Enzymatic Characterization of c-Met Receptor Tyrosine Kinase Oncogenic Mutants and Kinetic Studies with Aminopyridine and Triazolopyrazine Inhibitors

酶动力学 自磷酸化 突变体 激酶 受体酪氨酸激酶 生物化学 化学 背景(考古学) 酪氨酸激酶 生物 分子生物学 受体 活动站点 蛋白激酶A 基因 古生物学
作者
Sergei Timofeevski,Michele McTigue,Kevin Ryan,Jean Cui,Helen Y. Zou,Jeff Zhu,Fannie Chau,Gordon Alton,Shannon Karlicek,James G. Christensen,Brion W. Murray
出处
期刊:Biochemistry [American Chemical Society]
卷期号:48 (23): 5339-5349 被引量:95
标识
DOI:10.1021/bi900438w
摘要

The c-Met receptor tyrosine kinase (RTK) is a key regulator in cancer, in part, through oncogenic mutations. Eight clinically relevant mutants were characterized by biochemical, biophysical, and cellular methods. The c-Met catalytic domain was highly active in the unphosphorylated state (kcat = 1.0 s−1) and achieved 160-fold enhanced catalytic efficiency (kcat/Km) upon activation to 425000 s−1 M−1. c-Met mutants had 2−10-fold higher basal enzymatic activity (kcat) but achieved maximal activities similar to those of wild-type c-Met, except for Y1235D, which underwent a reduction in maximal activity. Small enhancements of basal activity were shown to have profound effects on the acquisition of full enzymatic activity achieved through accelerating rates of autophosphorylation. Biophysical analysis of c-Met mutants revealed minimal melting temperature differences indicating that the mutations did not alter protein stability. A model of RTK activation is proposed to describe how a RTK response may be matched to a biological context through enzymatic properties. Two c-Met clinical candidates from aminopyridine and triazolopyrazine chemical series (PF-02341066 and PF-04217903) were studied. Biochemically, each series produced molecules that are highly selective against a large panel of kinases, with PF-04217903 (>1000-fold selective relative to 208 kinases) being more selective than PF-02341066. Although these prototype inhibitors have similar potencies against wild-type c-Met (Ki = 6−7 nM), significant differences in potency were observed for clinically relevant mutations evaluated in both biochemical and cellular contexts. In particular, PF-02341066 was 180-fold more active against the Y1230C mutant c-Met than PF-04217903. These highly optimized inhibitors indicate that for kinases susceptible to active site mutations, inhibitor design may need to balance overall kinase selectivity with the ability to inhibit multiple mutant forms of the kinase (penetrance).
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