RAF二聚体抑制剂在RAF和RAS驱动的癌症中具有治疗潜力。这类药物的有效性依赖于其能够占据RAS-RAF信号复合物中的两个RAF亚基的能力。在这里，我们描述了一种在细胞中有条件地定量测定选定RAF亚基上药物与靶标的结合率的方法。在任何突变的KRAS等位基因存在与否的情况下，都可以测定RAF靶标的结合状态，从而可以在细胞介质中量化RAF二聚体抑制剂的高亲和力状态。临床阶段II型RAF抑制剂的细胞内亚基选择性研究显示，ARAF亚基结合，而BRAF或CRAF的结合与各种突变RAS细胞系中MAPK信号的抑制相符。我们的研究结果支持ARAF在突变RAS信号传导中的基本作用，并揭示了一群正在临床评估中的RAF抑制剂对ARAF亚基的易感性较差。版权所有©2023作者。由Elsevier Ltd.出版。保留所有权利。

RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers. The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex. Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF complex in cells. RAF target engagement can be measured in the presence or absence of any mutant KRAS allele, enabling the high-affinity state of RAF dimer inhibitors to be quantified in the cellular milieu. The intracellular protomer selectivity of clinical-stage type II RAF inhibitors revealed that ARAF protomer engagement, but not engagement of BRAF or CRAF, is commensurate with inhibition of MAPK signaling in various mutant RAS cell lines. Our results support a fundamental role for ARAF in mutant RAS signaling and reveal poor ARAF protomer vulnerability for a cohort of RAF inhibitors undergoing clinical evaluation.Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.