lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccccc1[C@](C)(O)c1ccc(-c2nc([C@@H]3CC[C@H]4CCC(=O)N4C3)n3ccnc(N)c23)cc1
InChIKey: BOWDMXQRNQPGIR-NSZUCLEHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated A5 peptide (Biotin-EQEDEPEGDYFEWLE-NH2) (SEQ.ID.NO.: 1), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 481.6, ALogP 4.41, HBA 6, HBD 2, PSA 96.75, QED 0.45
Compound (SMILES): COc1ncc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)CC(C)C)C[C@@H]1F
InChIKey: BWRSJNQRECUNKD-JKSUJKDBSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 7100 nM (pChEMBL 5.150000095367432)
Descriptors: MW 523.49, ALogP 2.73, HBA 8, HBD 2, PSA 127.74, QED 0.48
Compound (SMILES): O=C(O)CCCOc1c(F)cc(-n2ccc3cc(F)ccc32)cc1F
InChIKey: JQTZAUJKUWQYBT-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Measurement of Activity of GPR120 Agonist (Cell-Based Assay): CHO-K1 cells expressing Ga16 and hGPR120 were dispensed into each well of a 96-well plate (3×104 cells/100 μl/well) and then incubated in 5% CO2, 37° C. incubator for 18 hours. Each well was treated with 100 μl of Calcium 5 dye (Molecular Devices) solution including 2% DMSO and then incubated in 5% CO2, 37° C. incubator for 1 hour. Serially diluted GPR120 agonists were prepared to a final concentration of 0.5% DMSO in a 96-well plate. Each well was treated with 50 μl of the agonist compounds using Plexstation II, and then fluorescence was measured at Ex 485 nm and Em 525 nm.Fluorescence increased by the serially diluted GPR120 agonists is calculated as a relative percent (%) value based on the fluorescence represented by the treatment of 1% DMSO only. EC50 refers to the concentration of agonist which shows 50% of maximum fluorescence increased by the treatment of agonist. The calculation of measurement was carried out by using statistical software (Prizm).
Measurement: EC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 349.31, ALogP 4.29, HBA 3, HBD 1, PSA 51.46, QED 0.68
Compound (SMILES): CNC(c1nc2cc(C#N)ccc2[nH]1)c1c(OC)cc(C)cc1OC
InChIKey: ZMCDQOWTGRFTTD-UHFFFAOYSA-N
Target: Complement factor B (SINGLE PROTEIN), Homo sapiens
UniProt: P00751
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. IC50 values were calculated from percentage of inhibition of CVF-Bb activity as a function of test compound concentration.
Measurement: IC50 = 13900 nM (pChEMBL 4.860000133514404)
Descriptors: MW 336.4, ALogP 3.07, HBA 5, HBD 2, PSA 82.96, QED 0.75
Compound (SMILES): COc1ccc(Cl)c(S(=O)(=O)Nc2ccc(-c3nc(OC)c4c(N)n[nH]c4n3)cc2)c1
InChIKey: DKWVKDLGUSPTBF-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Sgk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00141
Assay: Enzymatic Activity Assay: The compounds were tested for serum and glucocorticoid-regulated kinase 1 (SGK-1) inhibitory activity in a substrate phosphorylation assay designed to measure the ability of the isolated enzyme to catalyze the transfer of phosphate from ATP to serine/threonine residues in a fluorescein-labeled substrate peptide, using recombinant human SGK-1 enzyme produced in a baculovirus system (Biomol, Hamburg, Germany, Cat. No. 4-331). The synthesized fluorescent labeled peptide substrate contained (5(6)-Carboxyfluorescein)-RPRAATF-NH2. The phosphorylated substrate peptide and non-phosphorylated substrate peptide were separated with caliper life science's lab-chip technology based on a micro fluidics method. All fluid flow was established on the chip by applying a vacuum of a few psi to the waste well transporting fluid from various sources through interconnecting channels. Because the phosphoryl group is doubly negatively charged, under the pressure-driven hydrodynamic flow and the voltage-driven flow within the electric field, the fluorescent labeled peptide substrate and its phosphorylation product appear at different times in the detection window to the detection point. Substrate turnover can thus be determined as the ratio of the product peak area and the sum of substrate peak area and product peak area.The enzyme reaction was carried out in a buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM MnCl2, 2 mM DTT, and 0.03% bovine serum albumine. The enzyme was pre-incubated with the test compound for 30 min at 24° C. The kinase reaction was initiated by addition of the substrate mixture containing the peptide substrate (final concentration 1 μM) and ATP (final concentration 10 μM). After 60 min incubation at 37° C., the enzyme reaction was terminated by adding a buffer containing 100 mM Hepes (pH 7.4) and 35 mM EDTA.
