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 在所有黑色素瘤中，约有一半的黑色素瘤是由BRAF突变引起的，BRAF是该途径中的一种激酶，也是导致 许多其他癌症也是如此。尽管几十年来人们对BRAF进行了密切的关注和调查，但BRAF的监管并不顺利- 明白了。此外，靶向RAS/MAPK途径的化合物表现出很少的理解 药理作用。BRAF抑制剂，如维莫拉非尼，有效地抑制V600E BRAF，但它们 自相矛盾地激活野生型BRAF。MEK是BRAF下游的一种激酶，其抑制剂的疗效不同 这取决于该通路是否被KRAS和BRAF的突变所激活。总体而言， 这些药物的混乱药理反映了我们对其调节和生化的不完全认识。 这一途径的工作机制限制了我们开发针对BRAF和RAS/MAPK的靶向治疗的能力 路径。 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 他们在癌症中的失调，以及抗癌药物的发现。我们已经应用了我们的基本生物物理、生物化学 以及对野生型和突变型EGFR的结构洞察，以发现新的药理制剂类别 靶向突变受体，包括突变选择性共价和变构抑制剂，可以 克服阻力机制。我们现在正在应用类似的结构和机械方法来 揭开BRAF调节和药理学的神秘面纱。我们的目标是了解BRAF在结构方面的监管 详细地，破译BRAF和MEK抑制剂的复杂药理，并开发新的药物 以突变选择性的方式靶向该途径。为了实现这些目标，我们将确定 冷冻电子显微镜下的自抑制和活性BRAF络合物。我们将从以下方面重建这条路径 KRAS到ERK的纯化组分，以剖析BRAF和MEK的激活机制和探针 以该途径为靶点的药理制剂的作用。此外，我们将一起使用这些重组 利用我们的结构洞察力，发现以突变选择性方式靶向该途径的新试剂。这些 研究将提供对BRAF监管的根本性新理解，从长远来看，它们应该会产生 对这一途径的突变激活所驱动的癌症的更有效和更耐受性的治疗。

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.