lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@@]1(c2sc(-c3cccc(C#N)c3)cc2Cl)CS(=O)(=O)C2(CCOCC2)C(=N)N1
InChIKey: OWXWGPFRXDZKJL-IBGZPJMESA-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 = 113 nM (pChEMBL 6.949999809265137)
Descriptors: MW 449.99, ALogP 3.7, HBA 6, HBD 2, PSA 103.04, QED 0.73
Compound (SMILES): CC(C)Oc1ccccc1N1CCCN(CCCCOc2ccc3ccc(=O)[nH]c3c2)CC1
InChIKey: OKJIXAFKUUQQCX-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Radioligand Binding Assay: Membranes prepared as above were resuspended to 1 ug protein/ul (measured by Bradford assay using BSA as standard), and 50 ul were added to each well of a polypropylene 96-well plate containing (per well): 50 ul of buffer (20 mM HEPES, 10 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 100 mM N-methyl-D-gluconate, pH 7.4), 50 ul of 1.5 nM [3H]N-methylspiperone (final concentration 0.3 nM) and reference or D2 test ligand at various concentrations ranging from 50 uM to 50 uM (final concentrations ranging from 10 uM to 10 uM, triplicate determinations for each concentration of D2 test ligand). After a 1.5-hr incubation in the dark at room temperature, the reactions were harvested onto 0.3% PEI-soaked Filtermax GF/A filters (Wallac) and washed three times with ice-cold 50 mM Tris, pH 7.4 using a Perkin-Elmer Filtermate 96-well harvester. The filters were subsequently dried, placed on a hot plate (100° C.), and Melitilex-A (Wallac) scintillant was applied.
Measurement: Ki = 0.93 nM (pChEMBL 9.029999732971191)
Descriptors: MW 449.6, ALogP 4.69, HBA 5, HBD 1, PSA 57.8, QED 0.48
Compound (SMILES): C[C@H]1C[C@@]2(CC[C@@H]1O)Cc1ccc(-c3cccnc3)cc1[C@]21N=C(N)N(Cc2ncccn2)C1=O
InChIKey: WFLMKRDUHUVWHW-OUSDSJFCSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Fluorescence Quench Assay: The inhibitory activity of compounds was assessed by a fluorescence quench assay of BACE1 activity using commercially available substrate HiLyte Fluor488-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-(QXL 520)-OH (SEQ ID NO:1) (AnaSpec, San Jose, Calif.) and truncated human beta-secretase, BACE1 (amino acids 1-454) fused to a myc-his tag and secreted from HEK293/BACEetc. cells into OptiMEM (Invitrogen). The substrate was dissolved at 1 mg/ml in DMSO.The assay was performed in the presence of OptiMEM (supernatant collected over 24 h and cleared from cellular debris by centrifugation) containing the ectodomain of BACE1, 25 ul water containing the desired 2-fold concentration of test compound and 2% DMSO, 1 uM substrate peptide, 20 mM NaOAc, pH 4.4, and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were added to the plate followed by the addition of 10 ul of BACE1 containing OptiMEM.
