lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(Nc1ccc([C@H]2CCCNC2)cc1)c1cccc(Cl)c1
InChIKey: ZLZDTXYJVXUIRV-HNNXBMFYSA-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 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 314.82, ALogP 4.06, HBA 2, HBD 2, PSA 41.13, QED 0.9
Compound (SMILES): CCCCCCCc1nc2ccccc2n1Cc1ccc(-c2ccccc2C(=O)O)cc1
InChIKey: RNPXGHFYTXGGJG-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P37231
Assay: Transactivation at Gal4 fused PPARgamma LBD (unknown origin) expressed in African green monkey COS7 cells after 42 hrs by luciferase assay
Measurement: EC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 426.56, ALogP 6.96, HBA 3, HBD 1, PSA 55.12, QED 0.28
Compound (SMILES): O=C1COc2ccc(C3(O)c4ccccc4C(=O)N3c3ccc(Cl)c(Cl)c3)cc2N1
InChIKey: HEBSEJYDCRBKHO-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 441.27, ALogP 4.18, HBA 4, HBD 2, PSA 78.87, QED 0.63
Compound (SMILES): Cc1c(N(Cc2ccc(F)c(C(F)(F)F)c2)S(=O)(=O)c2ccc(-n3cccn3)cc2)sc2ccccc12
InChIKey: BERQZFVESIMOKZ-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 545.58, ALogP 6.95, HBA 5, HBD 0, PSA 55.2, QED 0.22
Compound (SMILES): COc1ccccc1/C=C/S(=O)(=O)Nc1cccc(F)c1S(N)(=O)=O
InChIKey: SXOSYTOXUGKPMP-MDZDMXLPSA-N
Target: Prostaglandin E synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7Z7
Assay: Biochemical Assay: A solution of test compound was added to a diluted microsome preparation containing human mPGES-1 and pre-incubated for 15 minutes in potassium phosphate buffer pH 6.8 with cofactor glutathione (GSH). Corresponding solutions without test compound were used as positive controls, and corresponding solutions without test compound and without microsomes were used as negative controls. The enzymatic reaction was then started by addition of the substrate PGH2 in an organic solution (dry acetonitrile).The typical reaction conditions of the enzymatic reaction were thus: Test compound: ranging from 60 uM to 0.002 uM, or zero in positive and negative controls; potassium phosphate buffer pH 6.8: 50 mM; GSH: 2.5 mM; mPGES-1-containing microsomes: 2 ug/mL (sample and positive controls) or 0 ug/mL (negative control); PGH2: 10.8 uM; Acetonitrile: 7.7% (v/v); DMSO: 0.6% (v/v). The reaction was stopped after one minute by adding an acidic solution (pH 1.9) of ferric chloride.
Measurement: IC50 = 620 nM (pChEMBL 6.210000038146973)
Descriptors: MW 386.43, ALogP 1.89, HBA 5, HBD 2, PSA 115.56, QED 0.79
Compound (SMILES): O=C(Nc1ccc(C(F)(F)F)cn1)N1CCC(F)(c2ncc(-c3noc(CO)n3)cc2Cl)CC1
InChIKey: CQTXHSUWZCVQQT-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-Based Assay: Two days prior to performing this assay, cells are seeded on poly-D-lysine-coated 96-well clear-bottom black plates (commercially available from Becton-Dickinson) at 75,000 cells/well in growth media containing 5 uM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1x Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1.6 mM CaCl2 and 20 mM HEPES, pH 7.4 (wash buffer), and loaded using 0.1 mL of wash buffer containing Fluo-4 (3 uM final concentration, commercially available from Molecular Probes). After 1 h, the cells are washed twice with 0.2 mL wash buffer and resuspended in 0.05 mL 1x Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 3.5 mM CaCl2 and 10 mM Citrate, pH 7.4 (assay buffer). Plates are then transferred to a FLIPR for assay.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 500.84, ALogP 4.18, HBA 7, HBD 2, PSA 117.27, QED 0.52
Compound (SMILES): COc1cc2c(N)nc(N3CCN(C(=O)C[C@@H]4CCCCN4)CC3)nc2c(F)c1OC
InChIKey: DXHNVNOXACPDTI-ZDUSSCGKSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: ELISA Assay: CVF-Bb complex prepared from purified cobra venom factor (1 μM), recombinant human complement factor B (expressed in drosophila cells and purified using standard methods) and human complement factor D (expressed in E. Coli, refolded and purified using standard methods). CVF-Bb complex at 3 nM concentration was incubated with test compound at various concentrations for 1 hour at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human complement C3 substrate purified from plasma was added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction was stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, was quantified by means of an enzyme-linked-immunosorbent assay.