Measurement: IC50 < 1.5 nM (pChEMBL 8.824000358581543)
Descriptors: MW 460.9, ALogP 3.07, HBA 8, HBD 3, PSA 145.11, QED 0.4
Compound (SMILES): C[C@]1(O)CCCC[C@@H]1NC(=O)c1cc(Cc2ccc(Cl)nc2)c2ccccc2c1F
InChIKey: XWKJCKAMJNNLCF-RDPSFJRHSA-N
Target: Muscarinic acetylcholine receptor M1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11229
Assay: M1 PAM Assay: The assay is designed to select compounds that possess modulator activity at the acetylcholine muscarinic receptor expressed in CHO cells by measuring the intracellular calcium with a Fluorometric Imaging Plate Reader System (FLIPR, Molecular Devices). The assay study the effect of several concentrations of test compounds on basal or acetylcholine-stimulated Ca2+ levels using FLIPR.CHO human M1 are plated the day before the experiments at 2×105 cells/ml in PDL BioCoat 96 well black/clear plate (Becton 35 4640). The cells are grown at 37° C. and 5% CO2 in the following medium: F12 Nut Mix (Gibco 21765), 10% FCS heat inactivated (GIBCO 16000-044), 1% Pen Strep (Gibco, 15140) and 200 μg/ml Geneticin (Gibco 11811). On the day of the experiment, the medium was removed and replaced by 100 μl of dye loading buffer containing Hanks Balanced Salt solution (HBSS, 14065-049, Gibco) with 20 mM HEPES (Gibco 15630-056), 2 mM Probenicid (Sigma P8761), 2 mM Fluo-4AM ester (Molecular Probes F-14202), 10% Pluronic acid Molecular Probes P-3000) pH=7.4 and incubated at 37° C. After 60 minutes extracellular dye was removed and the cells were washed five times with FLIPR buffer containing HBSS (Gibco 14065-049) with 20 mM HEPES (Gibco, 15630-056), 2 mM Probenicid (Sigma P8761) pre-warmed at 37° C. using and Ebml cell washer leaving 100 μl of FLIPR buffer in each well. The cell plate and the diluted compounds (1% DMSO final concentration) are placed on the platform of the FLIPR and the door closed. A signal test to check background fluorescence and basal fluorescence signal is performed. Laser intensity is adjusted if necessary. Two minutes preincubation with the diluted test compounds is provide to determine any agonist activity on the M1 receptor by comparison to 30 nM Acetylcholine control. In order to determine any modulator activity the diluted compounds were added to cells and after two minutes preincubation, the EC20 of acetylcholine is added followed by another two minutes preincubation before the measurement of intracellular Ca2+ with a FLIPR (Molecular Devices).
Measurement: EC50 = 561 nM (pChEMBL 6.25)
Descriptors: MW 426.92, ALogP 5.04, HBA 3, HBD 2, PSA 62.22, QED 0.57
Compound (SMILES): Cc1cc(-c2ccc(CC(=O)Nc3ccc(N4C[C@@H](C)O[C@@H](C)C4)cn3)cc2)ccn1
InChIKey: GEAVZEGGEKRJRI-KDURUIRLSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: Wnt-Luc Reporter Assay: Human embryonic kidney 293 cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are cultured in DMEM medium (Gibco/Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS (Gibco/Invitrogen, Carlsbad, Calif.), 50 unit/mL penicillin and 50 μg/mL of streptomycin (Gibco/Invitrogen, Carlsbad, Calif.) at 37° C. with 5% CO2 in air atmosphere. 293 cells in a 10 cm dish are co-transfected with 8 μg of STF-reporter plasmid containing a luciferase gene driven by Wnt-responsive elements and 2 μg of pcDNA3.1-Neo (Gibco/Invitrogen, Carlsbad, Calif.) with 30 μL of FuGENE6 (Roche Diagnostics, Indianapolis, Ind.) following the manufacturer's protocol. Stable cell lines (293 Wnt-Luc) were selected with 400 μg/mL of G418 (Gibco/Invitrogen, Carlsbad, Calif.). The 293 Wnt-Luc cells and L-cell Wnt3a cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are trypsinized and co-cultured into a 384-well plate with DMEM medium supplemented with 2% FBS, and treated with different concentrations of a compound of the invention. After 24 hours, the firefly luciferase activities are assayed with the Bright-Glo Luciferase Assay System (Promega, Madison, Wis.). The IC50 is measured when the effect of the compound reduces the luminescence signal by 50%.
