Inhibitors of BRAF dimers using an allosteric site

Xiomaris M Cotto-Rios,Orsi Giricz,Bogos Agianian,Evripidis Gavathiotis,Emmanouil Zacharioudakis,Amit Verma,Nadege Gitego,Poulikos I Poulikakos,Yang Wu,Yiyu Zou

doi:10.1038/s41467-020-18123-2

Xiomaris M Cotto-Rios, Orsi Giricz + Show 8 more

Open Access

https://doi.org/10.1038/s41467-020-18123-2

Copy DOI

Abstract

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAFV600E signals as an active monomer in the absence of active RAS, however, in many tumors BRAF dimers mediate ERK signaling. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which leads to tumor resistance. We found that Ponatinib, an FDA-approved drug, is an effective inhibitor of BRAF monomers and dimers. Ponatinib binds the BRAF dimer and stabilizes a distinct αC-helix conformation through interaction with a previously unrevealed allosteric site. Using these structural insights, we developed PHI1, a BRAF inhibitor that fully uncovers the allosteric site. PHI1 exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors. This work shows that Ponatinib and BRAF dimer selective inhibitors will be useful in treating BRAF-dependent tumors.

Highlights

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers
To identify RAF dimer kinase inhibitors, we established an in-cell-western based screening assay using SKMEL239-C4 melanoma cells[31]. These cells were generated under Vemurafenib-induced selection pressure inhibiting BRAFV600E, allowing preferential growth of cells expressing p61BRAFV600E, a splice variant of BRAF that constitutively signals as a dimer in a RAS-independent manner[19] (Fig. 1a). p61BRAFV600E is resistant to Vemurafenib and is found in patients’ tumors[19]
Vemurafenib robustly inhibits ERK signaling in BRAFV600E expressing cells such as melanoma A375 cells at 0.3 μM, whereas in SKMEL239-C4 cells a similar effect required over 10 μM (Supplementary Fig. 1a, b)[11,12]

Summary

Introduction

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Several mechanisms of clinical resistance to RAF inhibitors have been identified, including feedback reactivation of receptor tyrosine kinases and RAS, RAS mutations, BRAF amplification and expression of BRAFV600E splice variants[16,17,18,19]. These resistance mechanisms commonly lead to reactivation of ERK signaling through dimerization of RAF proteins[16,17,18,19]. RAF dimers are poorly inhibited by the FDA-approved inhibitors[11,12] and have been recognized as an important target for limiting drug resistance and for more effective inhibition of ERK signaling in various tumors[20,21]

Objectives

Methods

Results

Conclusion