Discovery and in vitro evaluation of potent kinase inhibitors: Pyrido[1′,2′:1,5]pyrazolo[3,4-d]pyrimidines

Bioorganic and Medicinal Chemistry Letters

Volumn 15, Issue 16, 2005, Pages 3778-3781

  Alberti, Michael J ^a   Auten, Elizabeth P ^a   Lackey, Karen E ^a   McDonald, Octerloney B ^a   Wood, Edgar R ^a   Preugschat, Frank ^a   Cutler, Geoffrey J ^a   Kane Carson, Laurie ^a   Liu, Wei ^a   Jung, David K ^a  

^a GLAXOSMITHKLINE   (United States)

Author keywords

EGFR; ErB; GSK; Kinase inhibitor; VEGFR

Indexed keywords

CEP 1347; GEFITINIB; IMATINIB; LAPATINIB; PHOSPHOTRANSFERASE INHIBITOR; PYRIMIDINE DERIVATIVE;

ARTICLE; DRUG BINDING; DRUG SCREENING; DRUG STRUCTURE; DRUG SYNTHESIS; IC 50; STRUCTURE ACTIVITY RELATION;

HUMANS; HYDROGEN BONDING; MOLECULAR STRUCTURE; PROTEIN CONFORMATION; PROTEIN KINASE INHIBITORS; PROTEIN KINASES; PROTEIN STRUCTURE, TERTIARY; PYRIMIDINES; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 22844452590     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2005.05.100     Document Type: Article

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14. Compounds were tested for EGFR or ErbB-2 protein tyrosine kinase inhibitory activity in substrate phosphorylation assays using enzymes purified from a baculovirus expression system. Reagent production and assay methodology were conducted essentially as described (Brignola, P. S. et al. J. Biol. Chem. 2002, 277, 1576).
15. 33P]ATP per reaction, 2 μM peptide substrate, and 1 mM dithiothreitol. Reactions were initiated by adding 1 pmol (20 nM) per reaction of the indicated enzyme. The reaction was allowed to proceed for 15 min, terminated, and quantified using a scintillation proximity assay procedure as described (McDonald, O. B., Antonsson, B., Arkinstal, S., Marshall, C. J., and Wood, E. R. Anal. Biochem. 1999, 268, 318).
16. 2. The reaction was allowed to proceed for 40 min. Product was detected using a homogeneous time-resolved fluorescence procedure (Park, Y.-W., Cummings, R. T., Wu, L., Zheng, S., Cameron, P. M., Woods, A., Zaller, D. M., Marcy, A. I., and Hermes, J. D. Anal. Biochem. 1999, 269, 94). Briefly, the reactions were quenched by adding 100 μl of 100 mM HEPES, pH 7.5, 100 mM EDTA, 45 nM streptavidin-linked allophycocyanin (Molecular Probes, Eugene, OR), and 3 nM europium-conjugated anti-phosphotyrosine antibody (Wallac, Turku, Finland). The product was detected using a Victor plate reader (Wallac, Turku, Finland) with a time delay at 665 nm.
17. 33P labeled ATP, and 1.2 μg/mL GSK-3b protein. The plates are incubated at room temperature for 19 min prior to the addition of SPA stop solution.