Measurement: IC50 = 6.8 nM (pChEMBL 8.170000076293945)
Descriptors: MW 468.56, ALogP 2.81, HBA 7, HBD 2, PSA 117.59, QED 0.61
Compound (SMILES): Cc1cc(Nc2cc(C)c(C#N)c(NCCOc3ccncc3)n2)n[nH]1
InChIKey: MKIOKPWDAUFCOF-UHFFFAOYSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Inhibition of recombinant Aurora A kinase derived from human Hela cells using kemptide as substrate in presence of [c-32P]ATP
Measurement: Ki = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 349.4, ALogP 2.92, HBA 7, HBD 3, PSA 111.54, QED 0.56
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=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](Cc1ccccc1)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)N1CCC[C@H]1C(=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: BREYHDITNYIWLQ-PROASZILSA-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 2502.96
Compound (SMILES): [C-]#[N+]CC(=O)N1CCC(CCNC(=O)c2cnc(-n3ncc4cc(C#N)cnc43)cc2NC(C)C)CC1
InChIKey: RZSQQUFZWXJQLW-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 499.58, ALogP 2.79, HBA 8, HBD 2, PSA 133.19, QED 0.46
Compound (SMILES): NCCC1CNC(=O)c2cc(-c3cccc(OC(F)(F)F)c3)c(-c3ccccc3)n21
InChIKey: WTGCKCDNZFLRCX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Trans-Phosphorylation Assay: The inhibitory activity of putative kinase inhibitors and the potency of selected compounds were determined using a trans-phosphorylation assay. Specific peptide or protein substrates were trans-phosphorylated by their specific ser-thr or tyr kinase in the presence of ATP traced with 33P-gamma-ATP, and in the presence of their own optimal buffer and cofactors.At the end of the phosphorylation reaction, more than 98% unlabeled ATP and radioactive ATP were captured by an excess of the ion exchange dowex resin; the resin then settled down to the bottom of the reaction plate by gravity. Supernatant was subsequently withdrawn and transferred into a counting plate, then evaluated by beta-counting. Kinase Buffer (KB):The buffer for PIM1 assay was composed of HEPES 50 mM, at pH 7.5, with 10 mM MgCl2, 1 mM DTT, 3 uM NaVO3, and 0.2 mg/mL BSA.Â  Full-length human PIM1 was expressed and purified as described in Bullock A N, et al., J. Biol. Chem. 2005, 280, 41675-82.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 415.42, ALogP 4.35, HBA 4, HBD 2, PSA 69.28, QED 0.65
Compound (SMILES): O=C(COc1cccc2[nH]c(=O)cnc12)NCC(O)CN1CCc2ccccc2C1
InChIKey: BHHSWQSNBXDCGH-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 408.46, ALogP 0.84, HBA 6, HBD 3, PSA 107.55, QED 0.53
Compound (SMILES): COc1ccc(C[C@@H](NC(=O)c2ccccc2)C(=O)N[C@@H](Cc2ccccc2)C(=O)NCc2ccc(CN)cc2)cn1
InChIKey: WVHMGGCVTKVQNI-URLMMPGGSA-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 = 7540 nM (pChEMBL 5.119999885559082)
Descriptors: MW 565.67, ALogP 2.93, HBA 6, HBD 4, PSA 135.44, QED 0.2
Compound (SMILES): Cc1nc(N)cc(OCC(=O)C(C)(C)C)c1CNC(=O)c1cnn(Cc2ccc3ccccc3c2)c1
InChIKey: QXVKLXZLQIBRSP-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 = 7.8 nM (pChEMBL 8.109999656677246)
Descriptors: MW 485.59, ALogP 4.29, HBA 7, HBD 2, PSA 112.13, QED 0.38
Compound (SMILES): CC(C)(O)CNc1cc(C(N)=O)nc(-c2ccc(Oc3ccc(F)cc3)cc2)n1
