lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(-c2cn(CC(=O)Nc3cccc(Cl)c3Cl)nn2)ccc1-n1cnc(C)c1
InChIKey: XWKVYBKJUXFKMD-UHFFFAOYSA-N
Target: Probable G-protein coupled receptor 142 (SINGLE PROTEIN), Mus musculus
UniProt: Q7TQN9
Assay: Agonist activity at mouse GPR142 expressed in CHO cells measured after 30 to 60 mins by IP-One assay
Measurement: EC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 457.32, ALogP 4.39, HBA 7, HBD 1, PSA 86.86, QED 0.46
Compound (SMILES): Cc1ccc(C(=O)N2CC(c3ccc(C#N)cc3)C2)cc1-c1[nH]c(C2CCOCC2)nc1C#N
InChIKey: URNMDMNSTFPKSL-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 451.53, ALogP 4.26, HBA 5, HBD 1, PSA 105.8, QED 0.64
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#CC1CCCCC1)c1nc2cccc(Cl)c2c(=O)n1-c1ccccc1
InChIKey: XIJMBFHREMJADV-SFHVURJKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 499.02, ALogP 5.52, HBA 7, HBD 2, PSA 98.72, QED 0.36
Compound (SMILES): N#Cc1c(N)nc(N)nc1N[C@H](c1nc2c(F)ccc(Cl)c2c(=O)n1-c1ccc(N)nc1)C1CC1
InChIKey: VOKQIKXWIGKAPD-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy transfer (TR-FRET) Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoforms: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 10lps delay and 500 μs read window).
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 492.91, ALogP 2.54, HBA 11, HBD 4, PSA 187.44, QED 0.32
Compound (SMILES): Cc1nc(C(=O)N2Cc3cc4c(cc3C[C@H]2C(=O)N[C@@H](Cc2ccc(-c3ccnc(C)c3C)cc2)C(=O)O)OC[C@H](c2ccc(OCC3CCCCC3)cc2)O4)c(C)o1
InChIKey: ZFKSWEHSJKTDES-IWJNXRQDSA-N
Target: Glucagon-like peptide 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43220
Assay: cAMP Functional Assay: The efficacy of GLP-1 receptor agonists was studied in a cAMP functional assay using HEK-293 Cells expressing the cloned human GLP-1 receptor. GLP-1-expressing cells (10,000-20,000 cells per 0.1 mL) were plated in 96-well plates in Dulbecco's modified eagles media (DMEM) containing 10% fetal bovine serum, 2 mg/mL G418 and penicillin-streptomycin. Following overnight incubation of cells, media was removed, and compounds (at concentrations ranging from 0.0001 to 100 μM) were added to monolayer cells in Iscove's modified dulbecco's medium (IMDM), 100 μM RO 20-1724 PDE inhibitor, 0.1% BSA, 2% DMSO in a final volume of 100 μL, and incubated for 30 min at 37° C. 95%02, 5% CO2 in a humidified incubator. Alternatively, cells were harvested and combined with compounds in suspension using the buffer and protocol described above. cAMP was quantified using a homogenous time-resolved fluorescence detection system (cAMP dynamic, CIS bio International).