Measurement: IC50 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 432.5, ALogP 1.55, HBA 8, HBD 2, PSA 105.84, QED 0.73
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2ccc(Cl)cc2)C(=O)N[C@@H](Cc2ccccc2)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: WQZFDLDYUJSANS-AJQTZOPKSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Inhibition Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stiirzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 613.16, ALogP 4.58, HBA 5, HBD 4, PSA 122.55, QED 0.17
Compound (SMILES): c1ccc2c(c1)CC(Nc1nc(-c3ccsc3)nc3ccccc13)C2
InChIKey: JCDASFBLAYXAIQ-UHFFFAOYSA-N
Target: Lysosomal acid glucosylceramidase (SINGLE PROTEIN), Homo sapiens
UniProt: P04062
Assay: Inhibition of synthetic recombinant wild type GCase enzyme velaglucerase alfa (unknown origin) at pH 5.9 preincubated for 5 mins followed by addition of 4-Methylumbelliferyl beta-D-glucopyranoside as substrate measured after 30 mins by fluorescence analysis
Measurement: IC50 = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 343.46, ALogP 4.94, HBA 4, HBD 1, PSA 37.81, QED 0.57
Compound (SMILES): COc1cc(OC)cc(N(CCCN2CCC(C3C[C@@H](C)O[C@H](C)C3)C2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: IVHZIQUGHVXHHQ-ZTMFYPDUSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 47.86 nM (pChEMBL 7.320000171661377)
Descriptors: MW 584.77, ALogP 6.1, HBA 9, HBD 0, PSA 77.77, QED 0.22
Compound (SMILES): CC(C)=CCC[C@@]1(C)[C@H](CC=C(C)C)C[C@]2(CC=C(C)C)C(=O)[C@@]1(CC=C(C)C)C(=O)C(C(=O)c1cccc(O)c1)=C2O
InChIKey: BRKJGVUPNGKMRP-IIAGFODCSA-N
Target: DNA polymerase lambda (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGP5
Assay: Inhibition of recombinant human His-tagged DNA polymerase lambda assessed as reduction in [3H]dTTP incorporation using poly(dA)/oligo(dT)18 as template/primer preincubated for 10 mins followed by template/primer addition measured after 60 mins
Measurement: IC50 = 5700 nM (pChEMBL 5.239999771118164)
Descriptors: MW 586.81, ALogP 9.35, HBA 5, HBD 2, PSA 91.67, QED 0.12
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: SQIQCKDKPZSIRD-NKUBXJPTSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 2401.76
Compound (SMILES): CNC(=O)c1c(-c2ccc(C)nc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3C(CCN3CC(F)C3)C4)cc12
InChIKey: LTJXYYUXZWKIDA-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 13.02 nM (pChEMBL 7.880000114440918)
Descriptors: MW 710.81, ALogP 6.52, HBA 8, HBD 1, PSA 113.57, QED 0.19
Compound (SMILES): Cc1nn(C)c(C)c1N(C)S(=O)(=O)c1ccc(CCCCCCCCN(C)C)cc1
InChIKey: YLYZSKPIJSILNO-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
UniProt: Q388H8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 162 nM (pChEMBL 6.789999961853027)
Descriptors: MW 434.65, ALogP 4.31, HBA 5, HBD 0, PSA 58.44, QED 0.47
Compound (SMILES): Cn1c(SCCCN2CC[C@]3(C[C@H]3c3ccc(C(F)(F)F)cc3)C2)nnc1-c1cnccn1
InChIKey: GPDZHKOGLHALQL-AVRDEDQJSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]-spiperone from human dopamine D3 receptor expressed in CHO-K1 cell membranes after 90 mins by liquid scintillation counting
