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

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  • March 22, 2019
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  • Gonzalez-Perez, Vanessa
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • Molecularly targeted inhibitors are typically screened in cell-based assays for activity and target selectivity. However, early in vivo evaluation may improve characterization of specificity and/or detection of toxicity or off-target effects. In this dissertation, I describe use of the nematode C. elegans as an in vivo model to characterize both on- and off-target activity of novel putative Ras/Raf interaction inhibitors. In mammals, the Ras>Raf>MEK>ERK signaling cascade promotes cellular proliferation. Its aberrant activation is associated with oncogenesis and it is an attractive pharmaceutical target. In C. elegans, this pathway is highly conserved and regulates vulval development. Constitutive pathway signaling results in an easily scored Multivulva (Muv) phenotype that provides an accurate in vivo readout for activity of inhibitors targeting the pathway. I therefore validated the Muv phenotype for evaluation of the activity and specificity of known and novel pathway inhibitors, beginning with the well-characterized MEK inhibitor U0126. I then characterized its response to small molecule members of the MCP family of putative Ras/Raf interaction inhibitors. Analysis of a C. elegans strain expressing activated Ras (worm ortholog, LET-60) showed that MCP110 and MCP116 act downstream of Ras, causing significant dose-dependent reduction of Muv. Analysis of strains genetically activated downstream of Ras showed that these compounds act upstream of the ETS-like transcription factor (LIN-1) and the MAP kinases, MEK-2 and MPK-1. The best available strain expressing activated Raf (LIN-45AA) was unable to distinguish whether these compounds act at the level of Ras or of Raf, and may not be Ras-independent. I then turned to cell-based assays using NIH 3T3 mouse fibroblasts, and showed for the first time that MCP110 dose-dependently disrupts the physical interaction between Ras and Raf and impairs Ras recruitment of Raf to cellular membranes. I also narrowed the affected protein:protein interaction to that of Ras with the Ras binding domain (RBD) of Raf. Finally, I identified specific NPYR-like off-target effects of MCP compounds in C. elegans. Thus, C. elegans is a valuable in vivo genetic system to characterize on- and off-target activity of inhibitors targeting the Ras>Raf>MEK>ERK pathway and may be useful for other novel therapeutics.
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  • ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Genetics and Molecular Biology.
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  • Cox, Adrienne
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