Measurement: EC50 = 213 nM (pChEMBL 6.670000076293945)
Descriptors: MW 798.94, ALogP 7.82, HBA 9, HBD 2, PSA 153.32, QED 0.14
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cc4ccccc4s3)CC2)C1
InChIKey: CMBZNHSLRJYIHI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 522.64, ALogP 4.27, HBA 8, HBD 1, PSA 106.73, QED 0.37
Compound (SMILES): C[C@]1(N2CCc3c(-c4cnc(N)nc4)nc(N4CCOCC4)nc32)CCN(S(C)(=O)=O)C1
InChIKey: ROYQBLPXACEGTQ-FQEVSTJZSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Inhibition of recombinant human N-terminal Myr signal and C-terminal Flag-tagged PI3K p110delta expressed in rat Rat1 cells
Measurement: IC50 = 76 nM (pChEMBL 7.119999885559082)
Descriptors: MW 460.56, ALogP 0.14, HBA 10, HBD 1, PSA 130.67, QED 0.67
Compound (SMILES): Cc1cc(N=S(C)(C)=O)cc2ncnc(Nc3ccc(F)cc3OCCCO)c12
InChIKey: BZMZVZDMEVXDNM-UHFFFAOYSA-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 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 418.49, ALogP 3.94, HBA 7, HBD 2, PSA 96.7, QED 0.56
Compound (SMILES): O=C1NCC(c2cccc(Cl)c2Cl)N1c1ccc2nc[nH]c2c1
InChIKey: YVAVPYJCIDFSNA-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: Spectrophotometic Assay: Spectrophotometic Assay: This novel assay was used to determine the kinetic parameters for most of the QC substrates. QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R.C.J. 1989 J Neurosci Methods 30, 23-28).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 347.21, ALogP 4.14, HBA 2, HBD 2, PSA 61.02, QED 0.73
Compound (SMILES): COc1cccc(OC)c1C1CCCC(=O)N1Cc1ccc(-c2ccccc2)s1
InChIKey: GSZNHHZGYCSYMK-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements: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 g/ml G418, 100 U/ml penicillin, 100 g/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 M 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.
Measurement: IC50 = 117 nM (pChEMBL 6.929999828338623)
Descriptors: MW 407.54, ALogP 5.69, HBA 4, HBD 0, PSA 38.77, QED 0.53
Compound (SMILES): CS(=O)(=O)c1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: ZSQDUTTYOZJDEM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 410.5, ALogP 2.39, HBA 7, HBD 1, PSA 96.25, QED 0.55
Compound (SMILES): [C-]#[N+]c1c(F)ccc([C@H]2CN3CCN(C(=O)Cc4ccc(-n5cnnn5)nc4)C[C@H]3CO2)c1C1CC1
InChIKey: VWHYMPLXLPJHOT-GHTZIAJQSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: The ROMK channel functional thallium flux assay is performed in 384 wells, using the FLIPR-Tetra instrument. HEK-hKir1.1 cells are seeded in Poly-D-Lysine microplates and kept in a 37° C.-10% CO2 incubator overnight. On the day of the experiment, the growth media is replaced with the FluxOR™ reagent loading buffer and incubated, protected from light, at ambient temperature (23-25° C.) for 90 min. The loading buffer is replaced with assay buffer±test compound followed by 30 min incubation at ambient temperature, where the Thallium/Potassium stimulant is added to the microplate. The fluorescence intensity of wells containing 3 μM of a standard control ROMK inhibitor of the present invention is used to define the ROMK-sensitive component of thallium flux. Fluorescence in the presence of test compounds is normalized to control values to provide % fluorescence change. IC50 values represent the concentration of compound that inhibits 50% of the ROMK thallium flux signal.
Measurement: IC50 = 380 nM (pChEMBL 6.420000076293945)
Descriptors: MW 488.53, ALogP 2.45, HBA 8, HBD 0, PSA 93.63, QED 0.51
Compound (SMILES): COc1cc(C2CCNCC2)ccc1Nc1nc(Cc2cccc(Cl)c2Cl)n2cnnc2c1C(N)=O
