lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCOc1cccc(F)c1C1CC(C)C(=O)N1Cc1ccc(OC(F)(F)F)c(F)c1
InChIKey: YOPYIDHDUWIJLO-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 = 1494 nM (pChEMBL 5.829999923706055)
Descriptors: MW 429.39, ALogP 5.37, HBA 3, HBD 0, PSA 38.77, QED 0.58
Compound (SMILES): CCCN(CCC)C(=O)C1=Cc2ccc(C(=O)Nc3cccc(CCCCN)c3)cc2N=C(N)C1
InChIKey: LOFKWVOYEQTEKD-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Pharmacological Assay: For TLR8 and TLR7 activity testing, HEK-Blue human TLR8 or TLR7 cells (Invivogen, San Diego, Calif., USA) are used, respectively. These cells are designed for studying the stimulation of human TLR8 or TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-κB and AP-1-binding sites. Therefore the reporter expression is regulated by the NF-κB promoter upon stimulation of human TLR8 or TLR7 for 20 hours. The cell culture supernatant SEAP reporter activity was determined using Quanti Blue kit (Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple/blue in the presence of alkaline phosphatase.
Measurement: EC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 475.64, ALogP 4.64, HBA 5, HBD 3, PSA 113.81, QED 0.41
Compound (SMILES): CNC1=N[C@@H]2[C@@H](F)[C@H](O)[C@@H]([C@H](C)O)O[C@@H]2S1
InChIKey: DYOCXHLLDLBZHU-JMHNFOGYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Fluorescence-Based Assay: Enzymatic reactions were carried out in a reaction containing 50 mM NaH2PO4, 100 mM NaCl and 0.1% BSA (pH 7.0) using 2 mM 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide dihydrate (Sigma M2133) dissolved in ddH2O, as a substrate. The amount of purified human O-GlcNAcase enzyme used in the reaction was 0.7 nM. Test compound of varying concentrations was added to the enzyme prior to initiation of the reaction. The reaction was performed at room temperature in a 96-well plate and was initiated with the addition of substrate. The production of fluorescent product was measured every 60 sec for 45 min with a Tecan Infinite M200 plate-reader with excitation at 355 nM and emission detected at 460 nM, with 4-Methylumbelliferone (Sigma M1381) used to produce a standard curve.
Measurement: Ki = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 250.29, ALogP -0.52, HBA 6, HBD 3, PSA 74.08, QED 0.58
Compound (SMILES): CCC[C@@H]1NCCOc2ccccc2CCCNC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](CC(C)C)NC1=O
InChIKey: UPLQIOOHMDDLRG-GMQQYTKMSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki = 1000 nM (pChEMBL 6)
Descriptors: MW 536.72, ALogP 3.14, HBA 5, HBD 4, PSA 108.56, QED 0.45
Compound (SMILES): Cc1cccc2cc(CN3CCN(C(=O)Nc4ccccc4)CC3)c(Cl)nc12
InChIKey: RKUKDXDODIRJSH-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: Biological Assay: Assay Method 1: T84 frozen pellets (contents of 1-4x15 cm culture dishes) were homogenized in 300 mL of FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9). The assay mixture was prepared from 50 uL of the cell homogenate, 10 uL of the test compound, and 40 uL of anandamide [1-3H-ethanolamine] (3H-AEA; Perkin-Elmer, 10.3 Ci/mmol), which was added last, for a final tracer concentration of 200 nM. The reaction mixture was incubated at rt for 1 hour (h). During the incubation, 96-well Multiscreen filter plates (catalog number MAFCNOB50; Millipore, Bedford, Mass., USA) were loaded with 25 uL of activated charcoal (Multiscreen column loader, catalog number MACL09625, Millipore) and washed once with 100 uL of MeOH. Also during the incubation, 96-well DYNEX MicroLite plates (catalog number NL510410) were loaded with 100 uL of MicroScint40 (catalog number 6013641, Packard Bioscience, Meriden, Conn., USA).
