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)nc(N4CCCC4c4cccnc4)nc23)N1
InChIKey: ZGJIILUTKCTRRI-DHDCSXOGSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 431.46, ALogP 1.62, HBA 9, HBD 3, PSA 129.44, QED 0.41
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N(C)C)n2)n1
InChIKey: XIIRJPVKFJFUHB-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase Binding Assay: PIM-1, -2, and -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.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.727 nM (pChEMBL 9.140000343322754)
Descriptors: MW 331.38, ALogP 2.65, HBA 7, HBD 0, PSA 72.62, QED 0.57
Compound (SMILES): CC(C)C[C@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(N)=O)NC(=O)c1ccc(NC(=O)CNC(=O)CN2CCN(CC(=O)O)CCN(CC(=O)O)CCN(CC(=O)O)CC2)cc1)C(C)C)C(N)=O
InChIKey: ZJXVOWMCTPMTQE-ANJPDTGOSA-N
Target: Gastrin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30550
Assay: Displacement of [125I]-BBN from GRPR in human PC3 cells after 1 hr by compuGamma counting or microplate scintillation counting method
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 1518.7
Compound (SMILES): COC1CCN(c2cccc3c2CCN(C(=O)C2CCN(C(=N)N)CC2)C3C(=O)Nc2ccc(C(=O)O)cc2)CC1
InChIKey: BIRMYIMSFIENMI-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: In Vitro Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 75-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 5.68 nM (pChEMBL 8.25)
Descriptors: MW 562.67, ALogP 2.67, HBA 6, HBD 4, PSA 152.29, QED 0.31
Compound (SMILES): Cn1c(SCCCN2CC[C@]3(C[C@H]3c3ccc(C(F)(F)F)cc3)C2)nnc1-c1ccncn1
InChIKey: LKIZJVWLCHAZTJ-AVRDEDQJSA-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 = 5.129 nM (pChEMBL 8.289999961853027)
Descriptors: MW 474.56, ALogP 4.65, HBA 7, HBD 0, PSA 59.73, QED 0.37
Compound (SMILES): Cc1cc(CCCN2CCN(C)CC2)cc(C)c1S(=O)(=O)N(C)c1c(C)nn(C)c1C
InChIKey: AJMGORHICVNZCC-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
UniProt: Q388H8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 447.65, ALogP 2.66, HBA 6, HBD 0, PSA 61.68, QED 0.65
Compound (SMILES): Oc1ccc2c(c1)CCc1c-2noc1-c1cc(-c2ccccc2)on1
InChIKey: KTDFTMWYHOSQLG-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1^108cells/pellet) were suspended in buffer containing 20 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM EDTA and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at -80° C. after protein concentration determination.Membranes (2 ug/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA, 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes.
Measurement: IC50 = 15000 nM (pChEMBL 4.820000171661377)
Descriptors: MW 330.34, ALogP 4.47, HBA 5, HBD 1, PSA 72.29, QED 0.59
Compound (SMILES): NCc1ccc(Cl)cc1CNC(=O)[C@@H]1CCCN1C(=O)C1CNc2ccccc21
InChIKey: RUHSAAUEEQOTBI-IJHRGXPZSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: In Vitro Assay: Relevant in vitro assays are referenced in Morrissette, et al., Bioorg. Med. Chem. Lett. 2004, 14, 4161-4164 and described in Lewis, et al. Thromb. Res. 1993, 70, 173 (assays of human α-thrombin and human trypsin), and Lewis, et al. Thromb. Haemostasis 1995, 74, 1107-1112. The assays were carried out at 25° C. in 0.05 M TRIS buffer pH 7.4, 0.15 M NaCl, 0.1% PEG. Trypsin assays also contained 1 mM CaC12. In assays wherein rates of hydrolysis of a p-nitroanilide (pna) substrate were determined, a Thermomax 96-well plate reader was used was used to measure (at 405 nm) the time dependent appearance of p-nitroaniline. sar-PR-pna was used to assay human α-thrombin (Km=125 μM) and bovine trypsin (Km=125 μM). p-Nitroanilide substrate concentration was determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cm−1M−1.