InChIKey: LHQXVVWDVXUIIS-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Enzyme Inhibition Assay: ALK kinase assays were conducted with the indicated final concentrations unless otherwise specified. In 384 well black plates (Axygen), 8 ul of compound (2% DMSO) was incubated with 8 ul Lck-peptide substrate (0.5 uM, biotin-Ahx-GAEEEIYAAFFA-COOH) and 8 ul of a mixture of ALK (3 nM, Millipore) and ATP (50 uM) in reaction buffer (50 mM Hepes, pH 7.4; 10 mM MgCl2; 2 mM MnCl2; 0.1 mM sodium orthovanadate; 0.01% BSA and 1 mM DTT (added fresh before assay) for 1 h at room temperature. Reactions were then quenched by the addition of 30 ul quench solution (streptavidin-allophycocyanin and Europium-cryptate PT66 monoclonal antibody in 40 mM Hepes, pH 7.4; 480 mM KF; 66 mM EDTA; 0.01% Tween-20; and 0.1% BSA) at room temperature. Plates were read 1 h after quenching on an Envision Multilaber Reader and IC50 values were calculated using a sigmoidal fit of the concentration/inhibition response curves. These values were converted to apparent Ki values.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 526.43, ALogP 4.34, HBA 8, HBD 3, PSA 119.46, QED 0.33
Compound (SMILES): CC1(C)CCN(c2cccc(Nc3cc(-c4cccc(C(=O)O)c4)nn4ccnc34)n2)CC1
InChIKey: KOLZHRHCNWFURW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition Assay: Assay plates: 96-well MultiScreen 0.65 um filter plates (Millipore Cat. No.: MADVNOB10)Streptavidin coated beads: Streptavidin Sepharose, suspension 5.0 mL, in 50 mM EDTA/PBS diluted (1:100), (Amersham, Cat. No.: 17-5113-01)Compounds: 10 mM in 100% dimethylsulfoxide (DMSO), final conc.: compound 0.003-100 uM in 10% DMSOEnzyme: SYK RPA purified, truncated construct of Spleen Tyrosine Kinase aa 360-635, stock solution 1 mg/mL, MW: 31.2 KDa, final conc.:0.0005 uM. Peptide 1: biotinylated peptide is derived from a naturally occurring phosphor-acceptor consensus sequence (Biotin-EPEGDYEEVLE), special order from QCB, stock solution 20 mM, final conc.: 5.0 uM.ATP: Adenosine-5'-triphosphate 20 mM, (ROCHE Cat. No.: 93202720), final concentration: 20 uM Buffer: HEPES: 2-Hydroxyethyl piperazine-2-ethanesulfonic acid (Sigma, Cat. No.: H-3375)final concentration: 50 mM HEPES pH7.5BSA: Bovine Serum Albumin Fraction V, fatty acid free (Roche Diagnostics GmbH, Cat. No. 9100221).
Measurement: IC50 = 1383.6 nM (pChEMBL 5.860000133514404)
Descriptors: MW 442.52, ALogP 4.86, HBA 7, HBD 2, PSA 95.65, QED 0.46
Compound (SMILES): CCNC(=S)Nc1cc(-n2cnc(CNC3CCCCC3)c2)ncn1
InChIKey: RIYABXGLUCJQBX-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Rattus norvegicus
UniProt: P37136
Assay: Inhibition of rat cortex AChE using acetylthiocholine iodide as substrate measured after 20 mins by Ellman's method
Measurement: IC50 = 424 nM (pChEMBL 6.369999885559082)
Descriptors: MW 359.5, ALogP 2.39, HBA 6, HBD 3, PSA 79.69, QED 0.68
Compound (SMILES): O=C(Cn1c2ccccc2c2ccnc(CN(CCCC3CCNCC3)[C@H]3CCCc4cccnc43)c21)N1CCOCC1
InChIKey: DTWYHOJPBDJGOX-YTTGMZPUSA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P61073
Assay: Calcium Mobilization Assay: Functional modulation of CXCR4 was determined by calcium mobilization assay using leukemic lymphoid CEM cells, which naturally express high levels of CXCR4. Generally, the assay is carried out as follows: Cells are grown in vitro to confluency. On the day of the assay, cells are removed from the incubator, culture medium removed and replaced with a calcium sensitive dye (Calcium3 assay kit; Molecular Devices, Sunnyvale, Calif.). The cells are allowed to dye load for 45-60 min at 37 °C. in the cell culture incubator and then allowed to equilibrate to room temperature for no more than 15 min before assay. Compound plates were generated containing up to 3% dimethyl sulfoxide in media kept at room temperature. Test compounds were added to the cells at a 1:3 dilution, and calcium mobilization was measured using a Flex Station fluorescence imager (Molecular Devices). This first read was used to determine direct agonist activation of the CXCR4 receptor by the test compounds.
