lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COn1c(NC(C)c2ccc(C(F)(F)F)cc2)nc2c(c1=O)CN(C(=O)c1ccc(C#N)cc1)CC2
InChIKey: TTYPSQMIKGPQGE-UHFFFAOYSA-N
Target: Prolactin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P49683
Assay: Inhibition Assay: Receptor-expressing HEK 293 cell membrane fraction was analyzed by modifying the methods of C. J. Langmead et al. (see Langmead C J, Szekeres P G; Chambers J K, Ratcliffe S J, Jones D N C, Hirst W D, et al. Characterization of the binding of [125I]-human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor. Br J Pharmacol 2000; 131: 683-688.). To each well of a 96-well plate, 150 μl of assay buffer (20 mM HEPES, 10 mM EDTA, 1 μl/ml protease inhibiting agent cocktail, pH 7.4), 20 μl of cell membrane fraction, 10 μl of test compound and [3H]-PrRP (final concentration 1 nM) were added and incubated at room temperature for 90 minutes. After completion of the reaction, using a cell harvester, the cell-membrane fraction sample product was filtered under aspiration by a glass fiber filter plate (Unifilter; GF/B) previously treated with 0.5% polyethylene imine. The filter was washed with a 50 mM Tris hydrochloric acid buffer (pH 7.4) three times.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 497.48, ALogP 3.56, HBA 7, HBD 1, PSA 100.25, QED 0.58
Compound (SMILES): Cc1cc(CN2CCC(C(=O)NC(C)(C)C)CC2)cc(NC(=O)c2cncc(C)c2)c1
InChIKey: BWTWGWJBTWUAQM-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 76 nM (pChEMBL 7.119999885559082)
Descriptors: MW 422.57, ALogP 4.08, HBA 4, HBD 2, PSA 74.33, QED 0.76
Compound (SMILES): Cc1ccc(-c2noc(C3(CO)CC3)n2)cc1NC(=O)c1cnc2ccccn12
InChIKey: FFZMQGTUGMGYIF-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell Proliferation Assay: The compounds was tested for inhibition of SCF dependent proliferation using human Mo7e cells which endogenously express c-kit in a 384 well format. Three-fold serially diluted test compounds (Cmax=10 mM) were evaluated for their antiproliferative activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37° C., cell viability was measured by adding 25 uL of CellTiter Glo (Promega) to the cells and the luminescence was measured by a CLIPR CCD camera (Molecular Devices).
Measurement: IC50 = 137 nM (pChEMBL 6.860000133514404)
Descriptors: MW 389.42, ALogP 2.97, HBA 7, HBD 2, PSA 105.55, QED 0.54
Compound (SMILES): Cc1ccc(OCCCCOc2c3ccoc3cc3oc(=O)ccc23)cc1
InChIKey: GQFHTXXEYJJIFQ-UHFFFAOYSA-N
Target: Potassium voltage-gated channel subfamily A member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22001
Assay: Inhibition of Kv1.3 (unknown origin)
Measurement: EC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 364.4, ALogP 5.09, HBA 5, HBD 0, PSA 61.81, QED 0.34
Compound (SMILES): NC1CCN(Cc2n[nH]c3cnc(-c4cn[nH]c4)cc23)CC1
InChIKey: YBAPRVNDLDUEDL-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 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 297.37, ALogP 1.27, HBA 5, HBD 3, PSA 99.51, QED 0.68
Compound (SMILES): CCC(C)(C)NC(=O)C1CCN(Cc2cccc(NC(=O)c3ccc(Cl)c[n+]3[O-])c2)CC1
InChIKey: VFVAZWIGTDJXPJ-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 458.99, ALogP 3.74, HBA 4, HBD 2, PSA 88.38, QED 0.49
Compound (SMILES): COc1cc(OC)cc(N(CCCNCCC#N)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: WITKAEIQNFPTFS-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Inhibition of human FGFR1 using Btn-Flt3 as substrate after 60 mins by TR-FRET assay
Measurement: IC50 = 1.862 nM (pChEMBL 8.729999542236328)
Descriptors: MW 471.57, ALogP 4.08, HBA 9, HBD 1, PSA 101.12, QED 0.33
Compound (SMILES): CC(C)Oc1ccc(-c2nc(-c3cccc4c3CC[C@@H]4NC(=O)N3CCC(CO)CC3)no2)cc1C#N
InChIKey: NCNLNRARNCNSPD-DEOSSOPVSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 501.59, ALogP 4.46, HBA 7, HBD 2, PSA 124.51, QED 0.51
Compound (SMILES): O=C(c1ccncc1)N1CCN(C(=O)c2cc(Cc3n[nH]c(=O)c4c3NCCC4)ccc2F)CC1