Measurement: IC50 = 565 nM (pChEMBL 6.25)
Descriptors: MW 416.53, ALogP 4.25, HBA 5, HBD 1, PSA 67.35, QED 0.67
Compound (SMILES): CNCCN(C)c1ccc2c3ncc(-c4c(C)nnn4C)cc3n([C@H](c3ccccc3F)C3CCOCC3)c2n1
InChIKey: INWRSQYJZYOOAB-LJAQVGFWSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The binding of compounds to bromodomain BRD4 (44-168), BRD4 (333-460), and BRD4 (1-477 or 44-460) was assessed using a time resolved fluorescent resonance energy transfer binding assay (1), that measures the binding of a fluorescently labeled probe molecule to the bromodomain protein. The bromodomain protein, fluorescent probe molecule (either a biotinylated histone peptide or a fluorescently labeled small molecule), and dose-responded test compound are incubated together to reach thermodynamic equilibrium. In the absence of a test compound, the bromodomain and small molecule are bound, resulting in a high fluorescent signal. In the presence of a sufficient concentration of inhibitor, this intercation is disrupted resulting in a lost of fluorescent resonance energy transfer.All assay components were dissolved in buffer composition 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM DTT, 0.005% Tween 20, and 100 ug/ml BSA for BRD4 (1-477 and 44-460). The final concentrations of the bromodomain proteins are 1.6 nM BRD4(44-168), 1 nM BRD4(333-460), and 1 nM BRD4(1-477 or 44-460), and the fluorescent probe molecule is 100 nM, 50 nM, and 7.5 nM respectively. All proteins were biotinylated. A streptavidin labeled with terbium cryptate (Cisbio SA-Tb) was used as detection, and pre-mixed with the bromodomain protein at a final concentration of 0.2 nM. In some instances for BRD4 (44-460), anti-His terbium cryptate was used as a detection. 7.5 nl of dose-responded test compound or dmso vehicle (0.0375%) was pre-spotted in a black Corning 384 well plate and 10 ul each of bromodomain/detection reagent and fluorescent small molecule solution were added to the plate, and the reaction incubated for 60 min at room temperature. Plates were then read on EnVision plate reader, (λex=340 nm, acceptor λEm=520 nm, and donor λEm=615 nm, LANCE D400 mirror). Time resolved fluorescnce intensity measurements were made at both emissions, and the ratio of acceptor/donor was calculated and used for data analysis.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 542.66, ALogP 4.5, HBA 9, HBD 1, PSA 85.92, QED 0.31
Compound (SMILES): CN(CCC1CCN(c2ccccc2)CC1)C(=O)C1CCN(c2ccc(OC(F)(F)F)cc2)CC1
InChIKey: LRFHFGLDLYJZHC-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 489.58, ALogP 5.57, HBA 4, HBD 0, PSA 36.02, QED 0.51
Compound (SMILES): CCS(=O)(=O)c1ccc(Cl)cc1Cn1c(=O)[nH]c2c(Cl)c(CN3CCC[C@H](NS(C)(=O)=O)C3)c(C(F)(F)F)cc2c1=O
InChIKey: RHRPDYWAJATRRR-INIZCTEOSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: Binding Activity: DDR1 binding activity was measured using the LanthaScreen(Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen(Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 90 nM (pChEMBL 7.050000190734863)
Descriptors: MW 671.55, ALogP 3.37, HBA 8, HBD 2, PSA 138.41, QED 0.37
Compound (SMILES): O=C(O)[C@H](CCN(CCCCc1ccc2c(n1)NCCC2)CCOCC(F)(F)F)Nc1ncnc2ccccc12
InChIKey: ZVHVVZKZBSAJQM-DEOSSOPVSA-N
Target: Integrin alpha-5/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P18564
Assay: Binding Assay: Microplates were coated with recombinant human integrin αVβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 560.62, ALogP 4.54, HBA 8, HBD 3, PSA 112.5, QED 0.23
Compound (SMILES): C=CC(=O)N1CC[C@H](Nc2c(F)cc(C(N)=O)c3[nH]c(C)c(C)c23)C1
InChIKey: QBBWDANCZIQCDD-NSHDSACASA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Assay: To V-bottom 384-well plates were added test compounds, human recombinant Btk (1 nM, Invitrogen Corporation), fluoresceinated peptide (1.5 μM), ATP (20 μM), and assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij 35 surfactant and 4 mM DTT in 1.6% DMSO), with a final volume of 30 μL. After incubating at room temperature for 60 min., the reaction was 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 control reactions with no enzyme (for 100% inhibition) and controls with no inhibitor (for 0% inhibition). Dose response curves were generated to determine the concentration required for inhibiting 50% of Btk activity (IC50). Compounds were dissolved at 10 mM in DMSO and evaluated at eleven concentrations.