Measurement: IC50 = 124 nM (pChEMBL 6.909999847412109)
Descriptors: MW 580.78, ALogP 5.1, HBA 7, HBD 1, PSA 75.52, QED 0.3
Compound (SMILES): CC(C)N1CCC(c2cc(NC(=O)c3cnn4cccnc34)n(-c3ccc(C4CC4)cc3)n2)CC1
InChIKey: CYIHFXFWPCSXOH-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 469.59, ALogP 4.63, HBA 7, HBD 1, PSA 80.35, QED 0.45
Compound (SMILES): O=C(OC(c1ccc(Cl)c(Cl)c1)C1CNC1)c1ccccc1
InChIKey: XXXHDRXZALCTPU-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Binding Assay: 5-HT transporter binding assay for evaluating binding of the compound to the serotonin transporter was carried out using human recombinant serotonin transporter membrane (PerkinElmer Life and Analytical Sciences, USA) expressed in HEK293 cells and radioisotope [3H]Imipramine (PerkinElmer).That is, the test drug, 2 nM [3H]Imipramine, serotonin transporter membrane (9 ug/well), 120 mM NaCl and 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM KCl were added to obtain a reaction mixture with a final volume of 0.25 ml. After incubation for 30 minutes at 27° C., the mixture was quickly passed through a Filtermat A glass fiber filter (PerkinElmer) pre-soaked with 0.5% (w/v) PEI (polyethyleneimine) using Inotech Harvester (Inotech) to terminate the reaction. After washing with cold washing buffer (50 mM Tris-HCl, pH 7.4, 154 mM NaCl) solution, the filter was covered with MeltiLex and sealed in a sample bag. After drying in an oven, radioactivity was counted using MicroBeta Plus (Wallac).
Measurement: IC50 = 69.9 nM (pChEMBL 7.159999847412109)
Descriptors: MW 336.22, ALogP 4.11, HBA 3, HBD 1, PSA 38.33, QED 0.85
Compound (SMILES): COc1ccc(CC(=O)O)cc1-c1ccc(F)c2c1CN(C(=O)CC1COCc3ccccc31)CC2
InChIKey: YEVSJBYGDXMPRQ-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 22.3 nM (pChEMBL 7.650000095367432)
Descriptors: MW 489.54, ALogP 4.72, HBA 4, HBD 1, PSA 76.07, QED 0.54
Compound (SMILES): N#C[C@H](Cc1ccc(-c2ncc(C(F)(F)F)cn2)cc1)NC(=O)[C@@H]1CNCCCO1
InChIKey: PQGYESNKRDSLIY-IRXDYDNUSA-N
Target: Dipeptidyl peptidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53634
Assay: Inhibition of human recombinant DPP1 using Gly-Arg-AMC as substrate preincubated for 30 mins followed by substrate addition by fluorometric assay
Measurement: IC50 = 199.53 nM (pChEMBL 6.699999809265137)
Descriptors: MW 419.41, ALogP 2.09, HBA 6, HBD 2, PSA 99.93, QED 0.77
Compound (SMILES): O=S1(=O)CCC(CNc2ccc3ncc(-c4cccc(OC(F)(F)F)c4)n3n2)CC1
InChIKey: JABDFUFLQGAQCU-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase-Glo Assay: These assays are set up in duplicate 50 ul volumes in white, flat bottom 96 well plates. Inhibitors are added to the solution of 1× kinase buffer, 10 uM ATP, 100 uM Pim-1-specific substrate, 50 ng of active Pim-1 enzyme, and water in serial dilutions ranging from micromolar to nanomolar concentrations. This solution is incubated at 30 degrees Celsius at 360 rpm for two hours. Following the incubation, 50 ul of Kinase-Glo reagent is added to each well, including all positive and negative control wells, and incubated at room temperature for 15 minutes. The plate is then read by the Luminoskan Ascent instrument and the results displayed with the Ascent Software version 2.6.
