lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCC1CC(=O)NN=C1c1ccc2nc(-c3ccc(O[C@@H]4CCCC[C@H]4N(CC)CC)cc3)oc2c1
InChIKey: RLIXOECIKGKKEW-UCAJRPBESA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] were stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP-848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM beta-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37 C. 5% CO2. For the assay, the cells are placed in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37 C./5% CO2.After the medium has been sucked out of the wells completely, 10 ul of the test compound are added, the compounds are diluted beforehand with stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes' incubation at RT, the cAMP concentrations are determined using the AlphaScreen cAMP Assay.
Measurement: EC50 = 319 nM (pChEMBL 6.5)
Descriptors: MW 488.63, ALogP 5.78, HBA 6, HBD 1, PSA 79.96, QED 0.43
Compound (SMILES): C[C@@H]1COCCN1c1nc(N2CCOC[C@H]2C)c2nc(-c3ccc(N)nc3)[nH]c2n1
InChIKey: UPCZFZITQYXIIG-CHWSQXEVSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: mTOR ATP-Binding Assay: 1. 8-point serial dilutions of compounds (10 mM stock) are performed in 90% DMSO in a 384-well "masterplate" and 50 nL is transferred onto 384-well assay plates (white polystyrene small volume; Matrix/Thermo Scientific Cat. No. #4365). 2. The final volume of the assay is 10 μL and the order of addition is as follows: 50 nL of compounds dilution; 5 μL of a mixture of GST-mTOR and Europium anti-GST antibody with or without the PI3K/mTOR inhibitor P1-103 (3-(4-(4-morpholinyl)pyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl)phenol, Calbiochem); 5 μL of tracer-314. Incubated for 60 minutes at room temperature. TR-FRET measured in Biotek Synergy2 reader at: Excitation 340 nm/emission 665 nm; Excitation 340 nm/emission 620 nm. The assay buffer consists of 50 mM HEPES pH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.01% Pluronic F-127. Tracer-314 (Alexa Fluor® 647-labeled ATP-competitive kinase inhibitor; Cat. No. PV6087), Europium anti-GST antibody (Cat. No PV5594) and N-terminally GST-tagged truncated human mTOR (FRAP1) (Cat. No PV4754) are available from Invitrogen. The following final concentrations are used: 3 nM GST-mTOR; 1 nM Europium anti-GST antibody; +/− 10 μM P1-103; and 10 nM tracer-314. The final concentrations of diluted compounds are 9091; 2730; 910; 273; 91; 27; 9; and 3 nM. The final concentration of DMSO is 0.45%. The following controls are used: High signal: solvent vehicle, GST-mTOR, Eu-anti-GST antibody, tracer-314; Low signal: solvent vehicle, GST-mTOR, Eu-anti-GST antibody, P1-103, tracer-314.
Measurement: IC50 = 754 nM (pChEMBL 6.119999885559082)
Descriptors: MW 410.48, ALogP 1.45, HBA 9, HBD 2, PSA 118.31, QED 0.66
Compound (SMILES): Cc1c(CC(=O)N[C@H](CO[N+](=O)[O-])C(=O)O)cc(-c2ccc(S(C)(=O)=O)cc2)n1-c1cccc(F)c1
InChIKey: BNBDZFFKPLLERD-HXUWFJFHSA-N
Target: Prostaglandin G/H synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35354
Assay: In Vitro Inhibition Assay: The murine monocyte/macrophage cell line J774 is grown in Dulbecco's modified Eagle's medium (DMEM), enriched with glutamine (2 mM), HEPES (25 mM), penicillin (100 ug/mL), streptomycin (100 ug/mL), 10% of fetal bovine serum (FBS) and 1.2% of sodium pyruvate. The cells are distributed in 24-well plates at a density of 2.5x105 cells/mL or in culture dishes with a diameter of 10 cm (1x107 cells/10 mL/dish) and kept for 2 hours at 37° C. in a CO2 (5%)/O2 (95%) atmosphere. Just before the experiments, the culture medium is replaced with fresh medium without FBS to avoid interference during the radio-immunological phase and the cells are stimulated as described below.Evaluation of COX-1 Activity.The cells are pretreated with the test compounds for 15 minutes and then incubated for 30 minutes with arachidonic acid (15x10-6 m). At the end of incubation the supernatants are collected for evaluating, by means of radio-immunological assays, the amount of PGE2 produced.