Measurement: Ki = 10.72 nM (pChEMBL 7.96999979019165)
Descriptors: MW 474.56, ALogP 4.65, HBA 7, HBD 0, PSA 59.73, QED 0.37
Compound (SMILES): COc1nc(Oc2cc(F)ccc2F)c(Cl)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: PRYPWRYJDWGMQX-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 846 nM (pChEMBL 6.070000171661377)
Descriptors: MW 560.92, ALogP 4.91, HBA 9, HBD 1, PSA 125.55, QED 0.3
Compound (SMILES): OCc1ccc2c(c1)CCc1c-2noc1-c1cccc(C(F)(F)F)c1
InChIKey: DRISCVGHZVOGDW-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Binding Assay: Membranes were prepared from CHO cells expressing human S1P1. Cells pellets (1^108cells/pellet) were suspended in buffer containing 20 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM EDTA and Protease Inhibitor cocktail (Roche), and disrupted on ice using the Polytron homogenizer. The homogenate was centrifuged at 20,000 rpm (48,000 g) and the supernatant was discarded. The membrane pellets were resuspended in buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, 2 mM EDTA and stored in aliquots at -80° C. after protein concentration determination.Membranes (2 ug/well) and 0.03 nM final concentration of 33P-S1P ligand (1 mCi/ml, American Radiolabeled Chemicals) diluted in assay buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid free BSA, 1 mM NaF) were added to the compound plates (384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Binding was performed for 45 minutes at room temperature, terminated by collecting the membranes.
Measurement: IC50 = 3200 nM (pChEMBL 5.5)
Descriptors: MW 345.32, ALogP 4.62, HBA 3, HBD 1, PSA 46.26, QED 0.74
Compound (SMILES): CCC1CCC(c2cc(C(C)C)ccc2OC)=C(CN(Cc2cc(C(F)(F)F)cc(C(F)(F)F)c2)c2ncc(-c3cnn(C)c3)cn2)C1
InChIKey: DAMPJTQFWCMSBL-UHFFFAOYSA-N
Target: Cholesteryl ester transfer protein (SINGLE PROTEIN), Homo sapiens
UniProt: P11597
Assay: Inhibition of recombinant CETP (unknown origin) assessed as inhibition of BODIPY-labeled cholesteryl transfer to fluorescent substrate by fluorimetric assay
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 671.73, ALogP 9.72, HBA 6, HBD 0, PSA 56.07, QED 0.16
Compound (SMILES): N#Cc1cc(F)cc(C(=O)N2CCn3nc(COc4cccc(F)c4)cc3C2)c1
InChIKey: DRTKCRBMELMEQA-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Cell-Based Functional Assay: HEK cells transfected with the human mGluR5a receptor (H10H cell line) were plated at 15,000 cells/well in clear-bottomed poly-D-lysine-coated assay plates (BD Falcon) in glutamate-glutamine-free growth medium and incubated overnight at 37° C. and 5% CO2. The following day, the growth medium was removed and the cells were washed with assay buffer containing 1× Hank's balanced salt solution (Invitrogen, Carlsbad, Calif.), 20 mM HEPES, 2.5 mM probenecid, pH 7.4 and left with 20 μL of this reagent. Following this step, the cells were loaded with calcium indicator dye, fluo-4 AM, to a final concentration of 2 μM and incubated for 40-45 min at 37° C. The dye solution was removed and replaced with assay buffer.
