Probing RAS Function Using Monobody and NanoBiT Technologies

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 inhibitorsKRAS Cys 12KRAS mutationsG12C 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.