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 394.91, ALogP 4.55, HBA 3, HBD 1, PSA 48.47, QED 0.66
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3cccnc3)n2)CCN(C(=O)OCC(F)(F)F)CC1
InChIKey: QHWQAHGNFCIKJD-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 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 451.41, ALogP 2.52, HBA 8, HBD 2, PSA 139.16, QED 0.69
Compound (SMILES): CC[C@H]1COCCN1c1nc2c(c(-c3ccc(NC(=O)Nc4ccc(C)nc4)c(F)c3)n1)C(=O)N(CC)CC(C)(C)O2
InChIKey: JHCHFWUWAGEFIR-NRFANRHFSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The test detects the phosphorylation of the substrate GFP-4EBP1 using a terbium-labelled antibody that is specific for the phospho-Thr46 epitope. Binding of the Tb-antibody to the phosphorylated GFP peptide allows the transfer of energy from Tb, excited at 340 nm, to GFP with an increase in the fluorescence emission of the GFP at 520 nm. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 ul. To 1 ul of inhibitor in 100% DMSO are added (final concentrations) 400 nM of GFP-4EBP1 (Invitrogen PV4759), 8 uM of ATP, 200 ng/ml of enzyme (human recombinant aa mTOR 1360-2549, Invitrogen PV4753) in a buffer of HEPES 50 mM pH 7.5, EGTA 1 mM, MnCl2 10 mM, BSA 0.1 mg/ml, glycerol 0.5%, NV-10 0.1 mg/ml, DTT 2 mM. The mixture is incubated for 30 minutes at room temperature. 10 ul of solution are taken up and the reaction is quenched by adding 10 ul of Tb-antibody solution (Invitrogen PV4757)/EDTA.
Measurement: IC50 = 122 nM (pChEMBL 6.909999847412109)
Descriptors: MW 577.66, ALogP 4.88, HBA 8, HBD 2, PSA 121.81, QED 0.43
Compound (SMILES): Fc1ccc(Nc2n[nH]c3cccnc23)cc1C(F)(F)F
InChIKey: CXCIHOWTULOSBG-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Receptor Activity Assay: The disclosed compounds and compositions can be evaluated for their ability to act as a potentiator of metabotropic glutamate receptor activity, in particular mGluR4 activity, by any suitable known methodology known in the art. For example, Chinese Hamster Ovary (CHO) cells transfected with human mGluR4 or HEK cells co-transfected with rat mGluR4 and the G-protein regulated Inwardly Rectifying Potassium channel (GIRK) were plated in clear bottom assay plates for assay in a Hamamatsu FDSS Fluorometric Plate Reader. The cells were loaded with either a Ca2+-sensitive fluorescent dye or the thallium responsive dye and the plates were washed and placed into a suitable kinetic plate reader. For human mGluR4 assays, a fluorescence baseline was established for 3-5 seconds, the disclosed compounds were then added to the cells, and the response in cells was measured.