Measurement: Ki = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 412.92, ALogP 2.62, HBA 4, HBD 3, PSA 87.46, QED 0.7
Compound (SMILES): COc1ccc(C(C)C)cc1C1=C(CN(Cc2cc(C(F)(F)F)cc(C(F)(F)F)c2)c2ncc(-c3cnn(C)c3)cn2)CCCCC1
InChIKey: UPCUVHQXFLNFHQ-UHFFFAOYSA-N
Target: Cholesteryl ester transfer protein (SINGLE PROTEIN), Homo sapiens
UniProt: P11597
Assay: Inhibition of recombinant CETP (unknown origin) assessed as inhibition of BODIPY-labeled cholesteryl transfer to fluorescent substrate by fluorimetric assay
Measurement: IC50 = 1620 nM (pChEMBL 5.789999961853027)
Descriptors: MW 657.7, ALogP 9.47, HBA 6, HBD 0, PSA 56.07, QED 0.17
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1cnn(Cc2cccc(C(=O)Nc3ccccc3)c2)c1
InChIKey: MIRDRGNIUOZRES-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 152 nM (pChEMBL 6.820000171661377)
Descriptors: MW 454.53, ALogP 3.71, HBA 6, HBD 3, PSA 114.93, QED 0.39
Compound (SMILES): CC(=O)Nc1nc(C)c(C(=O)N[C@@H](C)c2ccc(CN3CCc4cc(OCC5CC5)ccc4C3)cc2)s1
InChIKey: LXWCJIRMZANBLQ-SFHVURJKSA-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 2000 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.Each assay microtiter plate contains wells with vehicle instead of compound as controls for the non-inhibited enzyme (100% CTL; HIGH) and wells without acetyl-CoA as controls for non-specific NADH degradation (0% CTL; LOW).
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 518.68, ALogP 5.25, HBA 6, HBD 2, PSA 83.56, QED 0.4
Compound (SMILES): Cc1ncc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cnc(OCCO)c(N2CCOCC2)c1
InChIKey: YQOYAEIODQJLFI-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 502.49, ALogP 3.93, HBA 7, HBD 2, PSA 96.81, QED 0.51
Compound (SMILES): NCCO.O=C(O)c1[nH]c(=O)[nH]c(=O)c1CCc1ccc2ccccc2c1
InChIKey: XKRQHMRWLAAMPC-UHFFFAOYSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Inhibition of human dihydroorotate dehydrogenase assessed as reduction in orotate production using L-dihydroorotate as substrate measured after 20 mins by spectrophotometric method
Measurement: Ki = 23100 nM (pChEMBL 4.639999866485596)
Descriptors: MW 310.31, ALogP 1.7, HBA 3, HBD 3, PSA 103.02, QED 0.68
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cc(F)cc(Br)c3)CC2)C1
InChIKey: CPDPOYFGOATSMC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 563.44, ALogP 3.95, HBA 7, HBD 1, PSA 106.73, QED 0.39
Compound (SMILES): O=C(NN1CCSCC1)c1cccc(S(=O)(=O)N2CCc3ccccc3C2)c1
InChIKey: UWTMOFZHDCRQND-UHFFFAOYSA-N
Target: Aldo-keto reductase family 1 member C3 (SINGLE PROTEIN), Homo sapiens
UniProt: P42330
Assay: Inhibition of human recombinant His-tagged AKR1C3 expressed in Escherichia coli BL21 (DE3) cells using 8-Acetyl-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one as substrate after 1 hr in presence of NADPH by fluorescence assay
Measurement: IC50 = 450 nM (pChEMBL 6.349999904632568)
Descriptors: MW 417.56, ALogP 2.13, HBA 5, HBD 1, PSA 69.72, QED 0.83
Compound (SMILES): COc1cc2c(N)nc(N3CCN(C(=O)c4cc5c(OC)cccc5[nH]4)CC3)nc2c(F)c1OC
InChIKey: KVDIYQOAWIQMBE-UHFFFAOYSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: ELISA Assay: CVF-Bb complex prepared from purified cobra venom factor (1 μM), recombinant human complement factor B (expressed in drosophila cells and purified using standard methods) and human complement factor D (expressed in E. Coli, refolded and purified using standard methods). CVF-Bb complex at 3 nM concentration was incubated with test compound at various concentrations for 1 hour at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human complement C3 substrate purified from plasma was added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction was stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, was quantified by means of an enzyme-linked-immunosorbent assay.
