lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1c(C(=O)C(=O)NC2(C#N)CC(F)(F)C2)c2n(c1C(=O)Nc1ccc(F)c(Cl)c1)CCC2
InChIKey: BTHWSSNMFYMFFF-UHFFFAOYSA-N
Target: Hepatic sodium/bile acid cotransporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q14973
Assay: HBV DNA Quantification Assay: A HepG2 cell line overexpressing the HBV virus attachment receptor sodium-taurocholate cotransporting polypeptide (NTCP) was grown to confluency in DMEM growth medium, Dulbecco's Modified Eagle Medium without sodium pyruvate (Life Technologies, Rockville, Md.) supplemented with 10% FBS (Thermo Scientific, Waltham, Md.), 1% penicillin/streptomycin (Life Technologies, Rockville, Md.) and 2 mM L-glutamine (Life Technologies, Rockville, Md.) in T175 flasks. Cells were infected with HBV AD38 viral particles (Texcell, Frederick, USA) at 4000 genome equivalents per cell. After allowing viral infection to take place for 4 days, the infected cells were harvested from the flasks by trypsinization, washed twice with OptiMEM (Life Technologies, Rockville, Md.) and re-suspended in DMEM containing 2% FBS and 1% DMSO at a density of 0.25E6 cells/ml. Infected cells were seeded on 384 well collagen coated plates (Greiner, Austria) at a density of 20,000 cells/well containing serially diluted compounds of the present disclosure or DMSO (0.5%) in a final volume of 80 μl. The assay plates were incubated for a period of 5 days and the antiviral activity of the test compounds were assayed by detecting the presence of HBV DNA in the culture supernatant using the QuantiGene 2.0 nucleic acid quantification kit (Affymetrix, Santa Clara, Calif.).The culture supernatant was harvested and treated with lysis buffer containing Proteinase K (Affymetrix, Santa Clara, Calif.). The supernatant was incubated with HBV viral DNA specific probes (Affymetrix, Santa Clara, Calif.) for 30 minutes at 55° C. This was followed by addition of 0.2M NaOH for 30 minutes at room temperature to denature the DNA, followed by addition of Neutralization buffer (Affymetrix, Santa Clara, Calif.). The resulting lysed and neutralized supernatant was then added to QuantiGene 2.0 384 well plates coated with capture oligonucleotides and incubated overnight at 55° C. The HBV specific probe set consists of Capture Extender oligonucleotides (CE's) and blocking probes. Following the overnight incubation, the wells were incubated for one hour sequentially with a Pre-Amplifier, Amplifier and Labeled probes conjugated to alkaline phosphatase with a wash step between incubations. After the final wash step, the alkaline phosphatase substrate (Luminol APS5) was added and the resulting luminescence signal was read in an EnVision Multilabel Plate Reader (PerkinElmer, Santa Clara, Calif.). The EC50 values were calculated from the fit of the doseresponse curves to a four-parameter equation. All EC50 values represent geometric mean values of a minimum of four determinations.
Measurement: EC50 = 10.4 nM (pChEMBL 7.980000019073486)
Descriptors: MW 478.86, ALogP 3.78, HBA 5, HBD 2, PSA 103.99, QED 0.5
Compound (SMILES): CN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(c1cnc(-c3ccc(C(N)=O)cc3)nc1)C(=O)N2
InChIKey: AXYZLKZNDFKJEW-MKPDMIMOSA-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 β-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 470.58, ALogP 3.54, HBA 5, HBD 2, PSA 104.45, QED 0.59
Compound (SMILES): COCCOCCOCCOCCOCCOCCOCCNc1ccc(S(=O)(=O)N(CC(C)C)CC(O)[C@H](Cc2ccccc2)NC(=O)O[C@H]2CO[C@H]3OCC[C@H]32)cc1
InChIKey: AAJXKCGGRTZZCZ-YRYHPRDQSA-N
Target: Protease (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: Q9YQ12
Assay: Enzyme Inhibition of Purified HIV-1 Protease: Protease inhibitors (PI) are used to treat HIV infection by preventing viral assembly and maturation through the inhibition of HIV-1 protease activity. Biochemical assays were performed to evaluate the potential of PEG-PI conjugates to inhibit HIV-1 protease activity, relative to their respective PI parent molecules. Activity assays were performed at using the SensoLyte 520 HIV-Protease Assay Kit (Anaspec Inc., San Jose, Calif.) and recombinant HIV-1 protease. Protease activity was monitored by the formation of a fluorescent reporter product generated during HIV-1 protease-mediated digestion of a quenched, fluorimetric substrate containing the p17/p24 Prgag cleavage site.