InChIKey: KNUSTXCZNIGYBZ-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: In vitro assays were performed in a recombinant cell line expressing cDNA encoding the alpha subunit (Nav1.7, SCN9a, PN1, NE) of human Nav1.7 (Accession No. NM002977). The cell line was provided by investigators at Yale University (Cummins et al, J. Neurosci. 18(23): 9607-9619 (1998)). For dominant selection of the Nav1.7-expressing clones, the expression plasmid co-expressed the neomycin resistance gene. The cell line was constructed in the human embryonic kidney cell line, HEK293, under the influence of the CMV major late promoter, and stable clones were selected using limiting dilution cloning and antibiotic selection using the neomycin analogue, G418. Recombinant beta and gamma subunits were not introduced into this cell line. Additional cell lines expressing recombinant Nav1.7 cloned from other species can also be used, alone or in combination with various beta subunits, gamma subunits or chaperones.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 396.42, ALogP 3.36, HBA 6, HBD 3, PSA 110.36, QED 0.56
Compound (SMILES): O=C(O)c1ccc2[nH]c(=O)n(-c3nc4ccccc4[nH]3)c2c1
InChIKey: RWISMQRIOIBWRA-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 = 3500 nM (pChEMBL 5.460000038146973)
Descriptors: MW 294.27, ALogP 1.89, HBA 4, HBD 3, PSA 103.77, QED 0.52
Compound (SMILES): CN(C)CC(=O)Nc1ccc(-c2n[nH]c3ccc(-c4nc[nH]n4)cc23)cc1
InChIKey: MYYLBBXQEOXPHT-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 361.41, ALogP 2.52, HBA 5, HBD 3, PSA 102.59, QED 0.51
Compound (SMILES): COCc1ccc(C(C)=O)c2sc(C(=O)Nc3cc(CN4CCCC4)c4ccccc4n3)c(C)c12
InChIKey: AMNWZJFKZRHXON-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Activity Assay: A human acute lymphoblastic lymphoma T cell line MOLT-4 (which can be purchased under ATCC No. CRL-1582 from ATCC) was cultured in an RPMI1640 medium containing 10% fetal bovine serum to obtain 5108 MOLT-4 cells. The cells were recovered by centrifugation and suspended in 10 ml of buffer solution A (25 mM Tris-HCl, 5 mM 2-mercaptoethanol, 2 mM benzamidine, 2 mM EDTA, and 0.1 mM 4-(2-aminoethyl)benzenesulfonyl hydrochloride, pH 7.5). The cells were homogenized using a Polytron homogenizer and centrifuged (4° C., 25,000 g, 10 minutes). Then, the supernatant was further ultracentrifuged (4° C., 100,000 g, 60 minutes). The obtained supernatant was filtered through a 0.2um filter to obtain a soluble fraction.HiTrap Q HP column (5 ml2, GE Healthcare Japan Corp.) equilibrated with buffer solution A was charged with the obtained soluble fraction. Phosphodiesterase was eluted with 300 ml of buffer solution A containing a linear gradient solution of 0 to 0.8 M NaCl to recover sixty 5-ml fractions. Each fraction was tested for cAMP-metabolizing phosphodiesterase activity. In each fraction, a fraction eluted as an active peak centered around 250 mM NaCl was collected from fractions that had cAMP metabolic activity and did not lose the metabolic activity by 10uM rolipram (PDE4 selective inhibitor) and 10uM milrinone (PDE3 selective inhibitor). In order to confirm whether or not PDE10A mRNA was expressed in the MOLT-4 cells, total RNA was prepared from the MOLT-4 cells according to a standard method and analyzed by RT-PCR using PDE10A gene-specific primers (PDE10A sense primer: 5'-TGCTCCATGGTGGAAGTGGA-3' (SEQ ID NO: 1) and PDE10A antisense primer: 5'-CAACTGGAAGCATGCGGTCA-3' (SEQ ID NO: 2)). As a result, PDE10A mRNA was detected from the total RNA of the MOLT-4 cells. On the other hand, total RNA was prepared from Jurkat cells, one type of human T cell, and similarly subjected to RT-PCR. As a result, PDE10A mRNA was rarely detected. An active peak centered around 250 mM NaCl was not observed in fractions obtained by the treatment of Jurkat cells in the same way as above. Eur J Biochem. 1999, 266 (3), 1118-2 has reported that PDE10A from the rat striatum and testis is eluted as an active peak centered around 250 mM NaCl.