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 440.45, ALogP 3.53, HBA 7, HBD 1, PSA 85.59, QED 0.65
Compound (SMILES): COc1ccc(S(=O)(=O)C2CCN(c3nc4c(nc3NC(C)C)CCN(C(C)=O)C4)CC2)c(F)c1
InChIKey: LJYOHQZAIKVRIW-UHFFFAOYSA-N
Target: G-protein coupled receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P46095
Assay: GPR6 In Vitro Assay (A.2): This cell based assay measures the ability of compounds to inhibit the constitutive cAMP activity of GPR6 receptor expressed in CHO-K1 cells. CHO cells were stably expressed with GPR6 receptor, whose expression is controlled by a tetracycline inducable element. The cells were cultured in medium containing F12K, 10% FBS, 1% PennStrep, 200 ug/mL Hygromycin. GPR6 receptor expression was induced for 20 hrs with 2 ug/ml doxycycline (sigma D9891) in growth media. After addition of doxycycline cells were plated at a density of 450-750 cells per well in 96-well half-volume black tissue culture plates (Costar) and placed in an incubator (37°, 5% CO2) for 20 hours prior to cAMP assays. Culture media was removed from cells and they were washed with 50 uL/well of Ringer's Buffer (MgCl2 0.047 mg/mL, NaH2PO4 0.18 mg/mL, Na2HPO4 0.1 mg/mL, KCl 0.34 mg/mL, NaHCO3 1.26 mg/mL, D-glucose 1.8 mg/mL, NaCl 7 mg/mL; pH=7.4). Compounds suspended in DMSO were diluted in Ringer's Buffer.
Measurement: IC50 = 0.2002 nM (pChEMBL 9.699999809265137)
Descriptors: MW 505.62, ALogP 2.79, HBA 8, HBD 1, PSA 104.73, QED 0.64
Compound (SMILES): NC1=N[C@@](c2cc(NC(=O)c3ccc(Cl)cn3)ccc2F)(C(F)F)C(F)(F)CC1
InChIKey: FGBKLHOHAIWKKJ-SFHVURJKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Binding Assay: The binding assay was performed as SPA-based assay using a biotinylated form of human BACE1 recombinantly expressed and subsequently purified from Freestyle HEK293 cells. The binding assay was run in a 50 mM sodium acetate buffer, pH 4.5 containing 50 mM NaCl and 0.03% Tween-20 in white clear bottom 384 plates (Corning #3653). 10 nM (final concentration) radioligand ([3H]−N-((1S,2R)-1-benzyl-3-cyclopropylamino-2-hydroxy-propyl)-5-(methanesulfonyl-methyl-amino)-N−((R)-1-phenyl-ethyl)-isophthalamide) (TRQ11569 purchased from GE Healthcare) was mixed with test compound at a given concentration, 6 nM (final concentration) human BACE1 and 25 μg Streptavidin coated PVT core SPA beads (RPNQ0007, GE Healthcare Life Sciences) in a total volume of 40 μl. Several concentrations of each test compound were tested in the assay for IC50 determination. The plates were incubated for one hour at room temperature and counted in a Wallac Trilux counter. Total and non-specific binding were determined using buffer and 1 μM (final concentration) of the high affinity BACE1 reference inhibitor (S)-6-[3-chloro-5-(5-prop-1-ynyl-pyridin-3-yl)-thiophen-2-yl]-2-imino-3,6-dimethyl-tetrahydro-pyrimidin-4-one, respectively.