Measurement: EC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 394.38, ALogP 3.27, HBA 5, HBD 0, PSA 71.15, QED 0.68
Compound (SMILES): CN1CN([C@H]2CC[C@H](CNCC#N)CC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: CBQUWKTZIMDXPX-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 352.44, ALogP 2.08, HBA 5, HBD 2, PSA 88.05, QED 0.65
Compound (SMILES): CC(=O)Nc1ccc(S(=O)(=O)N(Cc2ccc(F)cc2)CC2CCC(C(=O)O)CC2)cc1
InChIKey: SWKSLDOHMMTRJW-UHFFFAOYSA-N
Target: Taste receptor type 2 member 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYV8
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 842 nM (pChEMBL 6.079999923706055)
Descriptors: MW 462.54, ALogP 3.87, HBA 4, HBD 2, PSA 103.78, QED 0.62
Compound (SMILES): CC1(C)CC(=O)N(Cc2ccc3c(-c4ccc(F)cc4)cc(C#N)nc3c2)C(=O)C1
InChIKey: WSATUJJSARHJKO-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 475 nM (pChEMBL 6.320000171661377)
Descriptors: MW 401.44, ALogP 4.59, HBA 4, HBD 0, PSA 74.06, QED 0.6
Compound (SMILES): CC(C)(C)OC(=O)N1CCc2cc(CCc3c(C(=O)O)[nH]c(=O)[nH]c3=O)ccc21.NCCO
InChIKey: GYKVVZUAFSNEJO-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 = 389 nM (pChEMBL 6.409999847412109)
Descriptors: MW 401.42, ALogP 1.84, HBA 5, HBD 3, PSA 132.56, QED 0.72
Compound (SMILES): COc1ccc(CC(=O)O)cc1-c1ccc(F)c2c1CN(C(=O)CCCc1ccccc1F)CC2
InChIKey: LKNVTCITUPRECW-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 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 479.52, ALogP 5.18, HBA 3, HBD 1, PSA 66.84, QED 0.48
Compound (SMILES): O=c1[nH]c2ccccc2n1-c1nc2ccccc2[nH]1
InChIKey: ZSWQYSJNHWGDPD-UHFFFAOYSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Inhibition of His-tagged human Aurora A kinase (122 to 40 residues) expressed in Escherichia coli BL21 (DE3) Rosetta cells using biotinylated STK2 substrate incubated for 30 mins by HTRF assay
Measurement: IC50 = 6500 nM (pChEMBL 5.190000057220459)
Descriptors: MW 250.26, ALogP 2.2, HBA 3, HBD 2, PSA 66.47, QED 0.54
Compound (SMILES): CCC(Nc1ncnc2[nH]cnc12)c1cc2cccc(C)c2c(=O)n1-c1c(F)cccc1F
InChIKey: YTQXASSUPKUNMF-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 446.46, ALogP 4.81, HBA 6, HBD 2, PSA 88.49, QED 0.4
Compound (SMILES): Cn1c(=O)c2c(CC(=O)Nc3nc(-c4cccc(C(F)(F)F)c4F)cs3)coc2n(C)c1=O
InChIKey: MSQPVCFZSCZFMD-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 = 2.21 nM (pChEMBL 8.65999984741211)
Descriptors: MW 482.42, ALogP 3.29, HBA 8, HBD 1, PSA 99.13, QED 0.45
Compound (SMILES): Cc1c(Cl)cc(OCC(=O)N2CCCOC2)c2nc(CCc3nc(N4CCCC4)nn3C)nn12
InChIKey: NJDRFVHWGUDNLE-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Scintillation Proximity Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 μl contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. # A-7906), 50 nM cGMP (Sigma, cat. # G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. # TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat # SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 μl of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate solution (stop reagent). After 1 h under shaking, the plate was centrifuged one minute at 170 g to allow beads to settle. Afterwards, radioactive counts were measured on a Perkin Elmer Top-Count Scintillation plate reader.