Measurement: IC50 = 3.3 nM (pChEMBL 8.479999542236328)
Descriptors: MW 344.39, ALogP 2.22, HBA 3, HBD 3, PSA 91.22, QED 0.74
Compound (SMILES): COc1ccc(NC(=O)CN2C[C@]3(CC[C@](c4ccccc4)(N(C)C)CC3)N(CC3CCC3)C2=O)nc1
InChIKey: RMECZNJQCXTVLR-ISILISOKSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Human Nociceptin/Orphanin FQ Peptide (hNOP) Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4). The final assay volume (250 μl/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences, Inc. Boston, Mass., USA), and either test compound in dilution series or 1 μM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration, which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd., Buckinghamshire, UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences, Inc. Boston, Mass., USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ß-counter (PerkinElmer Life Sciences/Wallac, Turku, Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]nociceptin-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation, (Cheng and Prusoff, 1973).
Measurement: Ki = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 505.66, ALogP 4.34, HBA 5, HBD 1, PSA 78.01, QED 0.58
Compound (SMILES): O=C(O)c1ccc(N2CCC(CN[C@@H]3CC3c3ccccc3)(Cc3ccc(F)cc3)CC2)cn1
InChIKey: JTQLOLBCRLSAJV-JUERFOTFSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 histone demethylase biochemical assay: LANCE LSD1/KDM1A demethylase assay10 μL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 μL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 μL of assay buffer containing 0.4 μM Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO: 1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 μL 1× LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 459.57, ALogP 4.89, HBA 4, HBD 2, PSA 65.46, QED 0.5
Compound (SMILES): Cn1cc2c(C(=O)NN3CCC(CCN4CCc5sc(OCC(F)(F)F)nc5C4)CC3)ccc(F)c2n1
InChIKey: VNNNOJMMKZKFEA-UHFFFAOYSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Binding Inhibition for Dopamine D3 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl.6HO (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl) (067-31, NAKARAI CHEMICALS, LTD.) Radioligand: (final concentration) 2 nM [H]-Methylspiperone ([H���N-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 uM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma] SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well) Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 540.59, ALogP 3.92, HBA 8, HBD 1, PSA 75.52, QED 0.46
Compound (SMILES): COCCOc1c(C#N)cccc1-c1ccc2ncc(-c3cc(F)cc(F)c3)c(N3CCC(N)CC3)c2c1
InChIKey: XXVBCHGKMBJSCV-UHFFFAOYSA-N
Target: Somatostatin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P30874
Assay: cAMP Assay : Four days prior to the assay, 5,000 Chinese hamster ovary cells (CHO-K1, ATCC #CCL-61) stably expressing the human SSTR2 are plated in each well of a 96-well tissue culture-treated plate in Ham's F12 growth media (ThermoFisher #10-080-CM) supplemented with 10% donor bovine serum (Gemini Bio-Products #100-506), 100 U/mL penicillin; 100 ug/mL streptomycin; 2 mM L-glutamine (Gemini Bio-Products #400-110) and 0.2 mg/mL hygromycin B (GoldBio #31282-04-9). The cells are cultured at 37° C., 5% CO2 and 95% humidity. On the day of the assay, the media is aspirated and the cells are treated with 50 μL of 1.6 μM NKH477 (Sigma #N3290) plus various dilutions of compounds of the invention in assay buffer [1× Hank's Balanced Salt Solution (ThermoFisher #SH3058802), 0.5 mM HEPES pH 7.4, 0.1% bovine serum albumin, 0.2 mM 3-Isobutyl-1-methylxanthine (IBMX, VWR #200002-790)]. The cells are incubated for 20 minutes at 37° C. (the final concentration of the compounds of the invention are typically 0-10,000 nM). The cells are treated with 50 μL of lysis buffer (HRTF cAMP kit, Cisbio). The lysate is transferred to 384-well plates and cAMP detection and visualization antibodies are added and incubated for 1-24 hours at room temperature. The time-resolved fluorescent signal is read with a Tecan M1000Pro multiplate reader. The intracellular cAMP concentrations are calculated by regression to a standard curve and are plotted vs. the concentration of the compounds of the invention and the EC50 of the compounds are calculated using standard methods. All data manipulations are in GraphPad Prism v6.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 514.58, ALogP 5.67, HBA 6, HBD 1, PSA 84.4, QED 0.32
Compound (SMILES): Cc1c(N2CC3C(CC(=O)O)C3C2)nc(N2CC[C@@H]2C)nc1C(C)(F)F
InChIKey: SAZZPQRJVMUMRX-KODGCSJUSA-N
Target: Ketohexokinase (SINGLE PROTEIN), Homo sapiens
UniProt: P50053
Assay: Biological Assay: Assay A, a 384-well format on a Corning 3653 assay plate is used, and monitored by UV-vis spectroscopy in continuous mode at rt. Compounds were prepared in DMSO as 4 mM stocks, diluted using an 11-point half-log scheme on a Biomek FX (Beckman Coulter), and incubated at rt for 30 minutes with the reaction mixture containing 50 mM HEPES, pH 7.4, 140 mM KCl, 3.5 mM MgCl2, 0.8 mM fructose, 2 mM TCEP, 0.8 mM PEP, 0.7 mM NADH, 0.01% Triton X-100, 30 U/mL pyruvate kinase-lactate dehydrogenase, and 10 nM purified KHK-C. The compound concentration in each well ranged from 1 nM to 100 uM. The reaction was initiated with the addition of 0.2 mM ATP. The absorbance was measured for 30 minutes on a SpectraMax reader (Molecular Devices) after ATP was added. The concentrations provided are based on the final mixture volume of 40 uL (referred to as the final concentration).
