Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202.
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405.
Proc Natl Acad Sci U S A. 2020 Mar 31;117(13):7131-7139. doi: 10.1073/pnas.1913654117. Epub 2020 Mar 16.
Ral (Ras-like) GTPases are directly activated by oncogenic Ras GTPases. Mutant K-Ras (G12C) has enabled the development of covalent K-Ras inhibitors currently in clinical trials. However, Ral, and the overwhelming majority of mutant oncogenic K-Ras, are devoid of a druggable pocket and lack an accessible cysteine for the development of a covalent inhibitor. Here, we report that covalent bond formation by an aryl sulfonyl fluoride electrophile at a tyrosine residue (Tyr-82) inhibits guanine exchange factor Rgl2-mediated nucleotide exchange of Ral GTPase. A high-resolution 1.18-Å X-ray cocrystal structure shows that the compound binds to a well-defined binding site in RalA as a result of a switch II loop conformational change. The structure, along with additional high-resolution crystal structures of several analogs in complex with RalA, confirm the importance of key hydrogen bond anchors between compound sulfone oxygen atoms and Ral backbone nitrogen atoms. Our discovery of a pocket with features found on known druggable sites and covalent modification of a bystander tyrosine residue present in Ral and Ras GTPases provide a strategy that could lead to therapeutic agent targeting oncogenic Ras mutants that are devoid of a cysteine nucleophile.
Ral（Ras 样）GTPases 可被致癌性 Ras GTPases 直接激活。突变型 K-Ras（G12C）已促使目前处于临床试验阶段的共价 K-Ras 抑制剂的开发。然而，Ral 以及绝大多数突变致癌性 K-Ras 缺乏可成药口袋，且缺乏可用于开发共价抑制剂的易接近半胱氨酸。在这里，我们报告芳基磺酰氟亲电试剂与酪氨酸残基（Tyr-82）形成共价键可抑制 G 蛋白交换因子 Rgl2 介导的 Ral GTPase 的核苷酸交换。高分辨率 1.18 Å X 射线共晶结构显示，由于 II 型转换环构象改变，化合物结合到 RalA 中的一个明确的结合位点。该结构以及与 RalA 复合物的几种类似物的其他高分辨率晶体结构证实了化合物磺酰基氧原子与 Ral 骨架氮原子之间关键氢键锚的重要性。我们发现了一个具有已知可成药位点特征的口袋，并对 Ral 和 Ras GTPases 中存在的旁观者酪氨酸残基进行共价修饰，这为靶向缺乏半胱氨酸亲核基团的致癌性 Ras 突变体的治疗剂提供了一种策略。
