Genetic and functional characterization of putative Ras/Raf interaction inhibitors in C. elegans and mammalian cells

Our genetic analyses showed significant dose-dependent MCP-mediated reduction of Muv in C. elegans strains with activating mutations in orthologs of Ras (LET-60) or Raf (LIN-45), but not MAP kinases or an Ets-like transcription factor. Thus, these inhibitors selectively impair pathway function downstream of Ras and upstream of or at the level of Raf, consistent with disruption of the Ras/Raf interaction. Our biochemical analyses of MCP110-mediated disruption of Ras-Raf interactions in mammalian cells showed that MCP110 dose-dependently reduced Raf-RBD pulldown of Ras, displaced a fluorescently-tagged Raf-RBD probe from plasma membrane locations of active Ras to the cytosol and other compartments, and decreased active, phosphorylated ERK1/2.We have effectively utilized C. elegans as an in vivo genetic system to evaluate the activity and selectivity of inhibitors intended to target the Ras-Raf-MAPK pathway. We demonstrated the ability of MCP110 to disrupt, at the level of Ras/Raf, the Muv phenotype induced by chronic activation of this pathway in C. elegans. In mammalian cells, we not only demonstrated MCP-mediated blockade of the physical interaction between Ras and Raf, but also narrowed the site of interaction on Raf to the RBD, and showed consequent functional impairment of the Ras-Raf-MEK-ERK pathway in both in vivo and cell-based systems.Over the past two decades, there have been many attempts to isolate and characterize pharmacological inhibitors targeting Ras-dependent signaling pathways. The small GTPase Ras normally transmits signals downstream of diverse inputs and is a critical signaling node for many cellular activities. Aberrant Ras activity leads to the deregulation of numerous cellular processes including proliferation, survival, cell adhesion and migration, that in turn can contribute to cellular transformation, invasion and metastasis [1], and Ras is mutationally activated in ~30% of cancers [2]. Among the downstream effectors of Ras, the most well-c