Measurement: IC50 = 51.4 nM (pChEMBL 7.289999961853027)
Descriptors: MW 366.41, ALogP 2.65, HBA 5, HBD 1, PSA 69.56, QED 0.86
Compound (SMILES): CCc1nccn1Cc1cn(-c2ccc(Cl)c(Cl)c2)nn1
InChIKey: ACNAPZHYUZOZJN-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition Assay: Cells were split 2-3 times weekly between 1:3 and 1:4. For binding assays and membrane preparations the cell culture medium was removed, cells were washed with PBS. Crude membranes for radioligand binding experiments were prepared by scraping the cells off the dishes in ice cold 20 mM HEPES/0.1 mM KCl/pH 7.2. The cell suspension was homogenized on ice (Ultra turrax, 3×20 sec.) and the homogenate was spun for 10 min (1° C., 1000 g, OPTIMA, SW28, 2800 U/min). The supernatant was than centrifuged for 40 min at 100000 g (1° C., OPTIMA, SW28, 23000 U/min). The membrane pellet was resuspended in 20 mM HEPES/0.1 mM KCl/pH 7.2, frozen and stored at −80° C.After thawing on the day of the assay, the membrane suspension was diluted further with 20 mM HEPES/0.1 mM KCl/pH 7.2.The incubation mixture of 200 μl contained 1.5 nmol/l 3H-Dofetilide, optimized amount of membrane preparation, 20 mM HEPES/0.1 mM KCl/(pH 7.2) and inhibitor in 1% DMSO. Nonspecific binding was estimated in the presence of 10 M Dofetilide. The samples were incubated for 90 min. at RT.Binding was terminated by filtration of the incubated membrane preparations using Filtermat B (Pharmacia, Uppsala Sweden) and a Micro Cell Harvester (Skatron, Lier, Norway). The Filtermat B had been presoaked with 1% polyethylenimine and carefully washed with 0.05 M Tris/HCl-buffer pH=7.7 after the filtration to separate free and bound radioactivity. The filters were counted in a scintillation counter (Betaplate 1205, Berthold, Wildbad, Germany) in order to determine the specific binding of [3H]-Dofetilide.
Measurement: IC50 = 4.65 nM (pChEMBL 8.329999923706055)
Descriptors: MW 322.2, ALogP 3.38, HBA 5, HBD 0, PSA 48.53, QED 0.74
Compound (SMILES): Nc1ncccc1-c1ccc(S(=O)(=O)NC[C@@H]2CNCCN2)c(S(N)(=O)=O)c1-c1nn[nH]n1
InChIKey: CYDKMYNWSJUBBJ-JTQLQIEISA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: BIOLOGICAL ASSAYS: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1; for a review, see: Meine, M.-R.; Llarrull, L. I.; Vila, A. J. Antibiotics, 2014, 3, 285-316) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 μM to 0.00063 μM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 0.082 nM (pChEMBL 10.09000015258789)
Descriptors: MW 494.56, ALogP -2, HBA 11, HBD 6, PSA 223.76, QED 0.21
Compound (SMILES): C=CC(=O)N[C@@H]1CCCC[C@H]1NC(=O)c1sc2nccc3c2c1NC(=O)N3c1ccc(OC(C)C)cc1C
InChIKey: RKBGIFAUPKEZCL-RTBURBONSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 533.65, ALogP 5.42, HBA 6, HBD 3, PSA 112.66, QED 0.35
Compound (SMILES): Cc1cnc(-c2ccc3cc(C(=O)Nc4ccnnc4)[nH]c3c2)nc1Nc1ccc2[nH]ncc2c1
InChIKey: IXLKJDOEKCNYGP-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Biological Assay: The kinase IC50 was determined by a commercialized CISBIO kinase detection kit, HTRF KinEASE-STK S2 kit (62ST2PEC). ROCK2 (01-119) employed in the reaction was purchased from Carna Biosciences.Before the assay, the following working solutions as needed were formulated with corresponding reagents according to the instruction of the kinase detection kit: 1×kinase buffer, 5×STK-52 substrate working solution (1.5 μM) and 5×ATP working solution (1.5 μM), 5×ROCK2 kinase working solution, 4×Streptavidin-XL665 working solution, and 4×STK-Ab-Cryptate 2 detection solution. Then the assay was performed according to the following procedure.A solution of a compound at a concentration of 10000 nM was prepared with the 1×kinase buffer containing 2.5% DMSO. Gradient dilution of the solution of the compound was performed with the kinase buffer containing DMSO, so as to obtain solutions of a test compound at 9 different concentrations. In addition to wells of test compounds, a positive well (containing all the reagents except the compound) and a negative well (containing all the reagents except the test compound and kinase) were set. Except for the control wells (positive and negative wells), a solution of a test compound (4 μL) was added to each of the reaction wells, and a solution of 2.5% DMSO was added to the control wells. Then the substrate (2 μM, i.e., 2 μL 5×STK-S2 substrate