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 519.51, ALogP 2.32, HBA 8, HBD 2, PSA 157.84, QED 0.31
Compound (SMILES): C[C@@H]1CNC(=O)c2cc3ccc(C(=O)Nc4cnn(Cc5ccccc5)c4)cc3n2[C@H]1C
InChIKey: LZCWQKSZGMFDDP-SJORKVTESA-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 = 43.5 nM (pChEMBL 7.360000133514404)
Descriptors: MW 427.51, ALogP 4.08, HBA 5, HBD 2, PSA 80.95, QED 0.51
Compound (SMILES): Cc1nn(C)c(C)c1NS(=O)(=O)c1ccc(NCCN2CCN(C)CC2c2ccccc2)nc1
InChIKey: KJIKNIONOBEQAK-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 = 1000 nM (pChEMBL 6)
Descriptors: MW 483.64, ALogP 2.63, HBA 8, HBD 2, PSA 95.39, QED 0.51
Compound (SMILES): Cc1ccc([C@H]2C[C@@H]2COc2nc(C)ncc2-c2ccc(=O)n(C3CC3)c2)nc1
InChIKey: KZHAPZQQSBJAKP-MJGOQNOKSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: PDE10 Fluorescence Polarization (FP) Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAPU technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 uL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 uL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate.
Measurement: Ki = 0.519 nM (pChEMBL 9.289999961853027)
Descriptors: MW 388.47, ALogP 3.83, HBA 6, HBD 0, PSA 69.9, QED 0.64
Compound (SMILES): CC(C)C[C@H](CO)NC(=O)c1ccc(C2CC2)c(OCC2CC2)n1
InChIKey: PFQSGQULJACWGS-OAHLLOKOSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 332.44, ALogP 2.88, HBA 4, HBD 2, PSA 71.45, QED 0.73
Compound (SMILES): Cn1ccc(C(=O)Nc2cnn3ccc(N4CCC[C@@H]4c4cccc(F)c4)nc23)n1
InChIKey: ZWDXFAVAWPLHOJ-GOSISDBHSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: ELISA Assay: An enzyme-linked immunosorbant assay (ELISA) was used to assess TrkA kinase activity in the presence of inhibitors. Immulon 4HBX 384-well microtiter plates (Thermo part #8755) were coated with a 0.025 mg/mL solution of poly (Glu, Ala, Tyr; 6:3:1; Sigma P3899). Various concentrations of test compound, 2.5 nM TrkA (Invitrogen Corp., histidine-tagged recombinant human TrkA, cytoplasmic domain), and 500 uM ATP were incubated for 25 minutes at ambient temperature in the coated plates while shaking. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The reaction mixture was removed from the plate by washing with PBS containing 0.1% (v/v) Tween 20. The phosphorylated reaction product was detected using 0.2 ug/mL of a phosphotyrosine specific monoclonal antibody (clone PY20) conjugated to horseradish peroxidase in conjunction with the TMB Peroxidase Substrate System (KPL).
Measurement: IC50 = 8.5 nM (pChEMBL 8.069999694824219)
Descriptors: MW 405.44, ALogP 3.2, HBA 7, HBD 1, PSA 80.35, QED 0.56
Compound (SMILES): CCC(C)NC(=O)[C@@H](C)Oc1cc(F)ccc1Nc1ncnc2cc(N=S3(=O)CCCC3)cc(C)c12
InChIKey: VFMKAEVQTZGTPR-QRWMCTBCSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: In vitro Kinase Assay: The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled eIF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 513.64, ALogP 5.4, HBA 7, HBD 2, PSA 105.57, QED 0.42
Compound (SMILES): CCNC(=O)C(C)c1ccc(O[C@@H]2CCN(c3ccnc(N4CCC(F)(F)C4)n3)C2)cc1
InChIKey: MKOOZHAOPPEXPG-LRTDYKAYSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Malonyl CoA formation by acetyl CoA carboxylases is stoichometrically linked to the consumption of ATP. ACC2 activity is measured in a NADH-linked kinetic method measuring ADP generated during the ACC reaction using a coupled lactate dehydrogenase/pyruvate kinase reaction. 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, acetylCoenzyme 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.