Measurement: IC50 = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 870.07, ALogP 2.95, HBA 15, HBD 3, PSA 191.04, QED 0.09
Compound (SMILES): O=C(CCCCCCCN(c1ccc(F)cn1)c1nc2ccccc2o1)NO
InChIKey: FQNMAWUQAQHBRY-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC inhibitory assay: The assay is described in WO2008/062201.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 386.43, ALogP 4.35, HBA 6, HBD 2, PSA 91.49, QED 0.31
Compound (SMILES): C=CC(=O)N1C2CCC1[C@@H](c1nc(-c3ccc(C(=O)Nc4ccccn4)cc3)c3c(N)nccn13)C2
InChIKey: SXNGBNLNYTZLCG-GPJFCIFZSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro Assay for the Inhibition of BTK Kinase Activity: 1: Test Principle:Mobility-Shift Assay, which is microfluidic chip technology, applies the basic concept of capillary electrophoresis to microfluidic environments. The substrate used for the experiment is a polypeptide with a fluorescent label. Under the action of enzyme in the reaction system, the substrate is transformed into a product, and the charge the substrate carries also changes accordingly. The use of the charge difference between the substrate and the product involved in Mobility-Shift Assay achieves the separating of the two, and they are tested respectively. The test results are expressed by conversion rates.2: Test Method:(1) preparation of samples to be tested: diluted with 100% DMSO to 50 times the final concentration of the reaction, i.e. 25 μmol/L;(2) dilution: 25 μmol/L is the initial concentration, then diluted with 4 times the concentration and diluted with 10 concentration gradients;(3) 100% DMSO was added to the positive control well and the negative control well, respectively;(4) the prepared compounds with 10 concentrations were diluted 10-fold with 1 xkinase buffer, respectively; wherein, the kinase buffer contained hydroxyethyl piperazine ethanesulfonic acid at a concentration of 50 mmol/L and a pH of 7.5, 0.01% dodecyl polyethylene glycol ether, 10 mmol/L magnesium chloride, 2 mmol/L dithiothreitol;(5) preparation of 2.5×enzyme solution: the kinase was added to 1×kinase buffer to form 2.5×enzyme solution;(6) preparation of 2.5×substrate solution: FAM-labeled polypeptide and ATP were added to the 1 xkinase buffer to form 2.5×substrate solution; (7) addition of the enzyme solution to the 384-well plate: 5 μl of 5×compound dissolved in 10% DMSO contained in the 384-well reaction plate, then 10 μl of 2.5×enzyme solution was added, the obtained system was incubated for 10 minutes at room temperature;(8) addition of the substrate solution to the 384-well plate: 10 μl of 2.5×substrate solution was added to the 384-well reaction plate;(9) kinase reaction and termination: after incubation at 28° C. for 1 hour, 25 μl stop solution was added to terminate the reaction; wherein, the stop solution contained hydroxyethyl piperazine ethanesulfonic acid at a concentration of 100 mmol/L and a pH of 7.5, 0.015% dodecyl polyethylene glycol ether, 0.2% surface reagent No. 3, 20 mmol/L ethylenediaminetetraacetic acid; (10) Caliper data reading: conversion rate data was read from Caliper.
Measurement: IC50 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 479.54, ALogP 3.66, HBA 7, HBD 2, PSA 118.51, QED 0.42
Compound (SMILES): CCOc1nn(CC)cc1S(=O)(=O)N1c2cc(NC(=O)OC(C)(C)C(F)(F)F)ccc2O[C@@H](CNC(=O)[C@@H]2C[C@@H]2C#N)[C@H]1C
InChIKey: AWIRCPXKCQSZKJ-PZPFTHLGSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biochemical TR-FRET assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., a biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 μg/mL bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per μl in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 μl SF9 lysate per 15 μl of diluted RORγ-LBD.