Measurement: Ki = 18 nM (pChEMBL 7.75)
Descriptors: MW 432.78, ALogP 4.37, HBA 4, HBD 2, PSA 80.37, QED 0.7
Compound (SMILES): CN(C)C(=O)c1cccc(COc2ccc(CNC(=O)c3cc4cnccc4[nH]3)cc2)c1
InChIKey: HVZNSUOYEMWTPR-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Inhibition of human full length C-terminal His6-tagged NAMPT expressed in Escherichia coli Rosetta (DE3) cells using nicotinamide as substrate incubated for 15 mins prior to substrate addition measured after 30 mins in presence of PRPP
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 428.49, ALogP 3.77, HBA 4, HBD 2, PSA 87.32, QED 0.47
Compound (SMILES): C/C=C(\C)c1cn2c(-c3ccc(C(=O)NC4CC4)c(C)c3)cnc2c(NCCC(F)(F)F)n1
InChIKey: TXWWMZFLVUVYRP-LNKIKWGQSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Inhibition of GST-tagged recombinant human MPS1 expressed in baculovirus expression system using biotin-Ahx-PWDPDDADITEILG as substrate preincubated for 15 mins followed by substrate and 10 uM ATP addition and measured after 60 mins by TR-FRET assay
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 457.5, ALogP 5.38, HBA 5, HBD 2, PSA 71.32, QED 0.5
Compound (SMILES): CC1(C)CC(=O)C2=C(C1)OC(N)=C(C#N)C2c1ccc(-c2nnn[nH]2)cc1
InChIKey: MXXHAOFWHDYGRH-UHFFFAOYSA-N
Target: Carbonic anhydrase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P43166
Assay: Inhibition of human recombinant carbonic anhydrase 7 expressed in Escherichia coli assessed as reduction in CO2 hydration preincubated for 15 mins prior to testing measured for 10 to 100 secs by stopped-flow assay
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 362.39, ALogP 2.32, HBA 7, HBD 2, PSA 130.57, QED 0.84
Compound (SMILES): NC(=O)[C@H](O)[C@@H](O)c1cccc(-c2ccc(Oc3ccc(Cl)nc3)cc2)n1
InChIKey: XAJNPHSIMCBHKT-ZWKOTPCHSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR TETRA Sodium Dye Assay: FLIPR or FLIPRTETRA Sodium Dye Assay with KCl and Test Article Pre-Incubation: Cells were prepared by plating the recombinant HEK293 cells or other host cells expressing either recombinant or non-recombinant, native, NaV1.7 alpha subunit, alone or in combination with various beta and gamma subunits at a density of 40,000 cells/well into a 96-well black, clear-bottom, PDL-coated plate. The assay can be adapted to 384-well or 1,536-well format, if desired, using proportionately less cells and media. The plate was then incubated in growth media, with or without selective antibiotic, overnight at 37° C. at 5% CO2, 95% humidity, in preparation for the assay. For counter-screens of other voltage-gated sodium channels, the procedure was very similar, though optimal densities of cells, media and subsequent assay components can be fine-tuned for the particular cell line or isoform.The next day, at the start of the assay, the media was flicked from the cells and the wells were washed once.
Measurement: IC50 = 804 nM (pChEMBL 6.090000152587891)
Descriptors: MW 385.81, ALogP 2.47, HBA 6, HBD 3, PSA 118.56, QED 0.56
Compound (SMILES): CCNC(=O)c1ccc2cc(CCc3c(C(=O)O)[nH]c(=O)[nH]c3=O)ccc2c1.NCCO
InChIKey: JSBJJAGWNVSNCI-UHFFFAOYSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Inhibition of human dihydroorotate dehydrogenase assessed as reduction in orotate production using L-dihydroorotate as substrate measured after 20 mins by spectrophotometric method
Measurement: IC50 = 472 nM (pChEMBL 6.329999923706055)
Descriptors: MW 381.39, ALogP 1.45, HBA 4, HBD 4, PSA 132.12, QED 0.51
Compound (SMILES): NC1=NC2(CCS1)c1cc(NC(=O)c3ccc(F)cn3)ccc1Oc1c2cc(-c2ccnc(F)c2)nc1F
InChIKey: FIRQOPFPZBHPRO-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Fluorescence Resonance Energy Transfer (FRET) Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The beta secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 534.52, ALogP 5.01, HBA 8, HBD 2, PSA 115.38, QED 0.36
Compound (SMILES): CCn1c(=O)c2cn[nH]c2c2ccc(-c3cccc(-n4cccn4)c3)cc21
InChIKey: OPCWMMNJFXCSIA-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Enzymatic Screening Assay: The enzymatic test is a test in two steps.In a first step, it consists in placing in contact the compound according to the invention, the dialysed MetAP2 protein and the substrate (Met-Pro-Arg-pNa peptide synthesized by Neosystem), the N-terminal methionine of which can be cleaved with MetAP2, and which bears at the C-terminal end a para-nitroaniline (pNa) chromophore, which can itself be released by another peptidase only when the N-terminal methionine has been cleaved beforehand.Consequently, the second step consists in reacting the peptides cleaved in the preceding step with a second peptidase in order to release the chromophore. The peptidase used in this second step is cathepsin, which comes from the TagZyme DAPase kit (Quiagen, 34366).The MetAP2 activity is proportional to the amount of para-nitroaniline released, which is measured by absorbance at 405 nm.