Measurement: IC50 = 9630 nM (pChEMBL 5.019999980926514)
Descriptors: MW 480.5, ALogP 2.82, HBA 8, HBD 2, PSA 118.83, QED 0.45
Compound (SMILES): COc1cccc2[nH]c3c(c12)CC1c2cc4c(cc2CCN1C3)OCO4
InChIKey: BUYJSSDFOQMYKS-UHFFFAOYSA-N
Target: Alpha-1A adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35348
Assay: Antagonist activity at human full-length N-terminal SNAP-tagged alpha1A adrenoceptor expressed in HEK293 cells assessed as inhibition of agonist-induced calcium mobilization preincubated for 10 mins followed by agonist addition by fluo-4 AM dye-based assay
Measurement: IC50 = 202.3 nM (pChEMBL 6.690000057220459)
Descriptors: MW 348.4, ALogP 3.56, HBA 4, HBD 1, PSA 46.72, QED 0.73
Compound (SMILES): CC(C)Nc1cc(-n2ccc3cc(C#N)cnc32)ncc1C(=O)N[C@H]1CC[C@H](C(C)(C)O)C1
InChIKey: URLMSHFMEYHYSO-OALUTQOASA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 3.7 nM (pChEMBL 8.430000305175781)
Descriptors: MW 446.56, ALogP 3.78, HBA 7, HBD 3, PSA 115.86, QED 0.53
Compound (SMILES): Cc1nn(C)c(C)c1S(=O)(=O)N1CCC(Oc2ccc(Cl)cc2C(F)(F)F)CC1
InChIKey: FKPHYHMWXCZHQK-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TCW9
Assay: Biological Assay: Prokineticin receptor 1 (PKR1) antagonists may be functionally assessed by measurement of change in intracellular calcium levels induced by Gq mediated increase in inositol triphosphate (IP3) levels. The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro. Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1xHanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1x Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 uM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following excitation at 485 nM. Readings from wells that do not contain antagonist enable percentage inhibition curves to be plotted using 4-parameter fit algorithm and IC50 values are calculated for each test compound.
Measurement: IC50 = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 451.9, ALogP 3.94, HBA 5, HBD 0, PSA 64.43, QED 0.71
Compound (SMILES): Nc1ccccc1NC(=O)CCCCCNC(=O)c1nccs1
InChIKey: BPTJIUSSFWIZDJ-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 973 nM (pChEMBL 6.010000228881836)
Descriptors: MW 332.43, ALogP 2.65, HBA 5, HBD 3, PSA 97.11, QED 0.51
Compound (SMILES): CCc1ccc(/N=C2\S/C(=C\c3ccncc3)C(=O)N2C)cc1
InChIKey: YFUMNIDYRFWCJZ-SOBIRXOSSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Inhibition of His-tagged human Aurora A kinase (122 to 40 residues) expressed in Escherichia coli BL21 (DE3) Rosetta cells using biotinylated STK2 substrate incubated for 30 mins by HTRF assay
Measurement: IC50 = 390 nM (pChEMBL 6.409999847412109)
Descriptors: MW 323.42, ALogP 3.88, HBA 4, HBD 0, PSA 45.56, QED 0.81
Compound (SMILES): CCOc1ccc(-c2nc3ccccn3c2-c2ccnc(Nc3cc(CC)c(N4CCC(N5CCN(S(C)(=O)=O)CC5)CC4)cc3OC)n2)cc1C(=O)Nc1c(F)cccc1F
InChIKey: VWJFHLBAKQXQFR-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human GST-rTEV-tagged IGF1R (957 to 1367 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 866.01, ALogP 7.25, HBA 12, HBD 2, PSA 146.53, QED 0.12
Compound (SMILES): O=C(Nc1cc(C2CCNCC2)nn1-c1ccc(F)cc1)c1cnn2cccnc12
InChIKey: IMFDMGAYUUWCHU-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 405.44, ALogP 2.77, HBA 7, HBD 2, PSA 89.14, QED 0.55
Compound (SMILES): Cc1cc(-c2nc3ccc(C4CCN(C)C(=O)C4)cc3n2[C@@H](C)c2ccccc2)cn(C)c1=O
InChIKey: NPBHLYLOEFJNCF-YDNXMHBPSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (LysS, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 454.57, ALogP 4.66, HBA 5, HBD 0, PSA 60.13, QED 0.45