working solution) was added to each of the reaction wells. The 5×ROCK2 kinase working solution (2 μL, containing 1.4 ng ROCK2 kinase) was added to each of the reaction wells except for the negative well, the volume of which was made up with the 1×kinase buffer (2 μL). The 5×ATP working solution (2 μL) was added to each of the reaction wells, and the mixtures were incubated at room temperature for 2 hours. After the kinase reaction was complete, the 4×Streptavidin-XL665 working solution was added to each of the reaction wells, the solutions were mixed, followed by immediate addition of the 4×STK-Ab-Cryptate 2 detection solution (5 μL), and the mixtures were incubated at room temperature for 1 hour. The fluorescence signal was read on ENVISION (Perkinelmer) (excitation wavelength: 320 nm, and emission wavelength: 665 nm and 615 nm). The inhibitory rate in each well was calculated based on the fluorescence intensity value: ER (Emission Ratio)=(fluorescence intensity at 665 nm/fluorescence intensity at 615 nm); inhibitory rate=(ERpositive−ERtest compound) (ERpositive−ERnegative)*100%. Curves were plotted and fitted to obtain the median inhibitory concentration (IC50) of each teat compound with the PRISM 5.0 software.
Measurement: IC50 = 208 nM (pChEMBL 6.679999828338623)
Descriptors: MW 461.49, ALogP 4.6, HBA 7, HBD 4, PSA 137.16, QED 0.29
Compound (SMILES): CCP(=O)(CC)c1cc(C(C)(C)C)cc(NC(=O)Nc2ccc(Oc3ccnc(Nc4ccccc4)c3)c3ccccc23)c1OC
InChIKey: WCFDATPHRXKKJB-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: Enzyme Inhibition Assay: Method 1: The inhibitory activities of compounds of the invention against the GSK 3α enzyme isoform (Invitrogen), are evaluated by determining the level of activation/phosphorylation of the target peptide. The GSK3-α protein (500 ng/mL, 2.5 μL) is mixed with the test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptide (8 μM, 2.5 μL), which is a phosphorylation target for GSK3α, and ATP (40 μM, 2.5 μL) are then added to the enzyme/compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 > 14677 nM (pChEMBL 4.833000183105469)
Descriptors: MW 636.73, ALogP 9.75, HBA 6, HBD 3, PSA 101.58, QED 0.13
Compound (SMILES): O=c1[nH]c(CSC2CCOCC2)nc2cc(OCC3CCNCC3)cc(F)c12
InChIKey: YYQHGXMWWCGFNC-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Inhibition Assay: Enzymatic inhibition of PARP14 was measured using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) monitoring the auto-modification of PARP14 by biotinylated nicotinamide adenine dinucleotide (biotin-NAD). 1 μL of a dose response curve of each test compound was spotted in 384-well nickel-coated white microplates (Thermo) using a Mosquito (TTP Labtech). Reactions were performed in a 50 μL volume by adding 40 μL of PARP14 in assay buffer (20 mM HEPES pH==8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%0 Tween20), incubating with test compound at 25° C. for 30 min, then adding 10 μL of biotin-NAD (Biolog). The final concentrations of PARP14 and biotin-NAD are 50 nM and 3 μM, respectively. Reactions proceeded at 25° C. for 3 h, then were quenched with 5 μL of 10 mM unmodified nicotinamide adenine dinucleotide (Sigma-Aldrich). The quenched reactions were washed 3 times with 100 μL of TBST wash buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween20). Next, to the washed and dried plate was added 25 μL of DELFIA Europium-N1 streptavidin (Perkin Elmer) diluted in DELFIA assay buffer (Perkin Elmer). After a 30 min incubation at 25° C., the plate was washed 5 times with TBST wash buffer. Finally, 25 μL of DELFIA enhancement solution was added. After a 5 min incubation the plate was read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 340 nm and emission of 615 nm to measure the amount of Europium present in each well, informing on the amount of biotin-NAD that was transferred in the automodification reaction.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 407.51, ALogP 2.85, HBA 6, HBD 2, PSA 76.24, QED 0.77
Compound (SMILES): CC1COCCN1C(=O)c1ccc2c(c1)N(c1ncc(-c3ccccc3F)cn1)CC21CC1