Measurement: IC50 = 2.07 nM (pChEMBL 8.680000305175781)
Descriptors: MW 488.98, ALogP 1.79, HBA 10, HBD 0, PSA 102.91, QED 0.5
Compound (SMILES): COc1cc(OC)cc(N(CC2CC2)c2ccc3ncc(-c4cnn(C(C)(C)C(=O)NCCO)c4)nc3c2)c1
InChIKey: FIFDRXCJFUTRRL-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 776.25 nM (pChEMBL 6.110000133514404)
Descriptors: MW 530.63, ALogP 3.9, HBA 9, HBD 2, PSA 114.63, QED 0.3
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1cccc(N)n1)c1nc2cccc(Cl)c2c(=O)n1-c1cc(F)cc(F)c1
InChIKey: CMGCVPSEBJVSNB-AWEZNQCLSA-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 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 544.95, ALogP 4.24, HBA 9, HBD 3, PSA 137.63, QED 0.29
Compound (SMILES): O=C(c1ccc2[nH]cnc2c1)N1CCN(C(=O)c2cc(Cc3n[nH]c(=O)c4c3CCCC4)ccc2F)CC1
InChIKey: FCFNMHXERCAEBJ-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 514.56, ALogP 2.85, HBA 5, HBD 2, PSA 115.05, QED 0.43
Compound (SMILES): CO[C@@H](CCNC(=O)c1nc(C#N)c2cc(Oc3ccccc3)ccc2c1O)C(=O)O
InChIKey: KFQULEWLVTWOGD-SFHVURJKSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 421.41, ALogP 2.82, HBA 7, HBD 3, PSA 141.77, QED 0.5
Compound (SMILES): CCC(=O)N1CCOC(COc2cc3ncnc(Nc4ccc(Cl)c(Cl)c4)c3cc2OC)C1
InChIKey: MAJPXTGDUCKEGK-UHFFFAOYSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 491.38, ALogP 4.71, HBA 7, HBD 1, PSA 85.81, QED 0.51
Compound (SMILES): Cc1ccc(C(C)C)cc1OC(=O)Nc1cccc([N+](=O)[O-])c1
InChIKey: IIRSYCMFEMYJHR-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Inhibition of electric eel AChE using acetylthiocholine iodide as substrate preincubated for 15 mins followed by substrate addition by Ellman's method
Measurement: IC50 = 5050 nM (pChEMBL 5.300000190734863)
Descriptors: MW 314.34, ALogP 4.64, HBA 4, HBD 1, PSA 81.47, QED 0.66
Compound (SMILES): COc1cc(Nc2ncc3c(n2)CCN(C(=O)c2cc(C)nn2C)C3)cc(OC)c1OC
InChIKey: HHSBCGXINLUMIN-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 ug/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 uL reaction volumes containing 2.7 uM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 ul of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 ul of 0-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 ul of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm. IC50 values for compound inhibition were calculated directly from graphs of optical density (arbitrary units) versus compound concentration following subtraction of blank values.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 438.49, ALogP 2.49, HBA 9, HBD 1, PSA 103.63, QED 0.63
Compound (SMILES): NC(=O)CC[C@@H]1N[C@H](CNC(=O)/C=C/c2ccc(Cl)cc2)CCN(CC(c2ccccc2)c2ccccc2)C1=O
InChIKey: XMJIJOQAKOBWKZ-PXPNINDPSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 559.11, ALogP 4.13, HBA 4, HBD 3, PSA 104.53, QED 0.31
Compound (SMILES): Cc1cnc(C)n2nc(CCc3nc(N4CCCC4C)nn3C)nc12
InChIKey: IDUJZUCJVUEIDD-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Scintillation Proximity Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 μl contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. # A-7906), 50 nM cGMP (Sigma, cat. # G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. # TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat # SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 μl of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate solution (stop reagent). After 1 h under shaking, the plate was centrifuged one minute at 170 g to allow beads to settle. Afterwards, radioactive counts were measured on a Perkin Elmer Top-Count Scintillation plate reader.