InChIKey: VFFIAEVQTYLPHP-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 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 476.51, ALogP 1.85, HBA 6, HBD 2, PSA 111.29, QED 0.59
Compound (SMILES): N#Cc1cccc(-c2cnc3nnn(C[C@@H]4CN(c5ncc(C6CCN(CCO)CC6)cn5)CCO4)c3n2)c1
InChIKey: SOJNPCDWUKUIHT-QHCPKHFHSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Lanthanide Fluoro-Immunoassay: The c-Met kinase inhibitory activity was analyzed using dissociation enhanced lanthanide fluoro-immunoassay (DELFIA, Perkin Elmer), which is a kind of time-resolved fluorescence (TRF). 10 mL of the compound prepared in the present invention, as a test compound, was put in a Greiner 96-well V-shaped bottom plate, and a tyrosin kinase buffer (20 mL) mixed with a c-Met enzyme was added thereto, and then the enzyme and the test compound were stirred for 15 minutes, followed by culturing. An ATP solution (10 mL) was added thereto to proceed with a kinase reaction at room temperature for 30 minutes, and then a 50 mM ethylene diamine acetic acid solution (EDTA, 40 mL) was added to stop the reaction. After that, the reaction mixture was transferred to a plate coated with Streptavidin, followed by culturing under shaking. After 2 hours, the cultured material was washed three times with a PBS-T buffer (PBS 0.05% Tween 20). The europium-labeled anti-phosphotyrosine antibody was diluted to 1:2,500, and 100 mL of the diluent was added per well, followed by culturing under shaking. After 1 hour, the cultured material was washed three times with a PBS-T buffer (PBS 0.05% Tween 20). 100 mL of an enhancement solution was added, followed by shaking culturing for 5 minutes, and then the reaction material was read out within a wavelength range of 615/665 nm using a Wallac Envision 2103 device.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 526.61, ALogP 1.63, HBA 12, HBD 1, PSA 142, QED 0.38
Compound (SMILES): CCC1CCC(=O)N1c1cc(-c2c(C)noc2C)cc2[nH]c(C3CC3)nc12
InChIKey: LRJYXRIINFMDRS-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Homogeneous Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM crytptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 As delay and 200 Î¼s read window). Data were normalized based on a positive (2 Î¼M I-BET) and negative (DMSO) controls and IC50values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 186.6 nM (pChEMBL 6.730000019073486)
Descriptors: MW 364.45, ALogP 4.62, HBA 4, HBD 1, PSA 75.02, QED 0.73
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H](C(=O)N1c2ccccc2C[C@H]1C(=O)Nc1ccccc1)c1ccccc1
InChIKey: SXGUMECDXUJBHY-NWVWQQAFSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 226 nM (pChEMBL 6.650000095367432)
Descriptors: MW 456.55, ALogP 3.05, HBA 4, HBD 3, PSA 90.54, QED 0.51
Compound (SMILES): Nc1ncc(Cl)cc1-c1ccc(Oc2ccc(NC(=O)Nc3cc(C(F)(F)F)ccc3-c3cccnc3)cn2)cc1
InChIKey: MSGLTAJCZDCMAD-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Activity Assay: On the day before the assay, CellSenser TrkA-NFAT-bla CHO-K1 cells were suspended in an assay medium (Opti-MEM1 Reduced Serum Medium (Invitrogen) containing 0.5% dialysed fetal bovine serum (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen) and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin (Invitrogen))) and plated at a density of 2.4x104 cells/40 uL/well in a 96-well clear bottom plate (Corning, Catalogue No.: 3882). In some wells were added only the assay medium at 40 uL/well (Cell-free). On the day of the assay, 10 mM of the present compound (DMSO solution) was distributed in a 96-well plate (Costar, Catalogue No.: 3363) and serially diluted with DMSO with the geometrical ratio of 3. The serial dilutions were diluted with the assay medium to 100-fold to prepare a solution of the present compound with a 10-fold concentration (DMSO concentration: 1%). To the plate where cells were plated was added the present compound at 5 L/well and the plate was incubated in a CO2 incubator with 5% CO2, 95% Air at 37° C. for 30 