InChIKey: RTCBEOJIEVNFNR-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: cAMP HTRF Assay to Determine the Activity of hPDE4B1: The inhibiting effect of the compounds on the enzyme activity of human PDE4B1 was measured by the quantification of 5′-adenosine monophosphate (5′-AMP), which is formed from 3′,5′-cyclic adenosine monophosphate (cAMP). Human recombinant enzyme, expressed in Sf9 cells, and the HTRF (homogeneous time-resolved fluorescence) detection method were used in the assay.The test compound or water (control) was mixed with the human recombinant PDE4B1 enzyme (4.8 U) in a buffer consisting of 44.4 mM tris-HCl, 5.28 mM MgCl2, 2.64 mM DTT and 0.044% Tween 20 (pH 7.8). After adding the cAMP enzyme substrate (final concentration 40 nM), the mixture was incubated for 30 minutes at room temperature. Then a fluorescence acceptor (Dye2 marked with cAMP), a fluorescence donor (anti-cAMP antibody marked with a europium cryptate) and the non-specific phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine; final concentration 1 mM) were added. After 60 min the fluorescence transfer, which correlates with the amount of remaining cAMP, was measured with a microplate reader (Rubystar, BMG) at λex=337 nm, λem=620 nm and λem=665 nm. The enzyme activity was calculated from the quotient formed from the measured signal at 665 nm and that at 620 nm. The result was expressed as the percentage inhibition of enzyme activity of the control (without PDE4 inhibitor). The enzyme was omitted for measurement of the basal control.[N. Saldou et al., Comparison of recombinant human PDE4 isoforms: interaction with substrate and inhibitors, Cell. Signal. Vol. 10, No. 6, 427-440, 1998].
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 444.51, ALogP 4.33, HBA 5, HBD 0, PSA 58.56, QED 0.6
Compound (SMILES): COC1(c2cccc(C(N)=O)c2)C2CCCC1CN(C1COC1)C2
InChIKey: WEEWGPJBNAQFDT-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Competitive Displacement Assay: Membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed one type of the cloned human opioid receptor were incubated with 12 different concentrations of the compound in the presence of 0.25 nM [3H]DAMGO, 0.2 nM [3H]naltrindole or 1 nM [3H]U69,593 in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO and [3H]U69,593. Because of a slower association of [3H]naltrindole with the receptor, a 3 h incubation was used with this radioligand. Samples incubated with [3H]naltrindole also contained 10 mM MgCl2 and 0.5 mM phenylmethylsulfonyl fluoride. Nonspecific binding was measured by inclusion of 10 μM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber filters using a Brandel 48-well cell harvester. The filters were subsequently washed three times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL Ecoscint A scintillation fluid. For [3H]naltrindole and [3H]U69,593 binding, the filters were soaked in 0.1% polyethylenimine for at least 60 min before use.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 330.43, ALogP 1.76, HBA 4, HBD 1, PSA 64.79, QED 0.91
Compound (SMILES): Cc1cc(Nc2nc(N[C@H]3CC[C@H](N)CC3)c3c(ccn3-c3cnn(C)c3)n2)sn1
InChIKey: QOJLZLOHLWMEDE-HDJSIYSDSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Activity Inhibition Assay: IRAK4 kinase (purchased from Life Technologies, Cat. No.: PR5612U) was diluted to 2 folds of the final concentration (the final concentration is 0.76 ng/4) with reaction buffer (40 mM Tris-HC1, pH 7.5; 20 mM MgCl2; 0.1 mg/mL BSA; 1 mM DTT), and added to a 384-well plate at 5 μL/well. The test drugs were 10-fold serial diluted from 10 μM to set six concentration points and added to the test wells of the 384-well plate at 2.5 μL/well. After incubation for 10 min at 25° C., 10 μM of ATP and 0.1 μg/μL of an enzyme reaction substrate, myelin basic protein MBP (purchased from Signal Chem, Cat. No.: M42-51N) were added at 2.5 μL/well, and reacted at 25° C. for 60 minutes. After the reaction was completed, the kinase activity assay was performed using the ADP-Glo kinase assay kit (purchased from Promega, Cat. No.: V9102) according to the manufacture's instruction, i.e., 10 μL of ADP-Glo reaction reagent was first added and reacted at 25° C. for 40 minutes, 10 μL of the reaction mixture was taken and mixed with 10 μL of ADP-Glo detection reagent, and reacted at 25° C. for 30 minutes.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 423.55, ALogP 3.34, HBA 10, HBD 3, PSA 111.5, QED 0.45
Compound (SMILES): CC1(C#N)CC(CC#N)(n2ncc(-c3cc(-c4c[nH]nc4N)cc4ncccc34)n2)C1
InChIKey: HZIMIOGOUOAKGP-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 844 nM (pChEMBL 6.070000171661377)