Measurement: IC50 = 18.44 nM (pChEMBL 7.730000019073486)
Descriptors: MW 340.44, ALogP 1.64, HBA 8, HBD 0, PSA 77.03, QED 0.72
Compound (SMILES): Cc1cc(OC2CC2)ccc1N1CC[C@@H](Oc2ccc([C@H](C)NC(=O)c3cnco3)cc2)C1
InChIKey: NLZDMJBZPTWNKA-FDDCHVKYSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 200 μM f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
Measurement: IC50 = 397 nM (pChEMBL 6.400000095367432)
Descriptors: MW 447.54, ALogP 4.67, HBA 6, HBD 1, PSA 76.83, QED 0.54
Compound (SMILES): CCn1ccc2cc(/N=c3\[nH]c(=O)n(CC(C)(C)C(=O)OC)c(=O)n3Cc3ccc(Cl)cc3)ccc21
InChIKey: YGKBHZHTBZHKRV-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683)) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37 C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 uM Fluo-3-AM solution (pH 7.5) containing 20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 uL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.).
Measurement: IC50 = 102 nM (pChEMBL 6.989999771118164)
Descriptors: MW 509.99, ALogP 3.45, HBA 8, HBD 1, PSA 103.38, QED 0.39
Compound (SMILES): Cc1ccc(-c2n[nH]c3cnc(-c4cnn(C)c4)cc23)nc1N1CC(N2CCNC(=O)C2)C1
InChIKey: QGRXFBMPBXBHHN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 0.618 nM (pChEMBL 9.210000038146973)
Descriptors: MW 443.52, ALogP 1.35, HBA 8, HBD 2, PSA 107.86, QED 0.49
Compound (SMILES): Cc1cc(Nc2cc(C(F)(F)F)ccn2)nc(-c2ccc(NC3CCN(C(=O)c4ccc5nc(C)sc5c4)CC3)nc2)c1
InChIKey: FBOWEBJAXWXXCB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSyk fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-Syk (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45x at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 603.67, ALogP 7.24, HBA 8, HBD 2, PSA 95.93, QED 0.21
Compound (SMILES): Cc1nc2cnc3cc(F)c(-c4ccc(Oc5ncccn5)cc4Cl)cc3c2n1C1CCN(C(=O)C(C)O)CC1
InChIKey: OCMLTNFXMKEHJW-UHFFFAOYSA-N
Target: Dual specificity mitogen-activated protein kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02750
Assay: Radioactive Binding Assay: The radioactive filter binding assay is standardized using recombinant human activated BRAF (V599E) kinase (Cat No. 14-557) and kinase dead MEK1 (K97R) (Cat No. 14-737) procured from Upstate. The incorporation of 32P into MEK1 (K97R) by BRAF (V599E) is measured with final assay buffer conditions of 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM DTT, 100 mM sucrose, 100 μM sodium orthovanadate, 5 μM ATP and 2 μCi [γ 32P] ATP and 500 mg MEK1 Kinase dead substrate. The enzymatic reaction is stopped after 120 minutes with 8N HCl (hydrochloric acid) and 1 mM ATP. The solution is spotted on P81 filter paper and washed 4 times with 0.75% orthophosphoric acid and lastly with acetone. The dried P81 filter papers are read in a Micro-beta Trilux scintillation counter. The final concentration of DMSO is 1% in the assay. Compounds are screened at 10 μM concentration with pre-incubation of the enzymes in the presence of test compound for 45 minutes. Compounds of the invention were found to be inactive in this assy, e.g. ex. 33 (13% inhibition at 10 uM), ex. 1A (0% inhibition at 10 uM).
Measurement: IC50 = 62 nM (pChEMBL 7.210000038146973)
Descriptors: MW 561.02, ALogP 5.48, HBA 8, HBD 1, PSA 106.26, QED 0.3
Compound (SMILES): C=CC(=O)Nc1cccc(-c2cnc3[nH]ccc3c2)n1
InChIKey: HUBFPPMICSBHGU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Biochemical Inhibition Assay: 1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine and 5H-pyrrolo[2,3-b]pyrazine series of compounds exhibited over >100 fold selective against Janus and TEC family of kinases except for the BMX and TXK it had activity of 30 and 45 nM respectively.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 264.29, ALogP 2.75, HBA 3, HBD 2, PSA 70.67, QED 0.71