Measurement: IC50 = 21.7 nM (pChEMBL 7.659999847412109)
Descriptors: MW 642.66, ALogP 3.81, HBA 10, HBD 2, PSA 164.88, QED 0.39
Compound (SMILES): CN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(c1cnc(N3CCCC3)nc1)C(=O)N2
InChIKey: QTCQAFPZJSXNIS-PSWAGMNNSA-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 β-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 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 420.56, ALogP 3.38, HBA 5, HBD 1, PSA 64.6, QED 0.82
Compound (SMILES): Cc1ccc(S(=O)(=O)NC23CCC(O)(C2)C3)cc1-c1cnc(N)c(-n2cncn2)n1
InChIKey: MZNMXJRWFYYHMO-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI3Kgamma Scintillation Proximity Assay: γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand island, NY), ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 427.49, ALogP 0.95, HBA 9, HBD 3, PSA 148.91, QED 0.54
Compound (SMILES): N#CC[C@H]1CC[C@H](n2/c(=N/C#N)[nH]c3cnc4ccsc4c32)CC1
InChIKey: OSYAMACCIMLMPP-HAQNSBGRSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), JAK2 (a.a. 828-1132) and JAK3 (a.a. 781-1124) were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 μL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hour and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a PHERA star plate reader (BMG, Cary, N.C.).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 336.42, ALogP 3.61, HBA 6, HBD 1, PSA 93.55, QED 0.72
Compound (SMILES): CNc1cnc2c(c1)ncn2Cc1cc(OC)c2c(c1)OCC(c1ccc(OC(F)F)cc1)O2
InChIKey: LWMIKFDFFMQGQS-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: In Vitro Studies: All assay reaction conditions for IC50 determinations were within the linear range with respect to time and enzyme concentration. In a 384 well polypropylene plate, c-FMS (0.14 nM, Carna 08-155) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 μM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 70 pM c-FMS, 1.5 μM peptide substrate and 500 μM ATP (ATP Km). The reaction was incubated at room temperature for 120 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 468.46, ALogP 4.64, HBA 8, HBD 1, PSA 79.66, QED 0.42
Compound (SMILES): Oc1nc(-c2ccccn2)nc2sccc12
InChIKey: CBIDWCOAELWUSK-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Inhibition Assay: Buffer-I:    50 mM Tris, pH 7.5    150 mM NaCl (optional)    1 mM MgCl2    1 mM DTT.    KRas G12D mutant protein was expressed as a His-tagged protein. Purified His-KRas G12D protein was diluted in buffer-I to a final concentration of 3-10 μg/ml.    200 μl of the diluted His-KRas G12D protein was added to a nickel coated 96 well plate and incubated overnight at 4° C.    The next day, wells were washed 3× in 200 μl of Buffer-I.    Then 200 μl of Buffer-I were added to each well in the presence of 1% DMSO.    Tested compounds were added to the protein-coated wells at a concentration of 20 μM, and incubated for 3 hours at room temperature. While performing IC50 measurements a serial dilution of all tested concentrations was prepared.Then 22 μl of Cy3-GTP or Cy5-GTP was added to each well. The labeled GTP was incubated for 45 min. at room temperature.    Following GTP incubation, wells were washed 3× in Buffer-I, and 200 μl of Buffer-I were added to each well.    Following washes, the amount of bound labeled-GTP was measured with an Eppendorf AF2200 plate reader.