Measurement: IC50 = 6.2 nM (pChEMBL 8.210000038146973)
Descriptors: MW 487.63, ALogP 5.96, HBA 6, HBD 1, PSA 71.53, QED 0.33
Compound (SMILES): Oc1ccccc1-c1n[nH]c2ccc(-c3nnn[nH]3)cc12
InChIKey: ZMZRGAIBPKTIJA-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 278.28, ALogP 2.12, HBA 5, HBD 3, PSA 103.37, QED 0.52
Compound (SMILES): Cc1nn(-c2ccnc(Nc3ccc4c(c3)c(C(=O)C3CC3)cn4C)n2)cc1CN1C[C@H](O)CO1
InChIKey: YCZUBLQESBVOSH-IBGZPJMESA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition Assay: Compounds of the invention were initially diluted to 10 mM in 100% DMSO (CALBIOCHEM) for storage and made into kinase buffer solution to create a compound concentration ranging from 1 uM and 10 uM. Serial dilutions of compounds of the invention were dispensed into a 96-well plate (GREINER BIOSCIENCES) at 6 uL each. Purified full-length human SYK (CARNA BIOSCIENCES) were diluted in kinase buffer and added to the compound solutions and pre-incubated for 30 minutes at room temperature. Next, ATP (TEKNOVA) of Km (15 uM) and substrate solution (suggested manufacture substrates of PerkinElmer, Ulight-TK peptide for SYK) was added (12 uL each) to the wells containing the compound solution and enzyme. The reaction mixture was incubated for 1 hour. Following the incubation, the stop solution made with EDTA, water, and Lance detection buffer (PERKINELMER) was added (12 uL each) to stop phosphorylation. Following the addition of the stop solution and 5 minutes of shaking.
Measurement: IC50 = 6.15 nM (pChEMBL 8.210000038146973)
Descriptors: MW 473.54, ALogP 2.91, HBA 10, HBD 2, PSA 110.33, QED 0.39
Compound (SMILES): O=C(NC[C@@H](O)CN1CCc2ccccc2C1)Oc1ccccc1
InChIKey: ISPNKABZQKKZLU-QGZVFWFLSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 326.4, ALogP 2.19, HBA 4, HBD 2, PSA 61.8, QED 0.88
Compound (SMILES): COc1cccc(F)c1[C@@H]1C[C@H](Cl)C(=O)N1Cc1ccc(OC(F)(F)F)cc1
InChIKey: NXXKGTAACDQFCQ-ZFWWWQNUSA-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 = 396 nM (pChEMBL 6.400000095367432)
Descriptors: MW 417.79, ALogP 4.81, HBA 3, HBD 0, PSA 38.77, QED 0.52
Compound (SMILES): CN(C)C1CCN(c2nc(-c3cc(Cl)ccc3OC(F)F)c(NC(=O)c3cnn4cccnc34)s2)CC1
InChIKey: IDOBDQFVANDESV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.26 nM (pChEMBL 8.899999618530273)
Descriptors: MW 548.02, ALogP 4.89, HBA 9, HBD 1, PSA 87.89, QED 0.35
Compound (SMILES): C/C(=C\c1cc(F)c(Oc2ccc(S(=O)(=O)NCCOCCOCCOCCNC(=O)C(O)C(O)C(=O)NCCOCCOCCOCCNS(=O)(=O)c3ccc(Oc4c(F)cc(/C=C(\C)C(=O)N=C(N)N)cc4F)cc3)cc2)c(F)c1)C(=O)N=C(N)N
InChIKey: DIDFVKCTQYVLNJ-SWMYPXFDSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P26433
Assay: Inhibition of rat NHE-3-mediated Na+ dependent H+ antiport expressed in opossum kidney cells in presence of NHE-1 antagonist EIPA by BCECF-AM dye based fluorescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 1285.32
Compound (SMILES): CC[C@H](NC(=O)Nc1cc2[nH]nc(N3CCCC3)c2cn1)c1ccccn1
InChIKey: KAWFWIHPXVOMDC-AWEZNQCLSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 13.8 nM (pChEMBL 7.860000133514404)
Descriptors: MW 365.44, ALogP 3.23, HBA 5, HBD 3, PSA 98.83, QED 0.64
Compound (SMILES): CCn1cc(-c2cc3c(-c4cc(C(F)(F)F)ccn4)n[nH]c3cn2)cn1
InChIKey: VFXUYKQSTNDXRJ-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 = 65.6 nM (pChEMBL 7.179999828338623)
