Biochemical and cellular effects of c-Src kinase-selective pyrido[2,3- d]pyrimidine tyrosine kinase inhibitors

Abstract

Increased expression or activity of c-Src tyrosine kinase has been associated with the transformed phenotype in tumor cells and with progression of neoplastic disease. A number of pyrido[2,3- d]pyrimidines have been characterized biochemically and in cells as part of an assessment of their potential as anti-tumor agents. The compounds were ATP-competitive inhibitors of c-Src kinase with ic 50 values < 10 nM and from 6 to >100-fold selectivity for c-Src tyrosine kinase relative to basic fibroblast growth factor receptor (bFGFr) tyrosine kinase, platelet-derived growth factor receptor (PDGFr) tyrosine kinase, and epidermal growth factor receptor (EGFr) tyrosine kinase. The compounds yielded ic 50 values < 5 nM against Lck. Human colon tumor cell growth in culture was inhibited, as was colony formation in soft agar at concentrations < 1 μM. Phosphorylation of the c-Src cellular substrates paxillin, p130 cas, and Stat3 was also inhibited at concentrations < 1 μM. Autophosphorylation of EGFr tyrosine kinase or PDGFr tyrosine kinase was not inhibited by c-Src inhibitors, thus showing the selective nature of the compounds in cells. In a mitogenesis assay measuring thymidine incorporation stimulated by specific mitogens, the c-Src tyrosine kinase inhibitors reduced incorporated thymidine in a manner consistent with previously reported roles of c-Src in mitogenic signaling. Progression through the cell cycle was inhibited at G 2/M in human colon tumor cells treated with two of the c-Src-selective compounds, which is also consistent with earlier reports describing a requirement for active c-Src tyrosine kinase for G 2 to M phase progression. The compounds described here are selective inhibitors of c-Src tyrosine kinase and have antiproliferative effects in tumor cells consistent with inhibition of c-Src.