minutes. For a control and a blank, the assay medium containing 1% DMSO was added at 5 L/well in place of the solution of the present compound. Subsequently the assay medium containing NGF (Mouse 2.5s, Natural, Invitrogen) was added to the plate at 5 L/well (final concentration of NGF: 50 ng/ml) and the plate was incubated in a CO2 incubator with 5% CO2, 95% Air at 37° C. for 5 hours. For the blank group, the assay medium was added in place of NGF at 5 L/well. A reporter assay detection reagent (10 L/well) was added to the plate which was then incubated in the dark at room temperature for 120 minutes. The reporter assay detection reagent was prepared from LiveBLAzer -FRET B/G Loading Kit (Invitrogen). On the Analyst GT (Molecular Devices Japan, K.K.) the wells were irradiated with excitation light at 405 nm and the fluorescence intensities at 460 nm and 530 nm were measured.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 576.97, ALogP 7.9, HBA 6, HBD 3, PSA 115.05, QED 0.19
Compound (SMILES): Cc1cc(N=S(C)(C)=O)cc2ncnc(Nc3ccc(F)cc3OC[C@@H]3CCC(=O)N3)c12
InChIKey: GDQXGDWNSPTSSK-HNNXBMFYSA-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 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 457.53, ALogP 3.84, HBA 7, HBD 2, PSA 105.57, QED 0.58
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCN(c2cccnc2)CC1
InChIKey: GXTNSYGCZLJIDS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 419.46, ALogP 3.17, HBA 7, HBD 2, PSA 112.86, QED 0.63
Compound (SMILES): COc1ccc(C2=NN(C3CCN(C(=O)CN4C(=O)CCC4=O)CC3)C(=O)C23CCCC3)cc1OC
InChIKey: YMWIFJZZVYOEDH-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Scintillation Proximity Assay (SPA): PDE4B1 activity was inhibited by the compounds according to the invention in a modified SPA (scintillation proximity assay) test, supplied by Amersham Biosciences (see procedural instructions phosphodiesterase [3H]cAMP SPA enzyme assay, code TRKQ 7090), carried out in 96-well microtitre plates (MTP's). The test volume is 100 ul and contains 20 mM Tris buffer (pH 7.4), 0.1 mg/ml of BSA, 5 mM Mg2+, 0.5 uM cAMP (including about 50,000 cpm of [3H]cAMP), 1 ul of the respective substance dilution in DMSO and sufficient recombinant PDE (1000x g supernatant, see above) to ensure that 10-20% of the cAMP is converted under the said experimental conditions. The final concentration of DMSO in the assays (1% v/v) does not substantially affect the activity of the PDE investigated. After a preincubation of 5 min at 37° C., the reaction is started by adding the substrate (cAMP) and the assays are incubated for a further 15 min.
Measurement: IC50 = 16.2 nM (pChEMBL 7.789999961853027)
Descriptors: MW 496.56, ALogP 1.95, HBA 7, HBD 0, PSA 108.82, QED 0.56
Compound (SMILES): COc1cc(N(c2ccc(Cl)cc2)C(C2CC2)C(F)(F)F)cnc1C(=O)C1CC1C(=O)O
InChIKey: LWAUMGSKFOFDCB-UHFFFAOYSA-N
Target: Leukotriene C4 synthase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16873
Assay: In Vitro hLTC4 Synthase Enzyme Assay (Test A): In the assay, LTC4 synthase catalyses the reaction where the substrate LTA4 is converted to LTC4. Recombinant human LTC4 synthase is expressed in Piccia pastoralis and the purified enzyme is dissolved in 25 mM tris-buffer pH 7.8 supplemented with 0.1 mM glutathione (GSH) and stored at −80 °C. In order to obtain IC50-values for the compounds, the following procedure was used: A volume (24 μL) of 0.25 g/mL LTC4 synthase in 75 mM tris (pH ~8.5), 0.5 mM MgCl2 and 3 mM GSH was preincubated for 10 min with 0.5 μL of compound of interest in DMSO at ten different concentrations, typically in the range 10^−9,5-10^−5 M as well as with DMSO only. The buffer is used as background. Runs are performed in duplicates. The enzymatic reaction is initiated by addition of 0.5 μL LTA4 in diglyme (final assay concentration 8 μM). The reaction is stopped after 1 min by addition of double the reaction volume of a stop solution (MeOH:H2O:acetic acid 70:30:1).