Measurement: EC50 = 385 nM (pChEMBL 6.420000076293945)
Descriptors: MW 296.23, ALogP 3.86, HBA 3, HBD 2, PSA 53.6, QED 0.71
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1n[nH]c(Cc2ccccc2)n1
InChIKey: CJTYZSXAGJVLIH-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 5800 nM (pChEMBL 5.239999771118164)
Descriptors: MW 336.4, ALogP 1.92, HBA 5, HBD 3, PSA 109.58, QED 0.66
Compound (SMILES): O=C(CCc1ccc(O)cc1)N/N=C/c1ccc(O)c(O)c1O
InChIKey: SHKRFRMXWHNKAJ-RQZCQDPDSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Coxsackievirus B3 (strain Nancy)
UniProt: P03313
Assay: Inhibition of recombinant Coxsackievirus B3 3C protease expressed in Escherichia coli BL21 (DE3) preincubated for 5 mins followed by addition of NMA-EALFQGPPVK-DNP-rrr-NH2 as substrate measured up to 2 hrs by FRET-based enzyme assay
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 316.31, ALogP 1.59, HBA 6, HBD 5, PSA 122.38, QED 0.33
Compound (SMILES): CCOc1cc(C2=NN(C3CCN(C(=O)CN4C(=O)CCC4=O)CC3)C(=O)C2(C)C)ccc1OC
InChIKey: PDYAPLCZXYJGQA-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 = 22.4 nM (pChEMBL 7.650000095367432)
Descriptors: MW 484.55, ALogP 1.81, HBA 7, HBD 0, PSA 108.82, QED 0.55
Compound (SMILES): O=C(O)Cn1nnc(-c2cnc(N3CCC4(CC3)NC(=O)c3ccccc3O4)s2)n1
InChIKey: ZWSJMIXLKNUYHN-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase (SINGLE PROTEIN), Homo sapiens
UniProt: O00767
Assay: Enzyme Activity Assay: The potency of compounds of formula I against the stearoyl-CoA desaturase was determined by measuring the conversion of radiolabeled stearoyl-CoA to oleoyl-CoA using rat liver microsome or human SCD1 following previously published procedures with some modifications (Joshi, et al., J. Lipid Res., 18: 32-36 (1977); Talamo, et al., Anal. Biochem, 29: 300-304 (1969)). Liver microsome was prepared from male Wistar or Sprague Dawley rats on a high carbohydrate diet for 3 days (LabDiet #5803, Purina). The livers were homogenized (1:10 w/v) in a buffer containing 250 mM sucrose, 1 mM EDTA, 5 mM DTT and 50 mM Tris-HCl (pH 7.5). After a 100,000xg centrifugation for 60 min, the liver microsome pellet was suspended in a buffer containing 100 mM sodium phosphate, 20% glycerol, 2 mM DTT, and stored at -78° C. Human SCD1 desaturase system was reconstituted using human SCD1 from a baculovirus/Sf9 expression system, cytochrome B5 and cytochrome B5 reductase.
Measurement: IC50 = 181 nM (pChEMBL 6.739999771118164)
Descriptors: MW 427.45, ALogP 1, HBA 10, HBD 2, PSA 135.36, QED 0.62
Compound (SMILES): CCOC(=O)c1[nH]c2cc(Cl)ccc2c1C(C(=O)NC(C)(C)C)N(C=O)Cc1cc(F)c(F)c(F)c1F
InChIKey: QJZQXXANQWJLGH-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Fluorescence Polarization (FP) Assay: Fluorescent polarization experiments were performed as follows. Briefly, the fluorescence polarization experiments were read on an Ultra Evolution 384-well plate reader (Tecan) with the 485 nm excitation and 535 nm emission filters. The fluorescence intensities parallel (Intparallel) and perpendicular (Intperpedicular) to the plane of excitation were measured in parallel perpendicular black 384-well NBS assay plates (Corning) at room temperature (20° C). The background fluorescence intensities of blank samples containing the references buffer were subtracted and steady-state fluorescence polarization was calculated using the equation: P=(Intparallel Gintperpendicular)/(Intparallel+Gintperpendicular), and the correction factor G (G=0.998 determined empirically) was introduced to eliminate differences in the transmission of vertically and horizontally polarized light. All fluorescence polarization values were expressed in millipolarization units (mP).
Measurement: Ki = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 541.93, ALogP 5.17, HBA 4, HBD 2, PSA 91.5, QED 0.14
Compound (SMILES): COc1cc2c(Cc3ccc4cccc(N)c4n3)cnc(C)c2cc1OC
InChIKey: HRHRVUZKLUVCKM-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Diaxonhit Phosphodiesterase Assay: The phosphodiesterase assay was developed using the LANCE cAMP kit (PerkinElmer). The assay buffer contained HBSS with 5 mM HEPES, 0.1% BSA, and 1.5 mM MgCl2, pH 7.4. PDE10A (BPS Bioscience) was used at 200 pg/well (with a specific activity of 3200 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min) and PDE4D3 (BPS Bioscience) was used at 100 pg/well (with a specific activity of 32713 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min). The Biotin-cAMP tracer, supplied in 10 mmol/L Tris-HCl buffered (pH 8.0) salt solution with 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1% bovine serum albumin (BSA), and 0.05% sodium azide, is used at a dilution of 1/375. The assay detection mixture contained the LANCE Eu-W8044 labeled streptavidin 1/2250 (supplied in 50 mmol/L Tris-HCl buffer).