Descriptors: MW 358.33, ALogP 3.92, HBA 5, HBD 1, PSA 72.28, QED 0.6
Compound (SMILES): [C-]#[N+]/C(=C/c1ccc(Nc2cnc3[nH]cc(C(=O)C(C)(C)C)c3n2)cc1)c1nccs1
InChIKey: OCFCCQSJSSUFII-GZTJUZNOSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Phosphorylation Assay: Phosphorylation of the appropriate peptide substrate (fluorescently labeled Srctide) by recombinant JAK3 (catalytic domain, aa 781-1124) was measured in the presence and absence of different concentrations of test compounds. Test compound, enzyme, fluorescently labeled substrate, and cofactors (ATP and Mg2+) were combined in a well of a microtiter plate and incubated for 2 hours at 25° C. The composition of the reaction buffer was 100 mM HEPES pH 7.5, 5 mM MgCl2, 0.01% Triton-X 100, 0.1% Bovine Serum Albumin, and 1% DMSO. Enzyme concentration in the final reaction mixture was 0.5 nM while peptide substrate was at 1 uM. ATP was used at 1xKm concentration corresponding to 2 uM. Serial dilutions (3-fold steps) of compounds were prepared in DMSO. At the end of the incubation, the reaction was stopped by the addition of 20 mM EDTA. Substrate and product (i.e. phosphorylated substrate) were separated electrophoretically and both were quantified by fluorescence intensity.
Measurement: IC50 = 2200 nM (pChEMBL 5.659999847412109)
Descriptors: MW 428.52, ALogP 5.8, HBA 6, HBD 2, PSA 87.92, QED 0.31
Compound (SMILES): CC(c1ccccc1)n1cc(-c2ccc(N3CCc4cccc(C(=O)Nc5nc6ccccc6s5)c4C3)nc2C(=O)O)cn1
InChIKey: FTJLNNCJPJCARI-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 600.7, ALogP 6.68, HBA 8, HBD 2, PSA 113.24, QED 0.21
Compound (SMILES): Nc1ccc(F)cc1NC(=O)CCCCCNC(=O)c1ccc(F)cc1
InChIKey: RCTFWBRKYUQTHJ-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 5700 nM (pChEMBL 5.239999771118164)
Descriptors: MW 361.39, ALogP 3.48, HBA 3, HBD 3, PSA 84.22, QED 0.5
Compound (SMILES): O=C(CCCCc1ccc(F)cc1)N/N=C/c1ccc(O)c(O)c1O
InChIKey: MBOKXSKNGHWALA-RGVLZGJSSA-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 = 470 nM (pChEMBL 6.329999923706055)
Descriptors: MW 346.36, ALogP 2.81, HBA 5, HBD 4, PSA 102.15, QED 0.27
Compound (SMILES): N#Cc1ccc(Oc2ccc3nc(C(=O)NCCN4CCCCC4)ccc3c2)cc1
InChIKey: MWZSXMAZYHEUEU-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 = 222 nM (pChEMBL 6.650000095367432)
Descriptors: MW 400.48, ALogP 4.11, HBA 5, HBD 1, PSA 78.25, QED 0.67
Compound (SMILES): CS(=O)(=O)c1cc(C#N)ccc1C1C2=C(CCC2=O)N(c2cccc(C(F)(F)F)c2)C(=O)N1CCCO
InChIKey: OJHJODRBJXXFBI-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 533.53, ALogP 3.96, HBA 6, HBD 1, PSA 118.78, QED 0.6
Compound (SMILES): COC(=O)c1cccc(-n2nc(C(C)(C)C)cc2NC(=O)Nc2cnc(Oc3ccc(-c4cnc(N5CCCC5=O)s4)cc3)nc2)c1
InChIKey: ODGQYNDBTMEAKZ-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: LanthaScreen Kinase Assay: TrkA-inhibiting activity was measured according to the following method using LanthaScreen kinase assay reagents (Invitrogen; Fluorescein-Poly GT, LanthaScreen Tb-PY20 and TR-FRET Dilution Buffer), TrkA (Invitrogen), ATP (Sigma-Aldrich), a kinase reaction buffer (50 mM HEPES buffer (Sigma-Aldrich) (pH 7.5) containing 0.01% Brij35 (Sigma-Aldrich), 10 mM MgCl2 (Sigma-Aldrich) and 1 mM EGTA (Sigma-Aldrich)) and 0.5 M EDTA (pH 8.0) (Invitrogen).The present compound was dissolved in dimethylsulfoxide (hereinafter abbreviated as DMSO) to prepare a 10 mM solution. The test compound at 10 mM was distributed into a 96-well plate and serially diluted with DMSO with the geometrical ratio of 3. The serial dilutions (DMSO solutions) were diluted with the kinase reaction buffer to 20-fold to prepare solutions of the present compound with 5-times concentrations (DMSO concentration: 5%). TrkA was diluted with the kinase reaction buffer to prepare a solution at 38.5 ng/mL (enzyme solution). Adenosine
