Abstract
BRAF inhibitors stabilize a closed, rigid RAF kinase conformation that facilitates dimerization.
Major finding: BRAF inhibitors stabilize a closed, rigid RAF kinase conformation that facilitates dimerization.
Approach: A high-throughput biosensor assay evaluated the effects of compounds on RAF dimerization.
Impact: Testing the effect of lead compounds on RAF dimerization can eliminate those likely to activate RAF.
The propensity of BRAFV600E-selective inhibitors to induce wild-type RAF protein dimerization is thought to contribute to both the emergence of acquired resistance and the development of secondary cancers, but the underlying mechanism is unclear. Lavoie and colleagues developed a high-throughput bioluminescence resonance energy transfer-based assay in which RAF kinase domains were used as biosensors to allow real-time monitoring of the effects of compounds on RAF dimerization in living cells. This approach revealed that RAF inhibitors could selectively induce homo- or heterodimerization of different RAF protein pairs, indicating that distinct mechanisms underlie wild-type RAF protein activation in response to individual inhibitors. Unexpectedly, inhibitors of several other kinases, including p38, BCR–ABL, and VEGFR, also induced RAF dimerization and downstream ERK activation, raising the possibility that RAF dimerization was caused by a shared feature of these diverse compounds. However, an analysis of available structures of RAF dimerization-inducing compounds in complex with RAF or other kinases showed no obvious similarities in the compound structures or binding modes. Instead, the common feature was that the N and C lobes of the bound kinase domains adopted a closed, inflexible conformation. Given that the side-to-side dimerization surface of RAF kinases spans the N and C lobes, these findings suggest that inhibitors that induce RAF dimers do so by stabilizing a closed kinase conformation that creates a relatively static interface conducive to dimerization. Consistent with this model, BRAF mutations that prevented lobe closure also reduced dimerization. RAF inhibitors that do not restrict kinase domain flexibility may therefore be less likely to promote dimerization and activation of wild-type RAF proteins. Screening lead compounds for their ability to promote RAF dimerization may also help to minimize the possibility of adverse events linked to RAF activation.
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