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 359.43, ALogP 4.28, HBA 5, HBD 1, PSA 70.26, QED 0.55
Compound (SMILES): CC(c1nc2ccccc2c(=O)n1-c1ccc(F)cc1)N(C)C(=O)Nc1cccc(C#N)c1
InChIKey: BWDNHBQECVGKLU-UHFFFAOYSA-N
Target: Sonic hedgehog protein (SINGLE PROTEIN), Mus musculus
UniProt: Q62226
Assay: Gli-Luc Assay: Compounds may be tested in the "Gli-Luc" assay below, using the cell line 10T(s12), wherein the cells contain a Hedgehog-responsive reporter construct utilizing Luciferase as the reporter gene. In this way, Hedgehog pathway signaling activity can be measured via the Gli-Luc response.10t1/2(s12) cells are plated in a 96-well micro-titer plate (MTP) at 20,000 cells/well in full medium [DMEM with 10% FBS]. Then plates are placed in the incubator for incubation overnight (O/N), at 37° C. and 5% CO2. After 24 h, the medium is replaced with Luciferase-assay medium (DMEM with 0.5% FBS). Compounds are thawed and diluted in assay medium at 3:1000 (about 300-fold) resulting in a starting concentration of about 30 uM.Subsequently, 150 ul of each 30 uM sample is added to the first wells (in triplicate). The MTP samples are then diluted at 3-fold dilutions to a total of seven wells, ultimately resulting in a regiment of seven dilutions in triplicate, for each compound.
Measurement: IC50 < 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 441.47, ALogP 4.62, HBA 5, HBD 1, PSA 91.02, QED 0.5
Compound (SMILES): CN1C(=O)C(c2ncc(C#Cc3cccc(F)c3)cn2)CC1(C)C
InChIKey: BIHSKGYQWXRSGG-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Calcium Mobilization Assay: A monoclonal HEK-293 cell line stably transfected with a cDNA encoding for the human mGlu5a receptor was generated; for the work with mGlu5 Positive Allosteric Modulators (PAMs), a cell line with low receptor expression levels and low constitutive receptor activity was selected to allow the differentiation of agonistic versus PAM activity. Cells were cultured according to standard protocols (Freshney, 2000) in Dulbecco's Modified Eagle Medium with high glucose supplemented with 1 mM glutamine, 10% (vol/vol) heat-inactivated bovine calf serum, Penicillin/Streptomycin, 50 mg/ml hygromycin and 15 ug/ml blasticidin (all cell culture reagents and antibiotics from Invitrogen, Basel, Switzerland).About 24 hrs before an experiment, 5x104 cells/well were seeded in poly-D-lysine coated, black/clear-bottomed 96-well plates. The cells were loaded with 2.5 uM Fluo-4AM in loading buffer (1xHBSS, 20 mM HEPES) for 1 hr at 37° C. and washed five times with loading buffer.