Measurement: IC50 = 399 nM (pChEMBL 6.400000095367432)
Descriptors: MW 445.51, ALogP 3.22, HBA 6, HBD 1, PSA 70.59, QED 0.71
Compound (SMILES): COCc1nc(NC(=O)N[C@H](C)c2ccccc2)cc2[nH]nc(N)c12
InChIKey: MDLOYNLHRSOSPZ-SNVBAGLBSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 340.39, ALogP 2.57, HBA 5, HBD 4, PSA 117.95, QED 0.57
Compound (SMILES): COc1ccc(C(=O)Nc2ccc([C@@H]3CCCNC3)cc2)cc1Cl
InChIKey: ZBNVJXGGHHKIFD-OAHLLOKOSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 60.3 nM (pChEMBL 7.21999979019165)
Descriptors: MW 344.84, ALogP 4.07, HBA 3, HBD 2, PSA 50.36, QED 0.88
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2cc(-c3nn[nH]n3)ccc2NC1=O
InChIKey: CVWBODGHVOOTTK-CPJLOUKISA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
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 25-200 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 = 1.34 nM (pChEMBL 8.869999885559082)
Descriptors: MW 590.03, ALogP 5.98, HBA 6, HBD 2, PSA 116.76, QED 0.28
Compound (SMILES): Cc1ccc(CNc2cc(OC[C@H]3C[C@@H]3c3ccc(F)cn3)nn(C)c2=O)nc1
InChIKey: UHIROTGGZUANPT-PBHICJAKSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product #R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215).
Measurement: Ki = 0.782 nM (pChEMBL 9.109999656677246)
Descriptors: MW 395.44, ALogP 2.81, HBA 7, HBD 1, PSA 81.93, QED 0.66
Compound (SMILES): CC[C@@H](NC(=O)N1CCc2cnc(NC3CCOCC3)nc2C1)c1ccc(OC(F)F)cc1
InChIKey: HEDQQMCTHYPWRU-LJQANCHMSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100x final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 1 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 uM S/T17 peptide substrate and 25 uM ATP (corresponding to the observed Km) for a total reaction volume of 25 uL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 461.51, ALogP 3.89, HBA 6, HBD 2, PSA 88.61, QED 0.65
Compound (SMILES): C[C@@H](O)C1O[C@@H](n2ccc3c(N)ncnc32)[C@](C)(O)[C@@H]1O
InChIKey: YBGIKDCDNIQILP-AYLOQUPNSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Polymerase Inhibition Assay: The ability of the nucleoside triphosphate derivatives to inhibit the enzymatic activity of the HCV NS5B RNA-dependent RNA polymerase was measured in a radiolabeled nucleotide incorporation assay modified from the method described in International Publication No. WO2002/057287. Briefly, 50 μL reactions containing 20 mM HEPES (pH 7.3), 7.5 mM DTT, 20 units/mL RNasIN, 1 μM each of ATP, GTP, UTP and CTP, 20 μCi/mL [33P]-CTP, 10 mM MgCl2, 60 mM NaCl, 100 μg/ml BSA, 0.021 μM DCoH heteropolymer RNA template and 5 nM NS5B (1b-BKΔ55) enzyme were incubated at room temperature for 1 hour. Assay was terminated by the addition of 500 mM EDTA (50 μL). The reaction mixture was transferred to a Millipore DE81 filter plate and the incorporation of labeled CTP was determined using Packard TopCount.
