Activating mutations in RAS are found in approximately 30% of human cancers, resulting in the delivery of a persistent signal to critical downstream effectors that drive tumorigenesis. RAS-driven malignancies respond poorly to conventional cancer treatments and inhibitors that target RAS directly are limited; therefore, the identification of new strategies and/or drugs to disrupt RAS signaling in tumor cells remains a pressing therapeutic need. Taking advantage of the live-cell bioluminescence resonance energy transfer (BRET) methodology, we describe the development of a NanoBRET screening platform to identify compounds that modulate binding between activated KRAS and the CRAF kinase, an essential effector of RAS that initiates ERK cascade signaling. Using this strategy, libraries containing synthetic compounds, targeted inhibitors, purified natural products, and natural product extracts were evaluated. These efforts resulted in the identification of compounds that inhibit RAS/RAF binding and in turn suppress RAS-driven ERK activation, but also compounds that have the deleterious effect of enhancing the interaction to upregulate pathway signaling. Among the inhibitor hits identified, the majority were compounds derived from natural products, including ones reported to alter KRAS nanoclustering (ophiobolin A), to impact RAF function (HSP90 inhibitors and ROS inducers) as well as some with unknown targets and activities. These findings demonstrate the potential for this screening platform in natural product drug discovery and in the development of new therapeutic agents to target dysregulated RAS signaling in human disease states such as cancer.

中文翻译：

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开发高通量 NanoBRET 筛选平台来识别 RAS/RAF 相互作用的调节剂


大约 30% 的人类癌症中发现了 RAS 的激活突变，从而向驱动肿瘤发生的关键下游效应器传递持续信号。 RAS 驱动的恶性肿瘤对传统癌症治疗反应不佳，并且直接针对 RAS 的抑制剂有限；因此，寻找新的策略和/或药物来破坏肿瘤细胞中的 RAS 信号传导仍然是迫切的治疗需求。利用活细胞生物发光共振能量转移（BRET）方法，我们描述了 NanoBRET 筛选平台的开发，以识别调节激活的 KRAS 和 CRAF 激酶之间结合的化合物，CRAF 激酶是启动 ERK 级联信号传导的 RAS 的重要效应器。使用该策略，对包含合成化合物、靶向抑制剂、纯化的天然产物和天然产物提取物的文库进行了评估。这些努力导致鉴定出抑制 RAS/RAF 结合并进而抑制 RAS 驱动的 ERK 激活的化合物，但也鉴定出具有增强相互作用以上调通路信号传导的有害作用的化合物。在已确定的抑制剂中，大多数是源自天然产物的化合物，包括据报道可以改变 KRAS 纳米簇（ophiobolin A）、影响 RAF 功能（HSP90 抑制剂和 ROS 诱导剂）的化合物，以及一些具有未知靶点和活性的化合物。这些发现证明了该筛选平台在天然产物药物发现和开发新治疗药物以针对癌症等人类疾病状态中失调的 RAS 信号传导的潜力。