Measurement: IC50 = 20500 nM (pChEMBL 4.690000057220459)
Descriptors: MW 229.26, ALogP 2.46, HBA 5, HBD 1, PSA 58.9, QED 0.7
Compound (SMILES): CCOc1nc2ccc(OC[C@H]3C[C@@H](OC[C@H](C)NC(C)=O)C3)nc2n1C
InChIKey: UWFZHERWMZROAU-AEGPPILISA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 315 nM (pChEMBL 6.5)
Descriptors: MW 376.46, ALogP 2.07, HBA 7, HBD 1, PSA 87.5, QED 0.72
Compound (SMILES): C[C@@H]1[C@H](c2nc(-c3ccc(C(=O)Nc4cc(C(F)(F)F)ccn4)cc3)c3c(N)nccn23)CCCN1C(=O)C1(C)COC1
InChIKey: WTKAFKUTHUGWNG-DYESRHJHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay B: BTK enzymatic activity was determined in an IMAP-FP assay (Immobilized Metal ion Affinity-based fluorescence Polarization; Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, final compound concentration range in assay from 10 μM to 0.508 nM) titration curve using the following outlined procedure. To each well of a black Corning 384-well microplate (Corning Catalog #3575), 200 nL of compound (100 fold dilution in final assay volume of 20 μL) was dispensed, followed by the addition of 10 μL of 1× kinase buffer (10 mM Tris pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM MnCl2, and 1 mM DTT) containing 0.16 ng/μL (2.1 nM) 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 10 μL kinase buffer containing 100 nM Blk/Lyntide IMAP substrate peptide (5-carboxyfluorescein-EFPIYDFLPAKKK-NH2; Molecular Devices Catalog #R7188), and 10 μM ATP. The final reaction in each well of 20 μL consists of 1.05 nM hBTK, 50 nM Blk/Lyntide IMAP substrate, and 5 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes and were immediately quenched by the addition of 40 μL IMAP detection beads resuspended in IMAP progressive binding buffer (beads were diluted 1:600 in 75% and 25% progressive binding buffer A and B, respectively; Molecular Devices). Plates were read on Packard microplate reader after 60 minutes binding equilibration using Fluorescence Polarization protocol. Specifically, fluorescence at 535 nm is measured using emission filters oriented both parallel and perpendicular to the polarized excitation filter. This allows detection of changes in Fluorescence Polarization due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmP) of the controls with and without hBTK enzyme. IC50 values are determined by curve fitting of the experimental results using Activity Base.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 593.61, ALogP 4.78, HBA 8, HBD 2, PSA 127.74, QED 0.34
Compound (SMILES): COc1ccc(C(=O)Nc2ccc(O)cn2)cc1
InChIKey: AWLCYIIVQLVZIV-UHFFFAOYSA-N
Target: Sphingolipid delta(4)-desaturase DES1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15121
Assay: Des1 Activity Assays: Jurkat clone E6-1 cells were grown and then seeded at 106 cells/mL in a 96-well plate (400 μL in each well. The cells were administered 100 μL of cell culture media containing 50-μM NBD-C6-dihydroceramide (Des1 substrate), affording a final concentration of substrate of 10 μM. The cells were incubated with substrate at 4° C. for 30 minutes. Following the incubation at 4° C., the cell suspension was centrifuged at 1200 rpm for 3 minutes, and the cell pellet is resuspended in 400 μL of fresh media containing various concentrations of either fenretinide (known Des1 inhibitor control compound) or test article. The final concentrations of control compound and test compounds were tested in a range from 0-10 μM. The cells and compounds were incubated at 37° C. for 3 hours. Following the 3-hour incubation, the plate was centrifuged at 2500 g for 3 minutes at 4° C., followed by collection and transfer of 200 μL of the supernatant to a new 96-well plate with 300 μL of methanol an containing appropriate internal standards for liquid chromatography/tandem mass spectroscopy (LC/MS/MS) analysis (internal standard: 500 nM labetalol and 100 nM alprazolam). The samples were vortexed for 2 minutes followed by centrifugation at 3,220 g for 20 minutes. Following centrifugation, 200 μL of the supernatant was transferred to a new 96-well plate for LC-MS/MS analysis to determine the amount of NBD-C6-ceramide (Des1 product) produced. The assay was typically performed in duplicate. A reduction of at least 30% compared to vehicle control (0 μM test article) is indicative of an active compound, and a reduction of 75% compared to vehicle control is preferred.