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 652.74, ALogP 6.43, HBA 11, HBD 2, PSA 153.46, QED 0.18
Compound (SMILES): O=C1C(N2CCCC(c3ccc(O)cc3)C2)CCCN1Cc1ccc(Cl)cc1
InChIKey: LUTJNLKVMPYVLM-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q00960
Assay: Radioligand Binding Assay: To perform the competition binding assay, thawed membrane homogenate was added to each well of a 96-well plate (20 ug/well). The experimental compounds were serially diluted in 100% DMSO and added to each row of the assay plate to achieve desired compound concentrations, keeping the DMSO concentration in the assay plate at 1.33% of the final reaction volume. Next, 3H Ro 25-6981 (4 nM) was added to the assay plate. After incubation for 1 hr at room temperature, the membrane bound radioligand was harvested on to GF/B filter plates (treated with 0.5% PEI for 1 hr at room temperature). The filter plates were dried at 50° C. for 20 mins, incubated with microscint 20 for 10 minutes and finally, the counts were read on TopCount (Perkin Elmer). Non-specific binding was determined using MK-0657 (the preparation of this compound is described as example 1 in WO 2004 108705 (40 uM).
Measurement: Ki = 720 nM (pChEMBL 6.139999866485596)
Descriptors: MW 398.93, ALogP 4.42, HBA 3, HBD 1, PSA 43.78, QED 0.83
Compound (SMILES): CC(C)CCN1C[C@@H](C)N(C(=O)CCc2cccnc2)[C@@H](C)c2c(F)cccc21
InChIKey: GFAZKXBHWPWIIM-MOPGFXCFSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1G (SINGLE PROTEIN), Homo sapiens
UniProt: O43497
Assay: Channel blocking activity at recombinant human CaV 3.1 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 397.54, ALogP 5, HBA 3, HBD 0, PSA 36.44, QED 0.69
Compound (SMILES): O=S(=O)(Nc1ccc(F)c(Nc2ncccc2-c2ncnc3[nH]cnc23)c1F)c1ccccc1Cl
InChIKey: FJDHTUKBHFXBLS-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Enzyme Assay: To check the B-Raf kinase inhibitory activity of the compounds of the present invention, the following experiments were conducted.(1) Serial Signaling: 20 ul of diluting solvent (20 mM MOPS, pH 7.2, 25 mM Î²-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol) and 10 ul of Mg/ATP mixed solution (500 uM ATP, 75 mM magnesium chloride) were added into a centrifuge tube, the derivative compound of Formula 1 was added or the compounds of the Examples were not added as a control group, and then 1 ng of activated B-Raf, 0.4 ng of inactivated MEK1, and 1 ug of inactivated MAPK2 were added. The solutions in the tube were collected on the bottom via centrifugation and were reacted at 30° C. for 30 minutes. After taking 4 ul of the mixed solution, the test proceeded to the next step.(2) Phosphorylation of Matrix Protein MBP by MAPK2: 10 ul of diluting solvent, 20 ug of MBP used as a matrix.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 513.92, ALogP 4.89, HBA 7, HBD 3, PSA 125.55, QED 0.29
