Chemically Linked Vemurafenib Inhibitors Promote an Inactive BRAF^V600E Conformation

Abstract

The BRAF kinase, within the mitogen activated protein kinase (MAPK) signaling pathway, harbors activating mutations in about half of melanomas and to a significant extent in many other cancers. A single valine to glutamic acid substitution at residue 600 (BRAFV600E) accounts for about 90% of these activating mutations. While BRAFV600E-selective small molecule inhibitors, such as debrafenib and vemurafenib, have shown therapeutic benefit, almost all patients develop resistance. Resistance often arises through reactivation of the MAPK pathway, typically through mutation of upstream RAS, downstream MEK or splicing variants. RAF kinases signal as homo- and hetero-dimers, and another complication associated with small molecule BRAFV600E inhibition is drug-induced allosteric activation of a wild-type RAF subunit (BRAF or CRAF) of the kinase dimer, a process called ‘transactivation’ or ‘paradoxical activation.’ Here we used BRAFV600E and vemurafenib as a model system to develop chemically linked kinase inhibitors to lock RAF dimers in an inactive conformation that cannot undergo transactivation. This structure-based design effort resulted in the development of Vem-BisAmide-2, a compound containing two vemurafenib molecules connected by a bis amide linker. We show that Vem-BisAmide-2 has comparable inhibitory potency as vemurafenib to BRAFV600E both in vitro and in cells, but promotes an inactive dimeric BRAFV600E conformation unable to undergo transactivation. The crystal structure of a BRAFV600E/Vem-BisAmide-2 complex and associated biochemical studies reveal the molecular basis for how Vem-BisAmide-2 mediates selectivity for an inactive over active dimeric BRAFV600E conformation. These studies have implications for targeting BRAFV600E/RAF heterodimers and other kinase dimers for therapy.

Biochemical Characterization of Full‐Length Oncogenic BRAFV600E together with Molecular Dynamics Simulations Provide Insight into the Activation and Inhibition Mechanisms of RAF Kinases

“…Both cell‐based co‐immunoprecipitation of FL‐BRAF V600E/R509H and in vitro biophysical characterization of the purified BRAF V600E/R509H kinase domain support the idea that the R509H mutation dissociates BRAF V600E dimer . To verify that purified FL‐BRAF V600E/R509H indeed has a disrupted dimer interface, we incubated FL‐BRAF V600E and FL‐BRAF V600E/R509H with 32 P‐labeled ATP and MEK substrate for 30 min at 30 °C to let phosphorylation occur. The incorporation of 32 P into BRAF and MEK was recorded simultaneously by autoradiograph.…”

Mechanism of BRAF Activation through Biochemical Characterization of the Recombinant Full‐Length Protein

“…It has been an enigma that the notorious paradoxical activation of BRAF by inhibitors in cancer cell lines and clinics has not been observed with in vitro kinase assays; this led us to speculate that intact BRAF might display actual in vivo mechanisms more precisely. We applied an ELISA to quantify the potency of RAF inhibitors against BRAF. The ELISA readout is phosphorylated MEK1, which is probed with phosphospecific antibody pMEK1 (Ser217/221).…”

BRAF Splice Variant Resistance to RAF Inhibitor Requires Enhanced MEK Association

“…Our data suggest that the effectiveness of these RAF inhibitors in this model system may additionally relate to their ability to disrupt BRAF-MEK association or sequester BRAF-MEK in an inactive conformation (Haling et al, 2014; Karoulia et al, 2017). Furthermore, although the disruption of BRAF dimerization has been associated with favorable results in preclinical models (Freeman et al, 2013; Grasso et al, 2016; Sieved: et al, 2013), directly testing how these treatments effect substrate binding is necessary to broadly apply such strategies.…”