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 244.25, ALogP 2.05, HBA 4, HBD 2, PSA 71.45, QED 0.87
Compound (SMILES): CCCS(=O)(=O)NC1CN(c2cc3c(cc2F)OC[C@@H](NC)[C@@H]3Cc2ccccc2)C1
InChIKey: NMQLFRQXUMLYPW-WIYYLYMNSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: 1. [3H]-Glycine uptake into recombinant CHO cells expressing human GlyT1: Human GlyT1c expressing recombinant hGlyT1c_5_CHO cells were plated at 20,000 cells per well in 96 well Cytostar-T scintillation microplates (Amersham Biosciences) and cultured to sub-confluency for 24 h. For glycine uptake assays the culture medium was aspirated and the cells were washed once with 100 μl HBSS (Gibco BRL, #14025-050) with 5 mM L-Alanine (Merck #1007). 80 μl HBSS buffer were added, followed by 10 μl inhibitor or vehicle (10% DMSO) and 10 μl [3H]-glycine (TRK71, Amersham Biosciences) to a final concentration of 200 nM for initiation of glycine uptake. The plates were placed in a Wallac Microbeta (PerkinElmer) and continuously counted by solid phase scintillation spectrometry during up to 3 hours. Nonspecific uptake was determined in the presence of 10 μM Org24598. IC50 calculations were made by four-parametric logistic nonlinear regression analysis (GraphPad Prism) using determinations within the range of linear increase of [3H]-glycine incorporation between 60 and 120 min.2. Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1:Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 447.58, ALogP 2.65, HBA 5, HBD 2, PSA 70.67, QED 0.65
Compound (SMILES): COc1cc2ncnc(Oc3ccc(NC(=O)Nc4ccccc4CN4CCN(C)CC4)c(Cl)c3)c2cc1OC
InChIKey: SDXJBVJNXXJYRE-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Binding Assay: A competition binding assay (DISCOVERX KINOMESCAN) was used to measure the ability of a compound to compete for binding of an immobilized adenosine triphosphosphate (ATP) site directed ligand using a DNA-tagged vascular endothelial growth receptor 2 (VEGFR2) as the target. The ability of the test compound to compete with the immobilized ligand was measured using quantitative polymerase chain reaction (qPCR) of the DNA tag (Fabian, M. A. et al., 23 Nature Biotechnology 329-336 (2005); Karaman, M. W. et al., 26 Nature Biotechnology 127-132 (2008)).A VEGFR2 tagged T7 phage strain was prepared in an Escherichia coli (E. coli) derived from the BL21 strain. The E. coli were grown to log-phase, infected with VEGFR2 tagged T7 phage and then incubated with shaking at 32° C. until lysis. The lysate containing the kinase was then centrifuged and filtered to remove cell debris. Affinity resin for the VEGFR2 assay was prepared by treating Streptavidin-coated magnetic beads with a biotinylated small molecule ligand for 30 minutes at room temperature. The beads were blocked with excess biotin and then washed with blocking buffer (SEABLOCK (PIERCE), 1% bovine serum albumin, 0.17% phosphate buffered saline, 0.05% TWEEN 20, 6 mM dithiothreitol). The binding reaction was initiated by combining in a well of a polystyrene 96-well plate, DNA tagged VEGFR2, liganded affinity beads and the serial diluted test compound in 1× binding buffer (20% SEABLOCK, 0.17× phosphate buffered saline, 0.05% TWEEN 20, 6 mM dithiothreitol) in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and then the beads were washed with wash buffer (1× phosphate buffered saline, 0.05% TWEEN 20). The beads were re-suspended in elution buffer (1× phosphate buffered saline, 0.05% TWEEN 20, 0.05 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The VEGFR2 concentration in the eluate was measured using qPCR.An 11-point dose response curve of 3-fold serial diluted test compound starting at 1 μM was used to determine the VEGFR2 binding constant (Kd). The compounds were prepared in 100% DMSO at 100× the final test concentration and the diluted to 1× in the assay for final DMSO concentration of 1%. Binding constants were calculated with standard dose-response curve using the Hill equation with Hill slope set to −1. Curves were fit using a non-linear least square fit with the Levenberg-Marquardt algorithm.