Compound (SMILES): Cc1cc(C)nc(-n2nnc3c2CCN(C(=O)c2cccc(Cl)c2Cl)C3C)c1
InChIKey: DKMRXVCUOIHSIP-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 498.9 nM (pChEMBL 6.300000190734863)
Descriptors: MW 416.31, ALogP 4.35, HBA 5, HBD 0, PSA 63.91, QED 0.62
Compound (SMILES): N#Cc1cccc(C(=O)NCCCCCCNC(=O)N2Cc3ccccc3C2)c1
InChIKey: ABEQGUCEYPQNGT-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 = 18.3 nM (pChEMBL 7.739999771118164)
Descriptors: MW 390.49, ALogP 3.57, HBA 3, HBD 2, PSA 85.23, QED 0.68
Compound (SMILES): CC(C)Cc1cc(CNS(=O)(=O)c2c(Cl)cccc2Cl)nn1-c1ccccc1
InChIKey: RAPRVFZTSKPDRU-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P50406
Assay: Antagonist activity at human 5-HT6R expressed in human HeLa cells assessed as inhibition of 5-HT-induced cAMP levels pre-treated for 10 mins before 30 mins stimulation with 5-HT by D2-dye based HTRF assay
Measurement: IC50 = 5650 nM (pChEMBL 5.25)
Descriptors: MW 438.38, ALogP 4.86, HBA 4, HBD 1, PSA 63.99, QED 0.57
Compound (SMILES): CC(C)(C)CN1CCC2(CC1)CN(c1ccccc1NC(=O)Nc1nc3ccc(Cl)cc3s1)c1c(O)ccc(-c3nc4cccc(Cl)c4s3)c12
InChIKey: YNQHGZRYYPZIHU-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 uL containing 15 fmol of P2Y1 receptor (1.7 ug of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 uM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium Chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 ug of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 uL of the aqueous soluttion was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKERSM (Amersham) Image Reader. Dose-response curves (IC50) were fit by non-linear regression (Toolset an in house data processing program) and binding constants (Ki) calculated using the Cheng-Prusoff relationship (Ki=IC50/(1+L/Kd) in which a Kd for 2MeS-ADP to the P2Y1 receptor was determined to be 14 nM.
Measurement: Ki = 51.5 nM (pChEMBL 7.289999961853027)
Descriptors: MW 743.79, ALogP 10.76, HBA 8, HBD 3, PSA 93.62, QED 0.16
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(c3)-c3nc5ccc(F)cn5c3CO4)cc12
InChIKey: IOYPXIBVGWWLQK-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: ELISA-Based Assay: Measurement of inhibition by compounds was performed using the HCV replicon system. Several different replicons encoding different HCV genotypes or mutations were used. In addition, potency measurements were made using different formats of the replicon assay, including different ways of measurements and different plating formats. See Jan M. Vrolijk et al., A replicons-based bioassay for the measurement of interferons in patients with chronic hepatitis C, 110 J. VIROLOGICAL METHODS 201 (2003); Steven S. Carroll et al., Inhibition of Hepatitis C Virus RNA Replication by 2'-Modified Nucleoside Analogs, 278(14) J. BIOLOGICAL CHEMISTRY 11979 (2003). However, the underlying principles are common to all of these determinations, and are outlined below.Stable neomycin phosphotransferase encoding replicons-harboring cell lines were used, so all cell lines were maintained under G418 selection prior to the assay. Potency was determined using a cell ELISA assay with an antibody to the replicons encoded NS3/4a protease. See Caterina Trozzi et al., In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor, 77(6) J. Virol. 3669 (2003).