Measurement: EC50 = 137 nM (pChEMBL 6.860000133514404)
Descriptors: MW 323.37, ALogP 2.74, HBA 3, HBD 0, PSA 46.09, QED 0.76
Compound (SMILES): O=C(c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1)N1CCNC2(CCC2)C1
InChIKey: DKCVGHXFHUUPKI-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 539 nM (pChEMBL 6.269999980926514)
Descriptors: MW 442.44, ALogP 4.41, HBA 5, HBD 1, PSA 67.35, QED 0.65
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CSc2c(NC(=O)CCCC(C)=O)cccc2N(Cc2cccc3ccccc23)C1=O
InChIKey: HVPMNTAMRQCXLQ-CPJSRVTESA-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 = 373 nM (pChEMBL 6.429999828338623)
Descriptors: MW 546.69, ALogP 4.27, HBA 6, HBD 3, PSA 107.61, QED 0.35
Compound (SMILES): Cc1ccc(C(C)C)c(OC(=O)Nc2cccc3ccccc23)c1
InChIKey: JXDUPHZQYZPGDN-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Equus caballus
UniProt: P81908
Assay: Inhibition of horse serum BuChE using butyrylthiocholine iodide as substrate preincubated for 15 mins followed by substrate addition by Ellman's method
Measurement: IC50 = 96100 nM (pChEMBL 4.019999980926514)
Descriptors: MW 319.4, ALogP 5.88, HBA 2, HBD 1, PSA 38.33, QED 0.65
Compound (SMILES): CC(C)CC(=O)N1CCC(Oc2ccc(NC(=O)N3Cc4ccccc4C3)cn2)CC1
InChIKey: UOHTTZGCBWTDAM-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 = 222 nM (pChEMBL 6.650000095367432)
Descriptors: MW 422.53, ALogP 4.05, HBA 4, HBD 1, PSA 74.77, QED 0.79
Compound (SMILES): c1ccc2nc(NC3=NC[C@@]4(CN5CCC4CC5)O3)ccc2c1
InChIKey: KLCVWZMJNQGSJE-SFHVURJKSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05941
Assay: Agonist activity at rat alpha7 nAChR expressed in HEK293 cells co-expressing human RIC3 measured for 2 mins by FLIPR assay
Measurement: EC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 308.39, ALogP 2.5, HBA 5, HBD 1, PSA 49.75, QED 0.88
Compound (SMILES): COc1cc(-c2cnn(C)c2)ccc1Nc1ncc2ccnc(-c3cnn(C(C)C)c3)c2n1
InChIKey: VXHXSDDXZINUPL-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Enzyme Inhibition Assay: The enzyme reaction (total volume 10 μl) was carried out in black 384-well low volume plates containing full length MPS1 (12.5 nM or 3 nM), fluorescent labelled peptide [known as H236, which has the sequence: 5FAM-DHTGFLTEYVATR-CONH2] (5 μM), ATP(10 μM), either DMSO (1% v/v) or the test compound (in the range 0.25 nM-100 μM in 1% DMSO) and assay buffer (50 mM HEPES (pH 7.0), 0.02% NaN3, 0.01% BSA, 0.1 mM Orthovandate, 10 μM MgCl2, 1 μM DTT, Roche protease inhibitor). The reaction was carried out for 60 min at room temperature and stopped by the addition of buffer (10 μl) containing 20 mM EDTA, 0.05% (v/v) Brij-35, in 0.1M HEPES-buffered saline (Free acid, Sigma, UK). The plate was read on a Caliper EZ reader II (Caliper Life Sciences).
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 440.51, ALogP 4.62, HBA 9, HBD 1, PSA 95.57, QED 0.41
Compound (SMILES): CCN(c1cc(OC2CN(C)C2)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1C)C1CCC(N(C)C)CC1
InChIKey: BKZXDKSKVFDSEJ-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 8.7 nM (pChEMBL 8.0600004196167)