Compound (SMILES): NC(=O)c1cc(NC(=O)c2cc(-c3ccccn3)c(C3CC3)cc2Cl)n(-c2ccccc2)n1
InChIKey: TUOHPXSGIUGQKB-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 93.3 nM (pChEMBL 7.03000020980835)
Descriptors: MW 457.92, ALogP 4.82, HBA 5, HBD 2, PSA 102.9, QED 0.43
Compound (SMILES): CCCCNc1nc(N)c2nc(-c3ccco3)sc2n1
InChIKey: OCYRRIUPJYARRC-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Displacement of [3H]DPCPX from human adenosine A1 receptor expressed in CHO cell membranes measured after 90 mins by scintillation counting method
Measurement: Ki = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 289.36, ALogP 3.14, HBA 7, HBD 2, PSA 89.86, QED 0.7
Compound (SMILES): Cc1cc(Nc2cc(C(F)(F)F)ccn2)nc(-c2ccc(N3CCC(NC(=O)OC(C)(C)C)C3)nc2)c1
InChIKey: QPGHCSXVVJJKLF-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 = 248 nM (pChEMBL 6.610000133514404)
Descriptors: MW 514.55, ALogP 5.71, HBA 7, HBD 2, PSA 92.27, QED 0.45
Compound (SMILES): CSCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(N)=O)NC(=O)CC[C@@H](C)[C@H]1CC[C@H]2[C@@H]3[C@H](O)C[C@@H]4C[C@@H](NC(=O)COCCOCCNC(=O)CN5CCN(CC(=O)O)CCN(CC(=O)O)CCN(CC(=O)O)CC5)CC[C@]4(C)[C@H]3C[C@H](O)[C@]12C)C(C)C)C(N)=O
InChIKey: IAEWKGIOFOUGRZ-WSFKVXFUSA-N
Target: Gastrin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30550
Assay: Displacement of [125I]-BBN from GRPR in human PC3 cells after 1 hr by compuGamma counting or microplate scintillation counting method
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 1861.29
Compound (SMILES): O=C(c1cc[nH]n1)N1CCN(C(=O)c2cc(Cc3n[nH]c(=O)c4c3CCCC4)ccc2F)CC1
InChIKey: YXLQMPQABXLLRQ-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 464.5, ALogP 1.7, HBA 5, HBD 2, PSA 115.05, QED 0.61
Compound (SMILES): Cc1ccoc1C(=O)Nc1cccc(-c2[nH]c3ccccc3c2C(N)=O)c1
InChIKey: RVUIAFIZHNHJIV-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 2.7 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 μl per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 359.39, ALogP 4.09, HBA 3, HBD 3, PSA 101.12, QED 0.51
Compound (SMILES): O=C(Nc1cc(C(=O)N2CCOC[C@@H]2CO)nn1-c1ccccc1)c1cc(-c2ccccn2)c(F)cc1Cl
InChIKey: VWIVOOAPIKMDMO-SFHVURJKSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 119 nM (pChEMBL 6.920000076293945)
Descriptors: MW 535.96, ALogP 3.81, HBA 7, HBD 2, PSA 109.58, QED 0.39
Compound (SMILES): CN(CCCCN1C(=O)COc2ccccc21)Cc1ccc(Cl)cc1
InChIKey: NSULTPVPXTUFGQ-UHFFFAOYSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Cavia porcellus
UniProt: Q60492
Assay: Displacement of [3H]-(+)-pentazocine from sigma1 receptor in guinea pig brain cortex membranes after 120 mins by scintillation counting method
Measurement: Ki = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 358.87, ALogP 3.98, HBA 3, HBD 0, PSA 32.78, QED 0.7
Compound (SMILES): Cc1ccc(C(C)C)cc1OC(=O)Nc1ccc(I)cc1
InChIKey: VFVBWNCOJYCYJY-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Equus caballus
UniProt: P81908
Assay: Inhibition of horse serum BuChE using butyrylthiocholine iodide as substrate preincubated for 15 mins followed by substrate addition by Ellman's method
Measurement: IC50 = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 395.24, ALogP 5.33, HBA 2, HBD 1, PSA 38.33, QED 0.71
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc(-c3cccc(-n4ccnc4)n3)cc2Cl)CS1(=O)=O
InChIKey: RVJGXFQMEGVSJN-KRWDZBQOSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 μl are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 429 nM (pChEMBL 6.369999885559082)
