Timofeevski Sergei L, McTigue Michele A, Ryan Kevin, Cui Jean, Zou Helen Y, Zhu Jeff X, Chau Fannie, Alton Gordon, Karlicek Shannon, Christensen James G, Murray Brion W
Pfizer Global Research and Development, La Jolla, Pfizer Inc., 10777 Science Center Drive, San Diego, California 92121, USA.
Biochemistry. 2009 Jun 16;48(23):5339-49. doi: 10.1021/bi900438w.
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 (k(cat) = 1.0 s(-1)) and achieved 160-fold enhanced catalytic efficiency (k(cat)/K(m)) upon activation to 425000 s(-1) M(-1). c-Met mutants had 2-10-fold higher basal enzymatic activity (k(cat)) 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 (K(i) = 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).
c-Met受体酪氨酸激酶（RTK）是癌症中的关键调节因子，部分原因是通过致癌突变。通过生化、生物物理和细胞方法对8种临床相关突变体进行了表征。c-Met催化结构域在未磷酸化状态下具有高活性（k(cat)=1.0 s(-1)），激活后催化效率提高160倍（k(cat)/K(m)），达到425000 s(-1) M(-1)。c-Met突变体的基础酶活性（k(cat)）高2-10倍，但除Y1235D外，其最大活性与野生型c-Met相似，Y1235D的最大活性有所降低。基础活性的小幅增强对通过加速自磷酸化速率获得的完全酶活性有深远影响。对c-Met突变体的生物物理分析显示，解链温度差异最小，表明这些突变未改变蛋白质稳定性。提出了一个RTK激活模型，以描述RTK反应如何通过酶学特性与生物学背景相匹配。研究了来自氨基吡啶和三唑并吡嗪化学系列的两种c-Met临床候选药物（PF-02341066和PF-04217903）。从生化角度来看，每个系列产生的分子对大量激酶具有高度选择性，PF-04217903（相对于208种激酶的选择性>1000倍）比PF-02341066更具选择性。尽管这些原型抑制剂对野生型c-Met具有相似的效力（K(i)=6-7 nM），但在生化和细胞环境中评估的临床相关突变体的效力存在显著差异。特别是，PF-02341066对Y1230C突变体c-Met的活性比对PF-04217903高180倍。这些高度优化的抑制剂表明，对于易发生活性位点突变的激酶，抑制剂设计可能需要在整体激酶选择性与抑制激酶多种突变形式的能力（穿透性）之间取得平衡。
