Promega Corporation, Madison, WI, USA.
Vibliome Therapeutics LLC, Bozeman, MT, USA.
Methods Mol Biol. 2024;2797:287-297. doi: 10.1007/978-1-0716-3822-4_21.
Dysfunction of the RAS/mitogen-activated protein kinase (MAPK) pathway is a common driver of human cancers. As such, both the master regulator of the pathway, RAS, and its proximal kinase effectors, RAFs, have been of interest as drug targets for decades. Importantly, signaling within the RAS/MAPK pathway is highly coordinated due to the formation of a higher-order complex called the RAS/RAF signalosome, which may minimally contain dimers of both RAS and RAF protomers. In the disease state, RAS and RAF assemble in homo- and/or heterodimeric forms. Traditionally, drug development campaigns for both RAS and RAF have utilized biochemical assays of purified recombinant protein. As these assays do not query the RAS or RAF proteins in their full-length and complexed forms in cells, potency results collected using these assays have often failed to correlate with inhibition of the MAPK pathway. To more accurately quantify engagement at this signaling components, we present a bioluminescence resonance energy transfer (BRET)-based method to conditionally measure target engagement at individual protomers within the RAS/RAF signalosome in live cells.
RAS/丝裂原活化蛋白激酶（MAPK）通路的功能障碍是人类癌症的常见驱动因素。因此，作为药物靶点，该通路的主调控因子 RAS 和其近源激酶效应物 RAF 已经引起了数十年的关注。重要的是，由于形成了一种称为 RAS/RAF 信号小体的高级别复合物，RAS/MAPK 通路内的信号传递高度协调，该复合物可能至少包含 RAS 和 RAF 单体的二聚体。在疾病状态下，RAS 和 RAF 以同源和/或异源二聚体形式组装。传统上，针对 RAS 和 RAF 的药物开发活动都利用了纯化重组蛋白的生化测定。由于这些测定未在细胞中查询全长和复合物形式的 RAS 或 RAF 蛋白，因此使用这些测定收集的效力结果往往与 MAPK 通路的抑制无关。为了更准确地定量评估该信号组分的结合情况，我们提出了一种基于生物发光共振能量转移（BRET）的方法，用于在活细胞中条件性地测量 RAS/RAF 信号小体中单个单体的靶标结合情况。
