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Probing RAS Function Using Monobody and NanoBiT Technologies
Michael Whaby1,2, Rakesh Sathish Nair1,2,3, John P O'Bryan4,5,6
1Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA.
Abstract:
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.
Insights
Targeting RAS oncogenes in cancer therapy is advancing with new KRAS inhibitors. This study uses monobodies and NanoBiT to develop novel anti-RAS therapeutics and screen for small molecules.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Missense mutations in RAS oncogenes (HRAS, KRAS, NRAS) are implicated in ~20% of human cancers, presenting a significant therapeutic target.
- Recent successes with KRASG12C inhibitors and ongoing trials for KRASG12D inhibitors have challenged the notion of RAS being undruggable.
- However, numerous RAS mutants driving cancer remain without effective therapeutic strategies, necessitating further research into RAS biology and novel drug development.
Purpose of the Study:
- To develop novel protein-based inhibitors targeting specific RAS isoforms and mutants using monobody (Mb) technology.
- To create tools for analyzing RAS protein-protein interactions and screening for RAS-binding small molecules.
- To identify new therapeutic vulnerabilities for the development of anti-RAS drugs.
Main Methods:
- Utilized monobody (Mb) technology to engineer specific protein inhibitors for RAS oncogenes.
- Combined monobodies with NanoLuc Binary Technology (NanoBiT) for live-cell assays.
- Employed high-throughput screening assays to analyze RAS protein-protein interactions and identify small molecule binders.
Main Results:
- Demonstrated the successful application of monobodies for targeting specific RAS mutants.
- Established a novel NanoBiT-based system for live-cell analysis of RAS interactions.
- Developed a high-throughput screening platform for identifying potential anti-RAS therapeutics.
Conclusions:
- Monobody technology offers a promising avenue for developing targeted anti-RAS therapeutics against challenging cancer mutations.
- The combined Mb and NanoBiT approach provides a powerful tool for dissecting RAS biology and accelerating drug discovery.
- This work contributes to overcoming the limitations of current RAS-targeted therapies and addresses the need for drugs against previously undruggable RAS mutants.
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