Measurement: IC50 = 149 nM (pChEMBL 6.829999923706055)
Descriptors: MW 468.86, ALogP 5.13, HBA 5, HBD 1, PSA 79.73, QED 0.54
Compound (SMILES): Cc1cccc(C)c1N1C[C@@](CN(C)C(C)C)(C(=O)Nc2cc(C3CC3)cc(C(F)(F)F)c2)[C@H](C)C1=O
InChIKey: RSOBIPLMXJTXOW-PIIWDFAUSA-N
Target: Chemerin-like receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99788
Assay: Displacement of [125I]-chemerin from ChemR23 (unknown origin) transfected in mouse BaF3 cells after 3 hrs by scintillation counting method
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 515.62, ALogP 6.15, HBA 3, HBD 1, PSA 52.65, QED 0.48
Compound (SMILES): Cc1c(-c2ccc(N3CCc4cccc(C(=O)Nc5nc6ccccc6s5)c4C3)nc2C(=O)O)cnn1CC1CCCOC1
InChIKey: GXSMBNWKCTYDEF-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 608.72, ALogP 5.8, HBA 9, HBD 2, PSA 122.47, QED 0.24
Compound (SMILES): Cc1ccc2c(c1)Oc1sc(Cl)cc1C(N1CCN(CC(C)(C)C(=O)O)CC1)=N2
InChIKey: PSYAJOBNPBYKRW-UHFFFAOYSA-N
Target: Histamine H1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35367
Assay: Scintillation Proximity Assay: [3H]-Pyrilamine binding experiments are carried out in SPA (scintillation proximity assay) 96-well format. Membranes used in this assay are prepared from HEK-293 cells stably expressing recombinant H1 receptor (human). The incubation is initiated by the addition of a mixture of WGA PVT SPA beads (1 mg/well, Perkin Elmer (MA, USA) RPNQ0001) and 3 μg membranes to assay buffer (67 mM Tris; pH 7.6) containing 3.5 nM [3H]-Pyrilamine and varying concentrations of the test compound (10 point concentration response curves). Non-specific binding is determined in the presence of 10 μM Triprolidine. Samples are incubated for 4 hr. at room temperature (22° C.) and then read in a Microbeta Trilux.
Measurement: Ki = 20.6 nM (pChEMBL 7.690000057220459)
Descriptors: MW 433.96, ALogP 4.62, HBA 6, HBD 1, PSA 65.37, QED 0.76
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4cnc(OCC5CCOCC5)c(Cl)c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: CPETXTUMYSFBBH-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki < 0.01 nM (pChEMBL 11)
Descriptors: MW 859.88, ALogP 8.77, HBA 10, HBD 2, PSA 138.98, QED 0.13
Compound (SMILES): O=C(COc1ccc2cc(C(O)(C(F)(F)F)C(F)(F)F)c(O)cc2c1)NC12CC3CC(CC(C3)C1)C2
InChIKey: JSMGISXPSWKNKU-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Inhibition of recombinant SHP2 (unknown origin) using pNPP as substrate by spectrophotometric method
Measurement: IC50 = 9000 nM (pChEMBL 5.050000190734863)
Descriptors: MW 517.47, ALogP 5.32, HBA 4, HBD 3, PSA 78.79, QED 0.47
Compound (SMILES): N#Cc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)cc3)cn2CCN2CCCC2)CC1
InChIKey: RBBGFPFJXKWNLT-UHFFFAOYSA-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.degree. 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 = 2.6 nM (pChEMBL 8.59000015258789)
Descriptors: MW 460.56, ALogP 3.41, HBA 8, HBD 1, PSA 99.89, QED 0.6
Compound (SMILES): COc1cncc(C(=O)Nc2cccc(CN3CCC(C(=O)N(C)CC(C)C)CC3)c2)c1
InChIKey: OHWXBAAFSYKRTN-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 438.57, ALogP 3.67, HBA 5, HBD 1, PSA 74.77, QED 0.68
Compound (SMILES): CCCOC(=O)c1ccc(NC(=O)c2c(O)c3cc(C)ccc3n(CCC)c2=O)cc1
InChIKey: CBDWGNBITJLFQB-UHFFFAOYSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Measurement: EC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 422.48, ALogP 4.24, HBA 6, HBD 2, PSA 97.63, QED 0.56
Compound (SMILES): CCc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(C(F)(F)F)c3)cn2CCN2CCCCC2)CC1
InChIKey: BBZZGHQVCVQLBB-UHFFFAOYSA-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.degree. 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 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 545.63, ALogP 5.51, HBA 7, HBD 1, PSA 76.1, QED 0.39
Compound (SMILES): N#Cc1ccc(Cl)cc1-c1cc(=O)n(CC(=O)Nc2ccc(C(=O)O)cc2)c2c1C(=O)CC2