Descriptors: MW 523.72, ALogP 3.62, HBA 6, HBD 2, PSA 80.91, QED 0.52
Compound (SMILES): O=C1c2ccc(C3CC3)cc2OCCN1c1cccc(-c2ccnc3[nH]c(-c4ccc(CN5CCN(CC(F)(F)F)CC5)cn4)cc23)c1CO
InChIKey: PAQFRRFVYRPQKX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Recombinant Human BTK Inhibition Assay: The inhibition assays were performed using the generic tyrosine kinase assay kit from C is Bio (HTRF KinEASE-TK #62TK0PEB). The assay is based on the principle of fluorescence resonance energy transfer (FRET) between the Europium Cryptate labeled anti-phosphotyrosine antibody and streptavidin labeled fluorophore, XL-665. In an uninhibited kinase reaction the biotinylated peptide substrate (Tk-Biotin) gets phosphorylated at an intermediate tyrosine site which is captured by anti-phosphotyrosine antibody which eventually acts as a FRET donor. The streptavidin labeled XL-665 binds strongly to the Tk-Biotin substrate and act as a FRET acceptor. The resultant FRET signal is measured at 665 nm. A ratio of this specific fluorescence (665 nm) to the background fluorescence (620 nm), conferred by the free antibody, is directly proportional to the kinase activity. Inhibition of the kinase activity by a pharmacological intervention such as a Btk inhibitor of the present disclosure will result into decreased FRET signal.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 682.75, ALogP 6.38, HBA 7, HBD 2, PSA 97.82, QED 0.2
Compound (SMILES): CC(C)c1nc2c(c(Nc3ccc(C(C)(C)C)cc3)n1)CCN(c1ncc([N+](=O)[O-])cc1C(F)(F)F)CC2
InChIKey: BNNWREFTKIGWPD-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 410 nM (pChEMBL 6.389999866485596)
Descriptors: MW 528.58, ALogP 6.57, HBA 7, HBD 1, PSA 97.08, QED 0.3
Compound (SMILES): Cn1c(=O)c2c(CC(=O)Nc3nc(-c4cc(F)cc(C(F)(F)F)c4)cs3)nsc2n(C)c1=O
InChIKey: ODCGQSLLUGLMSU-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 = 4.4 nM (pChEMBL 8.359999656677246)
Descriptors: MW 499.47, ALogP 3.16, HBA 9, HBD 1, PSA 98.88, QED 0.44
Compound (SMILES): COc1cncc(C2=CC[C@H]3[C@@H]4CC[C@H]5COCCC[C@]5(C)[C@H]4CC[C@]23C)c1
InChIKey: TXAFJJUIERCQGJ-DCVSVKONSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human testicular microsomes using 17alpha-hydroxypregnenolone as substrate assessed as decrease in DHEA levels after 90 mins by ELISA
Measurement: IC50 <= 11 nM (pChEMBL 7.959000110626221)
Descriptors: MW 381.56, ALogP 5.75, HBA 3, HBD 0, PSA 31.35, QED 0.65
Compound (SMILES): Fc1ccc(-c2n[nH]c3ccc(-c4nc(-c5ccco5)n[nH]4)cc23)cc1
InChIKey: YVKRJXRMZSFCNE-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 345.34, ALogP 4.41, HBA 4, HBD 2, PSA 83.39, QED 0.51
Compound (SMILES): CC(C)Oc1ccnc(NC(=O)c2ccc(-c3nc(N4CCn5c(nnc5C(F)(F)F)C4)n4ccnc(N)c34)cc2)c1
InChIKey: PBOGXWOJLHDIPM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: LANCE-TR-FRET Assay: Assay Method B: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated "A5" peptide (Biotin-EQEDEPEGDYFEWLE-NH2), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 μL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity).
