癌基因RAS家族中的错义突变(HRAS,KRAS,和NRAS)存在于大约20%的人类癌症中,使RAS成为有价值的治疗靶标(Prior等人。,癌症报告80:2969-2974,2020)。尽管数十年来开发RAS治疗性抑制剂的研究努力并不成功,近年来,随着FDA批准的KRASG12C特异性抑制剂进入临床,取得了成功(Skoulidisetal.,NEnglJMed384:2371-2381,2021;Jänne等人。,NEnglJMed387:120-131,2022)。此外,KRASG12D特异性抑制剂目前正在进行临床试验(Wang等人。,JMedChem65:3123-3133,2022)。这些等位基因特异性抑制剂的出现已经推翻了以前的观点,即RAS是不可用的。尽管RAS靶向疗法取得了这些进展,一些经常在癌症中出现的RAS突变体仍然没有易于处理的药物。因此,进一步了解RAS在细胞中的功能和生物学特性,以及开发识别新的治疗漏洞的工具,对于开发抗RAS疗法至关重要.要做到这一点,我们已经利用单体(Mb)技术来开发特定的RAS同工型和突变体的基于蛋白质的特异性抑制剂(Spencer-Smith等人。,NatChemBiol13:62-68,2017;Khan等人。,细胞代表38:110322,2022;Wallon等人。,ProcNatlAcadSci美国119:e2204481119,2022;Khan等人。,小GTPases13:114-127,2021;Khan等人。,癌基因38:2984-2993,2019年)。在这里,我们描述了我们结合使用Mbs和NanoLuc二进制技术(NanoBiT)来分析RAS蛋白质-蛋白质相互作用,并在活细胞中筛选RAS结合小分子,高通量测定。
Missense mutations in the RAS family of oncogenes (HRAS, KRAS, and NRAS) are present in approximately 20% of human cancers, making RAS a valuable therapeutic target (Prior et al., Cancer Res 80:2969-2974, 2020). Although decades of research efforts to develop therapeutic inhibitors of RAS were unsuccessful, there has been success in recent years with the entrance of FDA-approved KRASG12C-specific inhibitors to the clinic (Skoulidis et al., N Engl J Med 384:2371-2381, 2021; Jänne et al., N Engl J Med 387:120-131, 2022). Additionally, KRASG12D-specific inhibitors are presently undergoing clinical trials (Wang et al., J Med Chem 65:3123-3133, 2022). The advent of these allele specific inhibitors has disproved the previous notion that RAS is undruggable. Despite these advancements in RAS-targeted therapeutics, several RAS mutants that frequently arise in cancers remain without tractable drugs. Thus, it is critical to further understand the function and biology of RAS in cells and to develop tools to identify novel therapeutic vulnerabilities for development of anti-RAS therapeutics. To do this, we have exploited the use of monobody (Mb) technology to develop specific protein-based inhibitors of selected RAS isoforms and mutants (Spencer-Smith et al., Nat Chem Biol 13:62-68, 2017; Khan et al., Cell Rep 38:110322, 2022; Wallon et al., Proc Natl Acad Sci USA 119:e2204481119, 2022; Khan et al., Small GTPases 13:114-127, 2021; Khan et al., Oncogene 38:2984-2993, 2019). Herein, we describe our combined use of Mbs and NanoLuc Binary Technology (
NanoBiT) to analyze RAS protein-protein interactions and to screen for RAS-binding small molecules in live-cell, high-throughput assays.