InChIKey: KKZSAPVNMYAUBS-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: The factor XIa inhibition of the inventive substances is determined using a biochemical test system which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 uM to 0.0078 uM; resulting final concentrations in the test: 50 uM to 0.00013 uM). In each case 1 ul of the diluted substance solutions is placed into the wells of white microtitre plates from Greiner (384 wells). 20 ul of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 ul of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 ul of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 uM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 461.86, ALogP 3.51, HBA 6, HBD 2, PSA 129.26, QED 0.6
Compound (SMILES): CC(C)C[C@@H](CO)NC(=O)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: LXHVQOVXCAZNPJ-FQEVSTJZSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 837 nM (pChEMBL 6.079999923706055)
Descriptors: MW 381.48, ALogP 3.37, HBA 3, HBD 3, PSA 81.67, QED 0.72
Compound (SMILES): CC(=O)C1=C(C)N(c2cccc(C(F)(F)F)c2)C(=O)N(C)C1c1ccc(C#N)cc1C(=O)NC1CCN(C)C1
InChIKey: BIWIVXWIEPGHOB-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available.The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA. Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 uL, of these compound dilutions were mixed with 10 uL Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 uL, substrate solution in assay buffer were added (250 uM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 539.56, ALogP 4.49, HBA 5, HBD 1, PSA 96.75, QED 0.6
Compound (SMILES): O=C(NCCc1ccc(F)cc1)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: DFNGTSXITOYJOX-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 56.7 nM (pChEMBL 7.25)
Descriptors: MW 403.46, ALogP 4.35, HBA 2, HBD 2, PSA 61.44, QED 0.67
Compound (SMILES): COC(=O)N1CCN(c2ccc(Nc3ncc(Cl)c(Nc4ccc(N5CCN(C(C)=O)CC5)cc4OC)n3)c(OC)c2)CC1
InChIKey: RJXCNRGHQNDKBN-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 625.13, ALogP 4.19, HBA 11, HBD 2, PSA 124.63, QED 0.37
Compound (SMILES): Cc1cccc(-c2ocnc2C(=O)Nc2cnn(CCn3ccc(-c4ccccc4)c3)c2)c1
InChIKey: UPMHLEIQTUZINJ-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20'000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37 °C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM. Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 μM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37 °C. in 5% CO2 followed by equilibration at RT for 30 min before measurement. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μl/well, incubated for 120 min and finally 10 μl/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 864 nM (pChEMBL 6.059999942779541)
Descriptors: MW 437.5, ALogP 5.27, HBA 6, HBD 1, PSA 77.88, QED 0.38
Compound (SMILES): CC[C@H](C)[C@H](NC(=O)C[C@H](N)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)Cc1cccc(OC(F)(F)F)c1)C(C)C)C(=O)N[C@H](C(=O)O)C(C)C
InChIKey: GUIJFUFZFTUQTG-IIEJMTDKSA-N
Target: Cathepsin D (SINGLE PROTEIN), Homo sapiens
UniProt: P07339
Assay: In-Vitro Fluorescence Assay: In order to identify modulators of cathepsin D activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group (MCA=(7-methoxycoumarin-4-yl)acetyl) which is quenched by energy transfer from a Dpn (2,4 dinitrophenyl) group on the same molecule, in Greiner 384-well nb microtitre plates. Cleavage of the peptidic substrate by cathepsin D causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was pepstatin A (Sigma-Aldrich). The substrate used was MCAGKPILFFRLK(Dnp)d-R-NH2 (Enzo Life Sciences, Lorrach). The enzyme employed was cathepsin D isolated from the human liver (Sigma-Aldrich) in a final concentration of 1.4 nM. The test was carried out in 100 mM sodium acetate buffer, 1.25% (v/v) of DMSO, 0.25% (w/v) of Chaps, pH 5.5. 2 ul of each substance solution with serially diluted substance concentration were added to in each case 4 ul of cathepsin D solution and incubated at room temperature for 10 min. The reaction was started by addition of 2 ul of substrate solution (final concentration 5 uM). After carrying out a starting-point fluorescence measurement (excitation wavelength 340 nm/emission wavelength 450 nm) using an Envision multilabel reader (Perkin Elmer), the reaction was incubated at room temperature for 60 min. The amount of peptide fragment cleaved off during the reaction time was subsequently measured by determination of the increase in the fluorescence intensity at 450 nm (excitation wavelength 340 nm).