Descriptors: MW 436.95, ALogP 2.66, HBA 7, HBD 2, PSA 103.97, QED 0.66
Compound (SMILES): Cc1nccc(Oc2ccc(C[C@H](NC(=O)[C@@H]3Cc4cc5c(cc4CN3C(=O)NC(C)(C)c3ccccc3)O[C@@H](c3ccc(OCc4ccc(Cl)c(Cl)c4)cc3)CO5)C(=O)O)cc2)c1C
InChIKey: IAFSFCHATHZYKQ-QQDGYHEMSA-N
Target: Glucagon-like peptide 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43220
Assay: cAMP Functional Assay: The efficacy of GLP-1 receptor agonists was studied in a cAMP functional assay using HEK-293 Cells expressing the cloned human GLP-1 receptor. GLP-1-expressing cells (10,000-20,000 cells per 0.1 mL) were plated in 96-well plates in Dulbecco's modified eagles media (DMEM) containing 10% fetal bovine serum, 2 mg/mL G418 and penicillin-streptomycin. Following overnight incubation of cells, media was removed, and compounds (at concentrations ranging from 0.0001 to 100 μM) were added to monolayer cells in Iscove's modified dulbecco's medium (IMDM), 100 μM RO 20-1724 PDE inhibitor, 0.1% BSA, 2% DMSO in a final volume of 100 μL, and incubated for 30 min at 37° C. 95%02, 5% CO2 in a humidified incubator. Alternatively, cells were harvested and combined with compounds in suspension using the buffer and protocol described above. cAMP was quantified using a homogenous time-resolved fluorescence detection system (cAMP dynamic, CIS bio International).
Measurement: EC50 = 262 nM (pChEMBL 6.579999923706055)
Descriptors: MW 915.87, ALogP 10.07, HBA 8, HBD 3, PSA 148.55, QED 0.1
Compound (SMILES): Fc1ccccc1-c1n[nH]c2cnc(-c3cncc(NC4CCNCC4)c3)cc12
InChIKey: DDJLCMFGTPWOTJ-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 = 2.42 nM (pChEMBL 8.619999885559082)
Descriptors: MW 388.45, ALogP 3.99, HBA 5, HBD 3, PSA 78.52, QED 0.49
Compound (SMILES): COc1ccc2c(c1)CN(C(=O)CC(=O)O)CCS2
InChIKey: CXESNZHSMAWROP-UHFFFAOYSA-N
Target: Ryanodine receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92736
Assay: Induction of FKBP12.6 re-binding to PKA-phosphorylated-RyR2 (unknown origin) after 30 mins in presence of [35S]-FKBP12.6 by liquid scintillation counting
Measurement: EC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 281.33, ALogP 1.6, HBA 4, HBD 1, PSA 66.84, QED 0.85
Compound (SMILES): O=C(COc1ccc2cnn(C3CCNCC3)c2c1)NCC(O)CN1CCc2ccccc2C1
InChIKey: BPCBLCQMCNDJJR-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 463.58, ALogP 1.88, HBA 7, HBD 3, PSA 91.65, QED 0.47
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2=O)nc(N2CCOCC2)n1
InChIKey: VWZRDTLPMXJTFJ-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity: Raf and biotinylated Mek, kinase dead, were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 0.1 nM (pChEMBL 10)
Descriptors: MW 541.53, ALogP 3.92, HBA 7, HBD 1, PSA 96.89, QED 0.52
Compound (SMILES): C[C@]1(O)CC[C@@]2(C)C(CC[C@@H]3[C@@H]2CC[C@]2(C)C(c4cccnc4)=CC[C@@H]32)C1
InChIKey: MFZFIPYAPCDNNJ-BZLQTJNUSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human testicular microsomes using 17alpha-hydroxypregnenolone as substrate assessed as decrease in DHEA levels after 90 mins by ELISA
Measurement: IC50 <= 11 nM (pChEMBL 7.959000110626221)
Descriptors: MW 365.56, ALogP 5.87, HBA 2, HBD 1, PSA 33.12, QED 0.68
Compound (SMILES): Cc1cc(OC2CC2)ccc1N1CC[C@@H](Oc2ccc([C@H](C)NC(=O)CC#N)cc2)C1
InChIKey: KZGPGJZTTCGNQC-FDDCHVKYSA-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 = 680 nM (pChEMBL 6.170000076293945)
Descriptors: MW 419.53, ALogP 4.28, HBA 5, HBD 1, PSA 74.59, QED 0.69