Descriptors: MW 409.46, ALogP 3.4, HBA 8, HBD 2, PSA 145.88, QED 0.52
Compound (SMILES): CC1(C)C(N)=N[C@](C)(c2cc(C(=O)Nc3ccc(C#N)cc3O)ccc2F)CS1(=O)=O
InChIKey: AFLCZLQKLWUSJR-NRFANRHFSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vivo Inhibition of Beta-Secretase: Several animal models, including mouse, rat, dog, and monkey, may be used to screen for inhibition of beta-secretase activity in vivo following administration of a test compound sample. Animals used in this invention can be wild type, transgenic, or gene knockout animals. For example, the Tg2576 mouse model, prepared and conducted as described in Hsiao et al., 1996, Science 274, 99-102, and other non-transgenic or gene knockout animals are useful to analyze in vivo inhibition of Amyloid beta peptide (Abeta) production in the presence of inhibitory test compounds. Generally, 2 to 18 month old Tg2576 mice, gene knockout mice or non-transgenic animals are administered test compounds formulated in vehicles, such as cyclodextran, phosphate buffers, hydroxypropyl methylcellulose or other suitable vehicles. One to twenty-four hours following the administration of compound, animals are sacrificed, and brains as well as cerebrospinal fluid (CSF) and plasma are removed for analysis of A-beta levels and drug or test compound concentrations (Dovey et al., 2001, Journal of Neurochemistry, 76, 173-181) Beginning at time 0, animals are administered by oral gavage, or other means of delivery such as intravenous injection, an inhibitory test compound of up to 100 mg/kg in a standard, conventional formulation, such as 2% hydroxypropyl methylcellulose, 1% Tween80. A separate group of animals receive 2% hydroxypropyl methylcellulose, 1% Tween80 alone, containing no test compound, and serve as a vehicle-control group. At the end of the test period, animals are sacrificed and brain tissues, plasma or cerebrospinal fluid are collected. Brains are either homogenized in 10 volumes (w/v) of 0.2% diethylamine (DEA) in 50 mM NaCl (Best et al., 2005, Journal of Pharmacology and Experimental Therapeutics, 313, 902-908), or in 10 volumes of 0.5% TritonX-100 in Tris-buffered saline (pH at about 7.6). Homogenates are centrifuged at 355,000 g, 4° C. for 30 minutes. CSF or brain supernatants are then analyzed for the presence of A-beta peptide by specific sandwich ELISA assays based on ECL (Electrochemiluminescence) technology.
Measurement: IC50 = 2290 nM (pChEMBL 5.639999866485596)
Descriptors: MW 444.49, ALogP 2.43, HBA 7, HBD 3, PSA 145.64, QED 0.62
Compound (SMILES): CC(CC1(C)NC(=O)NC1=O)C(=O)N1CCN(c2cccc(F)c2)[C@@H](C)C1
InChIKey: LJPINOUZBONPNV-MLZJXEJWSA-N
Target: A disintegrin and metalloproteinase with thrombospondin motifs 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UNA0
Assay: In Vitro Assay: ADAMTS-5:Protocol 2: The basis for the assay is the cleavage of the substrate TBIS-1 (5 FAM-TEGEARGSVILLK (5TAMRA)K-NH2) (SEQ ID No 2) by human ADAMTS-5.For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, 1/5 dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM Hepes pH7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS 1) containing hADAMTS-5 (1 ng/μL, affinity purified, followed by overnight digestion of 6His tag by thrombin and dialysis) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate TBIS-1 (10 μL, 4.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 45 min at 37° C. (Excitation 485 nm, emission 530).
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 376.43, ALogP 1.49, HBA 4, HBD 2, PSA 81.75, QED 0.78
Compound (SMILES): COc1cccc(-c2ccc(CNC(=O)[C@@H]3C[C@H]3c3ccccc3)c3c2CCN(C(=O)NC(C)(C)CC(C)(C)C)C3)c1
InChIKey: LINXUEWSEIEPQT-IOWSJCHKSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: Enzymatic Activity Assay: DGAT2 activity was determined by measuring the amount of enzymatic product triolein (1,2,3-Tri(cis-9-octadecenoyl)glycerol) using the membrane prep mentioned above. The assay was carried out in deep well 384 plates in a final volume of 40 μL at room temperature. The assay mixture contained the following: assay buffer (100 mM Tris.Cl, pH 7.0, 20 mM MgCl2, 5% ethanol), 25 μM of diolein, 10 μM of oleoyl-CoA and 10 ng/μL of DGAT2 membrane.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 567.77, ALogP 7.06, HBA 3, HBD 2, PSA 70.67, QED 0.31