Measurement: IC50 = 332 nM (pChEMBL 6.480000019073486)
Descriptors: MW 355.4, ALogP 3.75, HBA 5, HBD 1, PSA 68.5, QED 0.54
Compound (SMILES): CN(CCO)C(=O)CN1CC=C(c2[nH]c3ncc(F)c4c3c2CN(C)c2ccc(F)cc2-4)CC1
InChIKey: SWAFXDQVRSHLJG-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay : CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were diluted in 100% DMSO then diluted with 1:10 in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. Equal volume of the compound dilutions was added to a final reaction mixture containing LANCE detection buffer (PerkinElmer CR97-100), 100 nM ULight MBP (PerkinElmer TRF0109M), 1000 μM ATP, and CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 1 hour before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer. The reaction was incubated for 1 hour and the signal was read in Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 467.52, ALogP 3, HBA 5, HBD 2, PSA 75.7, QED 0.6
Compound (SMILES): CCCOc1ccc(C2C3C=C(Cl)C(Cl)=CC3C(=O)N2c2ccc3[nH]cnc3c2)cc1
InChIKey: LJFFSFFUIMGSGS-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: Spectrophotometic Assay: Spectrophotometic Assay: This novel assay was used to determine the kinetic parameters for most of the QC substrates. QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R.C.J. 1989 J Neurosci Methods 30, 23-28).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 454.36, ALogP 5.93, HBA 3, HBD 1, PSA 58.22, QED 0.52
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1cnccn1)c1nc2cccc(S(C)(=O)=O)c2c(=O)n1-c1ccccc1
InChIKey: YNPOLVLGTODWDU-KRWDZBQOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 538.59, ALogP 2.52, HBA 11, HBD 2, PSA 158.64, QED 0.32
Compound (SMILES): Cc1c(Cl)cccc1NC(=O)c1cc(NC(=O)c2ccccc2C(F)(F)F)cc2[nH]c(N[C@@H](CO)C(C)C)nc12
InChIKey: QJDABEDPOIRCDK-QHCPKHFHSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition Assay: mPGES-1 microsome fractions were prepared from CHO-K1 cells transiently transfected with plasmid encoding the human mPGES-1cDNA. Microsomes were diluted with potassium phosphate buffer containing reduced glutathione (pH7.4), and DMSO containing test compound or DMSO alone was added (such that DMSO final concentration would be 1% in each) and incubated at 4° C. for 20 minutes. Then, the enzymatic reactions were initiated by the addition of PGH2 substrate (final concentration 1 μM) and incubated at 4° C. for 60 seconds. The reaction was terminated by the addition of a citrate solution (final citrate concentration 50 mM) containing ferric chloride (final concentration 1 mg/mL). PGE2 production in the enzyme reaction aliquot was measured using HTRF kit (Cisbio International, catalogue #62P2APEC).