Measurement: EC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 294.31, ALogP -0.6, HBA 8, HBD 4, PSA 126.65, QED 0.58
Compound (SMILES): Cn1ncc(NC(=O)c2nc(-c3c(F)cccc3Cl)sc2N)c1[C@@H]1CC[C@@H](N)[C@H](F)CO1
InChIKey: HINUPSPCANVDPP-BZPMIXESSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Assay: LC3K 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 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL 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.
Measurement: Ki = 0.036 nM (pChEMBL 10.4399995803833)
Descriptors: MW 482.94, ALogP 3.69, HBA 8, HBD 3, PSA 121.08, QED 0.52
Compound (SMILES): O=c1[nH]c(N2CCC3(CCN(Cc4ccccc4)C3)C2)nc(N[C@@H]2CCCNC2)c1-c1nc2ccccc2s1
InChIKey: SJVBXAJVBWWKRQ-VWRCBCJMSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IMAP technology (Molecular Devices): A 20 ul reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 uM 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 ul 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, LJL BioSystems).
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 541.73, ALogP 4.31, HBA 8, HBD 3, PSA 89.18, QED 0.34
Compound (SMILES): C=C(C)[C@H]1Cc2c(ccc3c2OC2COc4cc(OC)c(OC)cc4C2[C@@]32CO2)O1
InChIKey: JSZISECBWYAATI-IITSNUCMSA-N
Target: Pancreatic triacylglycerol lipase (SINGLE PROTEIN), Sus scrofa
UniProt: P00591
Assay: Inhibition Assay: Particularly, pancreatic lipase inhibiting activity was measured by the conventional method known to those in the art (Kim, J. H.; Kim, H. J.; Park, H. W.; Youn, S. H.; Choi, D. Y.; Shin, C. S. FEMS Microbiol. Lett. 2007, 276, 93.). To prepare the enzyme buffer, 30 mL (10 units) of pig pancreatic lipase solution (Sigma, St. Louis, Mo.) and 1 mM EDTA (pH 6.8) were mixed with 10 mM MOPS (morpholinepropanesulphonic acid), which was added to 850 mL of tris buffer (100 mM Tris-HCl and 5 mM CaCl2, pH 7.0). Then, 100 mL of the rotenoisin A or B of the present invention was mixed with the prepared enzyme buffer at the experimental concentration. As the positive control, 880 mL of Orlistat (Roche, Basel, Switzerland) was mixed with the enzyme buffer. 20 mL of substrate solution containing 20 mM p-nitrophenylbutyrate dissolved in dimethyl formaide was added to the enzyme buffer, followed by culture for 15 minutes at 37° C. that was the temperature adequate for enzyme reaction.
Measurement: IC50 = 6300 nM (pChEMBL 5.199999809265137)
Descriptors: MW 408.45, ALogP 3.75, HBA 6, HBD 0, PSA 58.68, QED 0.57
Compound (SMILES): CC(C)(F)[C@H](F)CNC(=O)c1cnc(-n2cnc3cc(C#N)cnc32)cc1Nc1cnn(CC(F)F)c1
InChIKey: MGMYLXLTGWUIKG-GOSISDBHSA-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 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 515.48, ALogP 3.71, HBA 9, HBD 2, PSA 126.34, QED 0.33
Compound (SMILES): COC(=O)C[C@@H](C)n1c(=O)[nH]/c(=N\c2ccc(OC(C)C)c(Cl)c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: PRRZDPJSCKGFTP-OAHLLOKOSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683)) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37 C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 uM Fluo-3-AM solution (pH 7.5) containing 20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 uL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.).
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 521.4, ALogP 3.84, HBA 8, HBD 1, PSA 107.68, QED 0.45
Compound (SMILES): Cc1cnccc1C(=O)Nc1cnn2ccc(N3CCC[C@@H]3c3cccc(F)c3)nc12
InChIKey: VSSOJTPOJHCKOA-HXUWFJFHSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: ELISA Assay: An enzyme-linked immunosorbant assay (ELISA) was used to assess TrkA kinase activity in the presence of inhibitors. Immulon 4HBX 384-well microtiter plates (Thermo part #8755) were coated with a 0.025 mg/mL solution of poly (Glu, Ala, Tyr; 6:3:1; Sigma P3899). Various concentrations of test compound, 2.5 nM TrkA (Invitrogen Corp., histidine-tagged recombinant human TrkA, cytoplasmic domain), and 500 uM ATP were incubated for 25 minutes at ambient temperature in the coated plates while shaking. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The reaction mixture was removed from the plate by washing with PBS containing 0.1% (v/v) Tween 20. The phosphorylated reaction product was detected using 0.2 ug/mL of a phosphotyrosine specific monoclonal antibody (clone PY20) conjugated to horseradish peroxidase in conjunction with the TMB Peroxidase Substrate System (KPL).
