Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA.
Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA.
Methods Mol Biol. 2024;2797:211-225. doi: 10.1007/978-1-0716-3822-4_15.
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 KRAS-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, KRAS-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.
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，这使得 RAS 成为一个有价值的治疗靶点（Prior 等人，Cancer Res 80:2969-2974，2020）。尽管几十年来研究人员一直致力于开发 RAS 的治疗抑制剂，但近年来，FDA 批准的 KRAS 特异性抑制剂进入临床使用取得了成功（Skoulidis 等人，N Engl J Med 384:2371-2381，2021；Jänne 等人，N Engl J Med 387:120-131，2022）。此外，KRAS 特异性抑制剂目前正在进行临床试验（Wang 等人，J Med Chem 65:3123-3133，2022）。这些等位基因特异性抑制剂的出现否定了以前 RAS 不可成药的观点。尽管在 RAS 靶向治疗方面取得了这些进展，但癌症中经常出现的几种 RAS 突变仍然没有可行的药物。因此，进一步了解 RAS 在细胞中的功能和生物学特性，并开发用于鉴定新的抗 RAS 治疗靶点的工具至关重要。为此，我们利用单域抗体（Mb）技术开发了针对选定 RAS 同工型和突变体的特定蛋白质抑制剂（Spencer-Smith 等人，Nat Chem Biol 13:62-68，2017；Khan 等人，Cell Rep 38:110322，2022；Wallon 等人，Proc Natl Acad Sci USA 119:e2204481119，2022；Khan 等人，Small GTPases 13:114-127，2021；Khan 等人，Oncogene 38:2984-2993，2019）。在此，我们描述了我们联合使用单域抗体和 NanoLuc 二元技术（NanoBiT）来分析 RAS 蛋白-蛋白相互作用，并在活细胞高通量测定中筛选 RAS 结合的小分子。