Measurement: Kd = 27000 nM (pChEMBL 4.570000171661377)
Descriptors: MW 563.06, ALogP 5.48, HBA 8, HBD 2, PSA 101.08, QED 0.29
Compound (SMILES): O=C(NCc1cccnc1)Nc1ccc(S(=O)(=O)Nc2ccccc2OC(F)(F)F)cc1
InChIKey: WGGALYQNERJVNI-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100× stock. Buffer A (89 μL) containing 33 nM of NAMPT protein was added to 1 μL of 100× compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 μL of 10× substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 μM NAM, 100 μM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5′-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 μL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 μM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 466.44, ALogP 4.1, HBA 5, HBD 3, PSA 109.42, QED 0.49
Compound (SMILES): Cc1c(C#N)ccnc1O[C@@H]1CC[C@@H](C)N(C(=O)c2c(F)cccc2-c2cscn2)C1
InChIKey: FVCYMLUEJSZXEI-RHSMWYFYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand binding assay: Radioligand binding assay (described in Bergman et. al. Bioorg. Med. Chem. Lett. 2008, 18, 1425-1430) in which the inhibition constant (Ki) is determined in membranes prepared from CHO cells expressing either the OX1 or OX2 receptor. The intrinsic orexin receptor antagonist activity of a compound which may be used in the present invention may be determined by these assays.
Measurement: Ki = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 436.51, ALogP 4.6, HBA 6, HBD 0, PSA 79.11, QED 0.6
Compound (SMILES): N#Cc1ccc(N2CCN(C(=O)C[C@H]3CC[C@@H]([C@@H]4CCCN4c4cn[nH]c(=O)c4C(F)(F)F)O3)CC2)nc1
InChIKey: QQILLBXEPQCETH-HBFSDRIKSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 531.54, ALogP 2.31, HBA 8, HBD 1, PSA 118.45, QED 0.62
Compound (SMILES): CCn1c(=O)[nH]/c(=N\c2ccc(Oc3ccc(F)c(C(=O)OC)c3)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: QNMXCCSTIADWRY-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 524.94, ALogP 4.01, HBA 8, HBD 1, PSA 107.68, QED 0.37
Compound (SMILES): COc1ccnc(Cn2c(=O)[nH]c3ncc(-c4ccc(C(C)(C)C)cc4)cc32)c1OC
InChIKey: LIMSLWSHINXEKC-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: HTS cell-based calcium flux assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 (HTS assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 > 29000 nM (pChEMBL 4.538000106811523)
Descriptors: MW 418.5, ALogP 4.15, HBA 6, HBD 1, PSA 82.03, QED 0.53
Compound (SMILES): C[C@H](CN)NS(=O)(=O)c1ccc(N2CCC(CCN)CC2)c(-c2nn[nH]n2)c1S(N)(=O)=O
InChIKey: DJDJBTVGPINQTN-LLVKDONJSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Enzyme Activity: Determination of IC50 : 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) 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 = 0.33 nM (pChEMBL 9.479999542236328)
Descriptors: MW 487.61, ALogP -1.3, HBA 10, HBD 5, PSA 216.07, QED 0.28
Compound (SMILES): CCNC(=O)c1noc(-c2cc(-c3ccc(F)cc3)c(O)cc2O)c1-c1ccc(F)cc1
InChIKey: FVSQNSXKZKWXDX-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Fluorescence Polarization Assay: Fluorescence polarization {also known as fluorescence anisotropy} measures the rotation of a fluorescing species in solution, where the larger molecule the more polarized the fluorescence emission.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 436.41, ALogP 5.11, HBA 5, HBD 3, PSA 95.59, QED 0.4
Compound (SMILES): Cc1c(C(=O)CN2CCCCO2)c2ncc(Br)cc2n1-c1ccc(C#N)cc1
InChIKey: MCEGSFZBNMYZOP-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: P54578
Assay: USP14 Inhibition Assay: Using previously described methodology [B. H. Lee et al. Nature 2010, 467 (9), 179, the contents of which are expressly incorporated by reference herein], select compounds described herein were found to inhibit USP14.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 439.31, ALogP 4.18, HBA 6, HBD 0, PSA 71.15, QED 0.57
Compound (SMILES): COCCN1C[C@@H](NC(=S)Nc2c(C)c(-c3ccc(=O)n(C)c3)nn2-c2ccccc2)[C@H](c2ccc(F)c(F)c2)C1
InChIKey: DJYTZWYPLNYGCM-JYFHCDHNSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 578.69, ALogP 4.23, HBA 7, HBD 2, PSA 76.35, QED 0.3
Compound (SMILES): CN(C)C(=O)CN1CCN(c2cc(-n3ncc4ccc(-c5cnn(C)c5)cc43)ccn2)CC1