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 578.56, ALogP 4.04, HBA 11, HBD 2, PSA 141.38, QED 0.31
Compound (SMILES): O=C(c1ccccc1-n1nccn1)N1CCC(F)(F)C(Oc2ccc3ccccc3n2)C1
InChIKey: VCYRJXZNOOUBSE-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Displacement of (2S)-N-(2-pyrrol-1-ylphenyl)-1-[2-[1-(tritritiomethyl)benzimidazol-2-yl]sulfanylacetyl]pyrrolidine-2-carboxamide from human OX2 receptor expressed on CHO cell membrane measured after 3 hrs by TopCount method
Measurement: Ki = 1137 nM (pChEMBL 5.940000057220459)
Descriptors: MW 435.43, ALogP 3.74, HBA 6, HBD 0, PSA 73.14, QED 0.49
Compound (SMILES): O=C(Nc1cccc(CN2CCC(C(=O)NC3CCCCC3)CC2)c1)c1cnc2[nH]ccc2c1
InChIKey: IZRNXMYOMVUMRS-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 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 459.59, ALogP 4.48, HBA 4, HBD 3, PSA 90.12, QED 0.51
Compound (SMILES): O=c1[nH]c([C@H]2CC[C@@H]2c2ncccn2)nc2c1cnn2C1CCC(F)(F)CC1
InChIKey: LRRFQGYMGYXVHJ-STQMWFEESA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Scintillation Proximity Assay: The PDE9A2 enzymatic activity assay was run as scintillation proximity assay (SPA), in general according to the protocol of the manufacturer (GE Healthcare, former Amersham Biosciences, product number: TRKQ 7100). As enzyme source, lysate (PBS with 1% Triton X-100 supplemented with protease inhibitors, cell debris removed by centrifugation at 13.000 rpm for 30 min) of SF 9 cell expressing the human PDE9A2 was used. The total protein amount included in the assay varied upon infection and production efficacy of the SF9 cells and lay in the range of 0.1-100 ng. In general, the assay conditions were as follows: total assay volume: 40 microlitre; protein amount: 0.1-50 ng; substrate concentration (cGMP): 20 nanomolar; ~1 mCi/l; incubation time: 60 min at room temperature; final DMSO concentration: 0.2-1%. The assays were run in 384-well format. The test reagents as well as the enzyme and the substrate were diluted in assay buffer. The assay buffer contained 50 mM Tris, 8.3 mM MgCl2, 1.7 mM EGTA, 0.1% BSA, 0.05% Tween 20; the pH of assay buffer was adjusted to 7.5. The reaction was stopped by applying a PDE9 specific inhibitor (e.g. compounds according to WO 2004/099210 or WO 2004/099211, like one of the enantiomers of example 37, e.g. 1-(2-Chlorophenyl)-6-[2R)-3,3,3-trifluoro-2-methyl-propyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one) in excess.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 386.41, ALogP 3.32, HBA 6, HBD 1, PSA 89.35, QED 0.75
Compound (SMILES): COc1ccc2c(c1Cl)C(=O)N(Cc1c(C)cc(C)[nH]c1=O)CCC2
InChIKey: FPRBNBCSBZOASW-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition of wild-type EZH2 (unknown origin) expressed in baculovirus infected SF9 cells co-expressing SUZ12/EED/RbAp48 complex using HeLa cells derived oligonucleosomes as substrate after 60 mins in presence of [3H] SAM by scintillation counting
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 360.84, ALogP 3.24, HBA 3, HBD 1, PSA 62.4, QED 0.91
Compound (SMILES): C[C@H]1C[C@@H](c2ccncc2NC(=O)c2ccc(F)c(-c3c(F)cccc3F)n2)C[C@@H](N)[C@@H]1[S@@+](C)[O-]
InChIKey: SDHRADPEFDLIKD-DIXKNXRNSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: AlphaScreen Assay: Pim 1, Pim 2 & Pim 3 AlphaScreen assays using high ATP (11-125.times. ATP Km) were used to determine the biochemical activity of the inhibitors. The activity of Pim 1, Pim 2, & Pim 3 is measured using a homogeneous bead based system quantifying the amount of phosphorylated peptide substrate resulting from kinase-catalyzed phosphoryl transfer to a peptide substrate. Compounds to be tested are dissolved in 100% DMSO and directly distributed to a white 384-well plate at 0.25 .mu.l per well. To start the reaction, 5 .mu.l of 100 nM Bad peptide (Biotin-AGAGRSRHSSYPAGT-OH (SEQ ID NO:1)) and ATP (concentrations described below) in assay buffer (50 mM Hepes, pH=7.5, 5 mM MgCl.sub.2, 0.05% BSA, 0.01% Tween-20, 1 mM DTT) is added to each well. This is followed by the addition of 5 .mu.l/well of Pim 1, Pim 2 or Pim 3 kinase in assay buffer (concentrations described below). Final assay concentrations (described below) are in 2.5% DMSO. The reactions are performed for .about.2 hours.