Measurement: IC50 = 320.2 nM (pChEMBL 6.5)
Descriptors: MW 416.46, ALogP 4.17, HBA 6, HBD 1, PSA 75.42, QED 0.54
Compound (SMILES): N#Cc1ccc(C2C3=C(CCC3=O)N(c3cccc(C(F)(F)F)c3)C(=O)N2C(=O)N[C@@H]2CCC[C@H]2O)cc1
InChIKey: DODGRUXHYKDDNK-MRBOTJLVSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Nuetrophil Elastase Acitivty Assay: Various concentrations of the neutrophil elastase inhibitor are incubated with plasma. Subsequently, the enzyme activity is measured using the fluorogenic substrate MeOSuc-Ala-Ala-Pro-Val-AMC (Bachem Cat. No. 1-1270, substrate concentration: 250 uM, pH 7.5, 25 mM TRIS buffer, 250 mM NaCl) in analogous fashion as described for the human neutrophil assay.
Measurement: EC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 524.5, ALogP 4.8, HBA 5, HBD 2, PSA 113.74, QED 0.6
Compound (SMILES): Cc1c(NS(=O)(=O)c2ccc(Br)cc2)cnn1C
InChIKey: ASAFYUOOEIFTBG-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 > 100000 nM (pChEMBL 4)
Descriptors: MW 330.21, ALogP 2.29, HBA 4, HBD 1, PSA 63.99, QED 0.94
Compound (SMILES): COC[C@@H](NC(=O)Nc1cc2[nH]nc(OC3CCOCC3)c2cn1)c1ccccc1
InChIKey: XPFZWMCAWLXKER-GOSISDBHSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 411.46, ALogP 3.02, HBA 6, HBD 3, PSA 110.39, QED 0.55
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N2CCCC2c2c(C)noc2C)n2c1COCC2)c1ccccc1
InChIKey: LHHPQQSCUGACJT-ZMFCMNQTSA-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 = 320 nM (pChEMBL 6.5)
Descriptors: MW 476.58, ALogP 4.48, HBA 6, HBD 1, PSA 89.6, QED 0.57
Compound (SMILES): Cc1cc(-c2cc(-c3cnnn3C)cnc2N)ccc1C(=O)N[C@H](CO)c1cccc(Cl)c1
InChIKey: WWQLJMHLZXRPDD-OAQYLSRUSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Kinase Assay (New): Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions are read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values are determined using a four-parameter fit.
Measurement: IC50 = 0.149 nM (pChEMBL 9.829999923706055)
Descriptors: MW 462.94, ALogP 3.55, HBA 7, HBD 3, PSA 118.95, QED 0.4
Compound (SMILES): CCc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCNC[C@@H](O)C3)n2)c1
InChIKey: QBMHBUSZNODSPS-LJQANCHMSA-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 = 0.056 nM (pChEMBL 10.25)
Descriptors: MW 415.5, ALogP 2.21, HBA 8, HBD 2, PSA 91.99, QED 0.53
Compound (SMILES): CCn1cc(-c2cnc(C)nc2OC[C@H]2C[C@@H]2c2ccc3ccccc3n2)ccc1=O
InChIKey: BPYLPYLSWFBRIH-UXHICEINSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: PDE10 Fluorescence Polarization (FP) Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAPU technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 uL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 uL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate.