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 614.63, ALogP 6.01, HBA 7, HBD 1, PSA 106.15, QED 0.26
Compound (SMILES): O=C(N1CC=C(c2cc3c(-c4cccc(N5CCOc6cc(C7CC7)cc(F)c6C5=O)c4CO)ccnc3[nH]2)CC1)N1CCOCC1
InChIKey: WMWMALWQLNDUID-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 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 637.71, ALogP 5.32, HBA 6, HBD 2, PSA 111.23, QED 0.31
Compound (SMILES): COc1cnc(-c2ccc(NC(=O)Nc3ccc(C(N)=O)cc3)c(F)c2)nc1N1CCOCC1
InChIKey: NUELHVXAHIGFKH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: mTOR Kinase Assay: The activity of the compound against mTOR was examined by measuring the incorporation of 33P from [γ-33P]-ATP into 4EBP1. His-tagged, recombinant human 4EBP1 was expressed in E. coli, purified by nickel-nitrilotriacetic acid (Ni-NTA) resins, and stored at −80 °C. Phosphorylation of 4EBP1 by mTOR was assayed in the presence or absence of the compound and performed in a final volume of 25 μl reaction buffer containing 300 ng 4EBP1, 50 ng recombinant mTOR (Invitrogen), 50 mM HEPES (pH 7.5), 1 mM EGTA, 0.01% Polysorbate 20, 10 mM MnCl2, 2.5 mM DTT, 10 μM ATP, and 0.5 μCi [Î³-33P]-ATP (PerkinElmir) for 30 min at 30 °C. The reactions were terminated by adding 3% phosphoric acid. The 33P labeled 4EBP1 was transferred onto UniFilter-96 GF/B plate (PerkinElmer) and quantified by Top Count Microplate Scintillation Counter (PerkinElmer). For primary screening of kinase activity inhibition, each test compound was evaluated at 10 M in duplicate.
Measurement: IC50 = 215 nM (pChEMBL 6.670000076293945)
Descriptors: MW 466.47, ALogP 2.87, HBA 7, HBD 3, PSA 131.7, QED 0.51
Compound (SMILES): COc1cc(/N=C/c2ccc([N+](=O)[O-])cc2)cc(OC)c1
InChIKey: NTFTWACNCJWKBJ-MHWRWJLKSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Inhibition of electric eel AChE using acetylthiocholine iodide as substrate preincubated for 20 mins followed by substrate addition by Ellman's method
Measurement: IC50 = 7010 nM (pChEMBL 5.150000095367432)
Descriptors: MW 286.29, ALogP 3.36, HBA 5, HBD 0, PSA 73.96, QED 0.48
Compound (SMILES): Cc1ccc(-n2nc(C(C)(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4ccc5c(c4)OCO5)n3)c3ccccc23)cc1
InChIKey: KMECUGNHSIJUNR-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: p38 MAPKα Enzyme Inhibition Assay: The inhibitory activities of test compounds against the p38 MAPKα isoform (MAPK14: Invitrogen), are evaluated indirectly by determining the level of activation/phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPK&dlpha; protein (80 ng/mL, 2.5 uL) is mixed with the test compound (2.5 uL of either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL or 0.004 ug/mL) for 2 hr at RT. The mix solution (2.5 uL) of the p38&dlpha; inactive target MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 uM; a phosphorylation target for MAPKAP-K2) is then added and the kinase reaction is initiated by adding ATP (40 uM, 2.5 uL). The mixture is incubated for 1 hr at RT. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 369 nM (pChEMBL 6.429999828338623)
Descriptors: MW 627.71, ALogP 8.33, HBA 9, HBD 3, PSA 124.45, QED 0.16