Measurement: IC50 = 4.7 nM (pChEMBL 8.329999923706055)
Descriptors: MW 574, ALogP 6.48, HBA 5, HBD 5, PSA 119.14, QED 0.17
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CSc2c(NC(=O)c3ccccc3)cccc2N(Cc2cccc3ccccc23)C1=O
InChIKey: QRUYAIQIKGZQJC-FNZWTVRRSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6x 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-(8-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, 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 = 1424 nM (pChEMBL 5.849999904632568)
Descriptors: MW 538.67, ALogP 4.82, HBA 5, HBD 3, PSA 90.54, QED 0.31
Compound (SMILES): O=C(CC1CCOC1)NC1CC=C(c2ccc(NC(=O)N3Cc4ccncc4C3)cc2)CC1
InChIKey: UTMYGBQASGHHPI-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 = 19.5 nM (pChEMBL 7.710000038146973)
Descriptors: MW 446.55, ALogP 4.11, HBA 4, HBD 2, PSA 83.56, QED 0.72
Compound (SMILES): O=C(Nc1ccc(CCC2CCCCN2)cc1)c1ccc(Cl)cn1
InChIKey: QGLMYIVKQBMWLF-UHFFFAOYSA-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 = 197.8 nM (pChEMBL 6.699999809265137)
Descriptors: MW 343.86, ALogP 4.06, HBA 3, HBD 2, PSA 54.02, QED 0.86
Compound (SMILES): O=C1NCC2(CCCC2)n2c1cc1cnc(Nc3ccc(N4CCC(N5CCOCC5)CC4)cn3)nc12
InChIKey: RHZYTLOZMHUSKU-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: Caliper Assay: Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK6/CycD3 CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Phosphoacceptor substrate peptide concentration used was 1 μM for all assays and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below:CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM; Incubation time: 3 hr.CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM; Incubation time: 1 hr.CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 10 hr.CDK6/CyclinD3: Enzyme concentration: 1 nM; ATP concentration: 300 μM; Incubation time: 3 hr.
Measurement: IC50 = 1350 nM (pChEMBL 5.869999885559082)
Descriptors: MW 502.62, ALogP 2.88, HBA 9, HBD 2, PSA 100.44, QED 0.56
Compound (SMILES): CCN(C(=O)Oc1ccc(F)cc1)[C@@H]1CN(C(=O)C2CCN(c3ccc(C(C)O)cn3)CC2)C[C@H]1c1ccc(Cl)cc1
InChIKey: TXIKULUYOUEHLY-BQTLCQCVSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: [3H]SR142801 Competition Binding Assay: hNK3 receptor binding experiment were performed using [3H]SR142801 (Catalog No. TRK1035, specific activity: 74.0 Ci/mmol, Amersham, GE Healthcare UK limited, Buckinghamshire, UK) and membrane isolated from HEK293 cells transiently expressing recombinant human NK3 receptor. After thawing, the membrane homogenates were centrifuged at 48,000×g for 10 mM at 4° C., the pellets were resuspended in the 50 mM Tris-HCl, 4 mM MnCl2, 1 μM phosphoramidon, 0.1% BSA binding buffer at pH 7.4 to a final assay concentration of 5 μg protein/well. For inhibition experiments, membranes were incubated with [3H]SR142801 at a concentration equal to KD value of radioligand and 10 concentrations of the inhibitory compound (0.0003-10 μM) (in a total reaction volume of 500 μl) for 75 min at room temperature (RT). At the end of the incubation, membranes were filtered onto unitfilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.3% PEI+0.3% BSA, Packard BioScience, Meriden, Conn.) with a Filtermate 196 harvester (Packard BioScience) and washed 4 times with ice-cold 50 mM Tris-HCl, pH 7.4 buffer. Nonspecific binding was measured in the presence of 10 μM SB222200 for both radioligands. The radioactivity on the filter was counted (5 min) on a Packard Top-count microplate scintillation counter with quenching correction after addition of 45 μl of microscint 40 (Canberra Packard S.A., Zürich, Switzerland) and shaking for 1 h.
Measurement: Ki = 4.7 nM (pChEMBL 8.329999923706055)
Descriptors: MW 595.12, ALogP 5.66, HBA 6, HBD 1, PSA 86.21, QED 0.38
Compound (SMILES): CN(C)C(CNc1ccc(C(N)=O)c2ncncc12)c1cccc(Cl)c1
InChIKey: BTLXOEZIMXLEQI-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 ul of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.50 0.1 ng/uL His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 uM substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338) 0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25° C, and then stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 369.86, ALogP 3.1, HBA 5, HBD 2, PSA 84.14, QED 0.7
Compound (SMILES): Cc1ccsc1CNc1nc(NC2CC2)n2ncc(/C=C3\NC(=O)NC3=O)c2n1
InChIKey: QNFQUBVOEPIUJN-SDQBBNPISA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 410.46, ALogP 1.86, HBA 9, HBD 4, PSA 125.34, QED 0.36