Measurement: Ki = 0.024 nM (pChEMBL 10.619999885559082)
Descriptors: MW 412.49, ALogP 4.36, HBA 6, HBD 0, PSA 69.9, QED 0.47
Compound (SMILES): O=C(Nc1cnc2ccc(N3CCC[C@@H]3c3cc(F)ccc3F)nn12)N1CCC(O)CC1
InChIKey: ZSQPJEXCTAOQSR-GOSISDBHSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: ELISA Assay: An enzyme-linked immunosorbant assay (ELISA) was used to assess TrkA kinase activity in the presence of inhibitors. Immulon 4HBX 384-well microtiter plates (Thermo part #8755) were coated with a 0.025 mg/mL solution of poly (Glu, Ala, Tyr; 6:3:1; Sigma P3899). Various concentrations of test compound, 2.5 nM TrkA (Invitrogen Corp., histidine-tagged recombinant human TrkA, cytoplasmic domain), and 500 uM ATP were incubated for 25 minutes at ambient temperature in the coated plates while shaking. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The reaction mixture was removed from the plate by washing with PBS containing 0.1% (v/v) Tween 20. The phosphorylated reaction product was detected using 0.2 ug/mL of a phosphotyrosine specific monoclonal antibody (clone PY20) conjugated to horseradish peroxidase in conjunction with the TMB Peroxidase Substrate System (KPL).
Measurement: IC50 = 13.8 nM (pChEMBL 7.860000133514404)
Descriptors: MW 442.47, ALogP 3.34, HBA 6, HBD 2, PSA 86, QED 0.65
Compound (SMILES): NC(=O)c1ccc(N2CC[C@H](CNC(=O)c3ccc(F)c(F)c3)C2)c2cncnc12
InChIKey: GPHJWJNDSJUYFY-GFCCVEGCSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 470 nM (pChEMBL 6.329999923706055)
Descriptors: MW 411.41, ALogP 2.26, HBA 5, HBD 2, PSA 101.21, QED 0.67
Compound (SMILES): Cc1nn(C)c(C)c1-c1cnc(N)c(-c2ccc(C(=O)N[C@H](CN)c3cccc(Cl)c3)c(F)c2)c1
InChIKey: AQHZDDFYRPYMHU-HSZRJFAPSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 (20 pM) Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 20 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.02 nM ERK2, 400 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 4 hours at ambient temperature. Reaction was terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 20 ug/mL of AlphaScreen Protein A Acceptor Beads, 20 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1:1000 dilution phospho-EGF Receptor (Thr669) antibody (Cat #8808, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values were determined using a four-parameter fit.
Measurement: IC50 = 0.0364 nM (pChEMBL 10.4399995803833)
Descriptors: MW 492.99, ALogP 4.57, HBA 6, HBD 3, PSA 111.85, QED 0.36
Compound (SMILES): C[C@@H]1C[C@@H]2C[C@H](C1)C[C@@H](N1[C@@H]3CC[C@H]1C[C@@H](n1c(=O)c(N4CC[C@@H]4C(=O)O)nc4ccccc41)C3)C2
InChIKey: ZHAKSJXRYXIJIF-FWVOXTDISA-N
Target: Opioid growth factor receptor-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5TC84
Assay: In Vitro ORL-1 Receptor Binding Assay: Radioligand binding assays (screening and dose-displacement) used 0.1 nM [3H]-nociceptin (NEN; 87.7 Ci/mmole) with 10-20 μg membrane protein in a final volume of 500 μL binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Non-specific binding was determined in the presence of 10 nM unlabeled nociceptin (American Peptide Company). All reactions were performed in 96-deep well polypropylene plates for 1 h at about 25 °C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard) presoaked in 0.5% polyethylenimine (Sigma-Aldrich). Harvesting was performed using a 96-well tissue harvester (Packard) followed by three filtration washes with 500 μL ice-cold binding buffer. Filter plates were subsequently dried at 50 °C. for 2-3 hours. Fifty μL/well scintillation cocktail (BetaScint; Wallac) was added and plates were counted in a Packard Top-Count for 1 min/well.
Measurement: Ki = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 490.65, ALogP 4.44, HBA 6, HBD 1, PSA 78.67, QED 0.68