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 721.82, ALogP 3.47, HBA 7, HBD 6, PSA 188.95, QED 0.13
Compound (SMILES): FC(F)(F)c1ccc(N2CCN(c3nnc(Cc4ccccc4)c4c3CCC4)CC2)nc1
InChIKey: GYFPNEDYUMLPDL-UHFFFAOYSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 439.49, ALogP 4.3, HBA 5, HBD 0, PSA 45.15, QED 0.61
Compound (SMILES): NC(=O)C1(c2ccccc2CCc2nc(Nc3ccnnc3)ncc2Cl)CC1
InChIKey: PPFXUUIFCFAGQI-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P35916
Assay: VEGFR3 Biochemical Assay: A biotin labelled peptide is used as substrate (amino acid sequence: Biotin-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2). VEGFR3 cytoplasmic domain (amino acids 798-1298) was purchased as N-terminal GST-fusion protein (the enzyme). The 15 uL assay reactions are run in Greiner brand white 384-well low volume plates. All reactions contained 10 mM HEPES pH 7.4, mM MgCl2, 0.01% (v/v) Tween-20, 50 uM Na3VO4, 0.01% (w/v) albumin from chicken egg white, 1 mM Dithiothreitol, 111 nM peptide substrate, 500 uM ATP, and 3.8 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 90 minutes at 30 degree Celsius.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 394.87, ALogP 2.97, HBA 6, HBD 2, PSA 106.68, QED 0.64
Compound (SMILES): COc1cc(F)ccc1Oc1nc2ccccc2nc1C(=O)Nc1nnn[nH]1
InChIKey: DAYWNRVVMMSBKX-UHFFFAOYSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q62968
Assay: Electrophysiology Assay: Patch clamp electrophysiology was used to assess the efficacy and selectivity of sodium channel blockers in dorsal root ganglion neurons. Rat neurons were isolated from the dorsal root ganglions and maintained in culture for 2 to 10 days in the presence of NGF (50 ng/ml) (culture media consisted of NeurobasalA supplemented with B27, glutamine and antibiotics). Small diameter neurons (nociceptors, 8-12 um in diameter) were visually identified and probed with fine tip glass electrodes connected to an amplifier (Axon Instruments). The "voltage clamp" mode was used to assess the compound's IC50 holding the cells at -60 mV. In addition, the "current clamp" mode was employed to test the efficacy of the compounds in blocking action potential generation in response to current injections.
Measurement: IC50 = 2200 nM (pChEMBL 5.659999847412109)
Descriptors: MW 381.33, ALogP 2.34, HBA 8, HBD 2, PSA 127.8, QED 0.54
Compound (SMILES): O=S1(=O)c2ccccc2CN1c1cccc(Oc2ccccn2)c1
InChIKey: YZSQMDOFTDXYBI-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Agonist activity at C-terminal YFP-tagged human FFA4 expressed in HEK293 cells in presence of coelentrazine h by BRET based beta-arrestin-2 interaction assay
Measurement: EC50 = 1548.82 nM (pChEMBL 5.809999942779541)
Descriptors: MW 338.39, ALogP 3.58, HBA 4, HBD 0, PSA 59.5, QED 0.73
Compound (SMILES): CCC(CC)C(=O)N1CCC(Oc2ccc(NC(=O)N3Cc4ccncc4C3)cc2)CC1
InChIKey: PTPQPADJFFVIJB-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 266 nM (pChEMBL 6.579999923706055)
Descriptors: MW 436.56, ALogP 4.44, HBA 4, HBD 1, PSA 74.77, QED 0.73
Compound (SMILES): O=C(CSC(c1ccccc1)c1ccccc1)N1CCN(CCCc2ccccc2)CC1
InChIKey: GDQGBNUPTWWVQR-UHFFFAOYSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Rattus norvegicus
UniProt: P23977
Assay: Displacement of [3H]WIN35,428 from DAT in Sprague-Dawley rat brain membranes incubated for 120 mins by radioligand binding assay
Measurement: Ki = 47.2 nM (pChEMBL 7.329999923706055)
Descriptors: MW 444.64, ALogP 5.29, HBA 3, HBD 0, PSA 23.55, QED 0.45