Measurement: IC50 = 12.4 nM (pChEMBL 7.909999847412109)
Descriptors: MW 502.56, ALogP 4.4, HBA 5, HBD 2, PSA 103.96, QED 0.5
Compound (SMILES): C[C@@]1(c2cc(NC(=O)c3ccc(S(C)(=O)=O)cn3)ccc2F)CS(=O)(=O)[C@]2(CCOC2)C(=N)N1
InChIKey: HHMXNQSEIIIPBO-SFTDATJTSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3x the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 ul) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol.
Measurement: Ki = 3166 nM (pChEMBL 5.5)
Descriptors: MW 510.57, ALogP 1.25, HBA 8, HBD 3, PSA 155.38, QED 0.55
Compound (SMILES): Cc1nc(C)c(-c2csc(Nc3ccc(C(=O)N4[C@@H]5CC[C@H]4C/C(=N/OCC(F)(F)F)C5)cn3)n2)s1
InChIKey: HRPZHVMUTDQPOZ-LPMRMCHTSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Antagonist activity at human TRPV4 assessed as inhibition of 4alpha-PDD-induced activation
Measurement: IC50 = 0.36 nM (pChEMBL 9.4399995803833)
Descriptors: MW 536.61, ALogP 5.72, HBA 9, HBD 1, PSA 92.6, QED 0.41
Compound (SMILES): Cc1cc(C(F)(F)F)n(-c2cc(N3CC(c4nc5ccccc5n4C)C3)nc(C)n2)n1
InChIKey: UNHNEQXZHGKGSX-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition is determined using a known PDE10 inhibitor, which can be any compound that is present at 5,000 times its Ki value in the assay described as follows, such as papaverine (see Siuciak, et al. Neuropharmacology (2006) 51:386-396; Becker, et al. Behav Brain Res (2008) 186(2):155-60; Threlfell, et al., J Pharmacol Exp Ther (2009) 328(3):785-795), 2-{4-[pyridin-4-yl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl]phenoxymethyl}quinoline succinic acid or 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]quinoline succinic acid (see Schmidt, et al. J Pharmacol Exp Ther (2008) 325:681-690; Threlfell, et al., J Pharmacol Exp Ther (2009) 328(3): 785-795). 0% of inhibition is determined by using DMSO (1% final concentrations). A Labcyte Echo 555 (Labcyte, Sunnyvale, Calif.) is used to dispense 200 mL from each well of the titration plate to the 384 well assay plate. A solution of enzyme (1/7000 dilution from aliquots; sufficient to produce 20% substrate conversion) and a separate solution of FAM-labeled cAMP PDE from Molecular Devices (product # R7506), at a final concentration of 50 nM are made in the assay buffer (10 mM Tris HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3 0.01% Tween-20, and 1 mM DTT). The enzyme is added to the assay plates by the addition of 10 μL of enzyme solution to each well, shaken to mix and incubated at room temperature for 60 minutes. The substrate is then added to the assay plates by the addition of 10 μÎ¼L of substrate solution to each well, shaken to mix, and incubated at room temperature for 60 minutes. A binding solution is then made from the kit components, comprised of 80% Solution A, 20% Solution B and binding reagent at a volume of 1/600 the total binding solution. The enzymatic reaction is stopped by addition of 60 lit of the binding solution to each well of the assay plates and the plates are sealed and shaken for 10 seconds. The plate was incubated at room temperature for at least one hour prior to determining the fluorescence polarization (FP).
Measurement: Ki = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 427.43, ALogP 3.79, HBA 7, HBD 0, PSA 64.66, QED 0.5
Compound (SMILES): O=S(=O)(Nc1cncc(Br)c1)c1ccccc1C(F)(F)F
InChIKey: NFHYBIRNRJMVFU-UHFFFAOYSA-N
Target: Solute carrier family 22 member 12 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96S37
Assay: Inhibition of human C-terminal eGFP-tagged URAT1 long isoform expressed in CHO cells assessed as decrease in [14C]-uric acid uptake preincubated for 15 mins followed by [14C]-uric acid addition and incubated in dark for 3 hrs by scintillation counting method
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 381.17, ALogP 3.66, HBA 3, HBD 1, PSA 59.06, QED 0.88