Structure, mechanism, and pharmacology of BRAF and its partners in the RAS/RAF/MAP kinase pathway

摘要 RAS/MAP 激酶通路在多种人类癌症中被异常激活。 V600E BRAF 突变（该通路中的一种激酶）导致大约一半的黑色素瘤，并且是黑色素瘤的驱动因素 还有许多其他癌症。尽管几十年来人们一直对 BRAF 产生了浓厚的兴趣和调查，但 BRAF 监管仍然不够完善—— 明白了。此外，针对 RAS/MAPK 通路的化合物尚不清楚 药理作用。 BRAF 抑制剂，例如维莫非尼，可有效抑制 V600E BRAF，但它们 矛盾的是激活野生型 BRAF。 MEK（BRAF 下游激酶）抑制剂的功效不同 取决于 KRAS 与 BRAF 中的突变是否激活该通路。总的来说， 这些药物令人困惑的药理学反映了我们对调节和生化的不完全了解 该通路的工作原理并限制了我们开发 BRAF 和 RAS/MAPK 靶向疗法的能力 途径。 在过去的二十年里，我的实验室一直专注于蛋白激酶的结构生物学和 他们在癌症和癌症药物发现方面的失调。我们应用了基本的生物物理、生物化学 以及对野生型和突变型 EGFR 的结构见解，以发现新类别的药物 针对突变受体，包括突变选择性共价抑制剂和变构抑制剂 克服抵抗机制。我们现在正在应用类似的结构和机械方法 揭秘 BRAF 调节和药理学。我们的目标是了解 BRAF 结构性监管 详细信息，破译 BRAF 和 MEK 抑制剂的复杂药理学，并开发新的药物 以突变选择性方式靶向该途径。为了实现这些目标，我们将确定 使用冷冻电子显微镜观察自抑制和活性 BRAF 复合物。我们将重建路径 使用纯化成分将 KRAS 转化为 ERK，以剖析 BRAF 和 MEK 激活和探测机制 针对该途径的药物的作用。此外，我们将一起使用这些重构 凭借我们的结构见解来发现以突变选择性方式靶向该途径的新药物。这些 研究将为 BRAF 调控提供基本的新认识，从长远来看，它们应该会产生 对于由该途径的诱变激活驱动的癌症，有更有效和更好耐受的疗法。

Abstract The RAS/MAP kinase pathway is aberrantly activated in a wide variety of human cancers. The V600E mutation in BRAF, a kinase in this pathway, causes approximately one-half of all melanomas and is the driver in many other cancers as well. Despite decades of intense interest and investigation, BRAF regulation is not well- understood. Furthermore, compounds targeting the RAS/MAPK pathway exhibit poorly understood pharmacologic effects. BRAF inhibitors, such as vemurafenib, potently inhibit V600E BRAF, but they paradoxically activate wild type BRAF. Inhibitors of MEK, a kinase downstream of BRAF, differ in their efficacy depending upon whether the pathway is activated by mutations in KRAS versus BRAF. Collectively, the confusing pharmacology of these agents reflects our incomplete knowledge of the regulation and biochemical workings of this pathway and limits our ability to develop targeted therapies for BRAF and the RAS/MAPK pathway. Over the last two decades, my laboratory has focused on the structural biology of protein kinases and their dysregulation in cancer, and on cancer drug discovery. We have applied our basic biophysical, biochemical and structural insights into wild-type and mutant EGFR to discover new classes of pharmacologic agents targeting the mutant receptor, including both mutant-selective covalent and allosteric inhibitors that can overcome resistance mechanisms. We are now applying an analogous structural and mechanistic approach to demystify BRAF regulation and pharmacology. Our objectives are to understand BRAF regulation in structural detail, to decipher the complex pharmacology of the BRAF and MEK inhibitors, and to develop new agents that target the pathway in a mutant-selective manner. To achieve these goals, we will determine the structure of autoinhibited and active BRAF complexes using cryo-electron microscopy. We will reconstitute the pathway from KRAS to ERK using purified components in order to dissect mechanisms of BRAF and MEK activation and probe the effects of pharmacologic agents that target the pathway. In addition, we will use these reconstitutions together with our structural insights to discover new agents that target the pathway in a mutant-selective manner. These studies will provide fundamental new understanding of BRAF regulation and, in the long term, they should yield more effective and better tolerated therapies for cancers driven by mutagenic activation of this pathway.