Mutant-selective degradation by BRAF-targeting PROTACs

Shanique Alabi,Zhan Yao,Yijun Gao,Neal Rosen,Craig M Crews,Kusal T G Samarasinghe,Lea Vogt,John Hines,Saul Jaime-Figueroa

doi:10.1038/s41467-021-21159-7

Abstract

Over 300 BRAF missense mutations have been identified in patients, yet currently approved drugs target V600 mutants alone. Moreover, acquired resistance inevitably emerges, primarily due to RAF lesions that prevent inhibition of BRAF V600 with current treatments. Therefore, there is a need for new therapies that target other mechanisms of activated BRAF. In this study, we use the Proteolysis Targeting Chimera (PROTAC) technology, which promotes ubiquitination and degradation of neo-substrates, to address the limitations of BRAF inhibitor-based therapies. Using vemurafenib-based PROTACs, we achieve low nanomolar degradation of all classes of BRAF mutants, but spare degradation of WT RAF family members. Our lead PROTAC outperforms vemurafenib in inhibiting cancer cell growth and shows in vivo efficacy in a Class 2 BRAF xenograft model. Mechanistic studies reveal that BRAFWT is spared due to weak ternary complex formation in cells owing to its quiescent inactivated conformation, and activation of BRAFWT sensitizes it to degradation. This study highlights the degree of selectivity achievable with degradation-based approaches by targeting mutant BRAF-driven cancers while sparing BRAFWT, providing an anti-tumor drug modality that expands the therapeutic window.

Highlights

Over 300 BRAF missense mutations have been identified in patients, yet currently approved drugs target V600 mutants alone
The utility of vemurafenib is limited to the treatment of tumors driven by BRAFV600 mutations, biochemical and binding studies show that these inhibitors interact with BRAFWT, Class 2, and Class 3 BRAF mutants[11,15,18,33]
Crystal structures of vemurafenib bound to BRAFV600E reveal a solvent-exposed chloride at the para-position on the phenyl ring, which we posited would be ideal for linker addition (PDB: 3OG7)[34] (Supplementary Fig. 1a,b)

Summary

Introduction

Over 300 BRAF missense mutations have been identified in patients, yet currently approved drugs target V600 mutants alone. We use the Proteolysis Targeting Chimera (PROTAC) technology, which promotes ubiquitination and degradation of neo-substrates, to address the limitations of BRAF inhibitor-based therapies. Using vemurafenib-based PROTACs, we achieve low nanomolar degradation of all classes of BRAF mutants, but spare degradation of WT RAF family members. Class 3 BRAF mutants such as G466V and D594N harbor low to no kinase activity and function by binding tightly to RAS recruiting CRAF into hyperactivated heterodimers[15,16,17]. Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional small molecules composed of a warhead that binds a protein of interest (POI), a flexible linker, and a ligand that binds an E3 ligase[20,21]. The modular design of PROTACs allows for additional selectivity to be tuned into the small molecule, making it ideal for addressing difficult targets such as BRAF

Methods

Results

Conclusion