InChIKey: QYYZAYNJGFOJLJ-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen Assay: LRRK2 Km ATP LanthaScreen Assaya) 400 nl of a 1:2.15 serial dilution of test compound (98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO.b) 10 μl of a 2.5 nM LRRK2 (G2019S mutation, GST-LRRK2 (amino acids 970-2527)) enzyme solution in 1× assay buffer (50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells.c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 186 μM ATP solution in 1× assay buffer to all wells.d) After a 60 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer (Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells.e) Plates are incubated at room temperature for 1 hour and read on an Envision multi-mode plate reader with LanthaScreen settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 444.54, ALogP 2.03, HBA 8, HBD 0, PSA 75.32, QED 0.47
Compound (SMILES): Cc1c(F)cc(C(=O)NC2CC2)cc1-c1ccc(C(=O)NCC(C)C)nc1
InChIKey: HAGQIVNMNILOER-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: Assay of p38 Kinase Activity: Experimental Method: 40 nM p38a was added into a reaction buffer, which include 20 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), 5 mM MgCl2 and 1 mM DTT (dithiothreitol) at pH 7.4. 40 μM phosphorylated peptide substrate was added, followed by different concentrations of drugs and 100 μM ATP (adenosine triphosphate), and reacted for 90 minutes in dark. The fluorescence values were determined at excitation and emission wavelengths of 544 nM and 590 nM respectively. The IC50 values of the compounds were obtained according to the fluorescence values.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 369.44, ALogP 3.47, HBA 3, HBD 2, PSA 71.09, QED 0.82
Compound (SMILES): FC(F)(F)c1ccc(CCN2CCC(Nc3ncnc4[nH]ccc34)C(F)(F)C2)cn1
InChIKey: BYPNHXZTICELBB-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: NR2B Antagonist Activity: HEK293 cell lines stably expressing cloned human NR1/NR2B and NR1/NR2A, respectively, were established according to standard previously described methods (Hansen et al., Comb. Chem High Throughput Screen. 11:304, 2008). Activation of the NR2A or NR2B subtype of NMDA receptor with glutamate as an agonist and glycine co-agonist on these cells results in calcium influx, which can be monitored with fluorescent indicator Fluo-4. A cell based assay has been implemented to evaluate the effect of a compound on NR2A and NR2B receptors by measuring the fluorescent changes (Hansen et al., Comb. Chem High Throughput Screen. 11:304, 2008).HEK293 cells stably expressing NR2A or NR2B receptors were cultured at 37° C. in a humidified CO2 incubator in DMEM supplemented with 10% fetal bovine serum (FBS) (Hyclone), 10 μM MK801 (Sigma-Aldrich) and 50 μM AP-5 (Tocris). For experiments, the cells were seeded onto poly-D-lysine-coated 96-well black plates with clear bottom (Corning) at a density of 50,000 cells/well. After overnight culture, the growth medium was removed from the wells and the cells were incubated at 37° C. for 60 min in Hanks buffer containing 4 μM fluo-4-AM (Invitrogen) and 0.1% bovine serum albumin (BSA). After dye-loading, the cells were washed three times with Hanks buffer and incubated for 10 min at room temperature with various concentrations of test compounds prepared in Hanks buffer with 0.1% BSA. The cell plates were placed onto FDSS μCell fluorescence reader (Hamamatsu). After 20 sec reading of background fluorescence, agonist glutamate at final 100 μM and co-agonist glycine at final 50 μM were added to the cells to activate the receptor, and the resulting fluorescence changes were recorded and quantified. Based on the changes in fluorescence intensity, the pharmacological effect of test compounds were analyzed and the IC50 values derived from a non-linear least squares fitting of the concentration-dependent response to a standard logistic equation using Prism (Graphpad, Inc):Amplitude=Max Amplitude/(1+(IC50/[antagonist])n).
Measurement: IC50 = 1740 nM (pChEMBL 5.760000228881836)
Descriptors: MW 426.39, ALogP 3.74, HBA 5, HBD 2, PSA 69.73, QED 0.61