Molecular probes for allele-specific interdiction of K-Ras G12D signaling

项目总结/文摘

Project Summary/Abstract Single amino acid activating mutations at G12, G13, or Q61 are known to impair the intrinsic GTPase activity of K-Ras which leads to its constitutive activation to drive tumor development and growth. Consequently, inhibition of K-Ras signaling offers an attractive strategy for therapeutic intervention in cancers. Apart from small molecule covalent modifiers of K-Ras G12C, efforts to develop reversible small molecule antagonists of K-Ras mutants have been unsuccessful due to the intractable nature of K-Ras proteins to reversible small molecule binders. Conversely, high affinity antagonism of K-Ras mutants have been demonstrated with antibodies and designer binding proteins. In particular, the 7-kDa protein R11.1.6 was recently shown to antagonize K-Ras G12D signaling when overexpressed relative to the amount of K-Ras G12D in cells. However, no inhibition was observed in more relevant cancer models where the intracellular concentration of R11.1.6 was insufficient to outcompete the high local effective molarity of K-Ras G12D–Raf interaction. Such stoichiometric bottleneck, in theory, could be overcome through exogenous administration of R11.1.6 to cells harboring K-Ras G12D. Furthermore, inhibition by degradation of K-Ras G12D could also circumvent the need for high intracellular concentration of R11.1.6. I will test the hypotheses that exogenously delivered R11.1.6 or an R11.1.6-derived chimeric degrader can circumvent the stoichiometric bottleneck to produce an effective antagonism of K-Ras G12D–Raf interaction. To test these hypotheses, I will prepare protein–small-molecule chimeras of R11.1.6 as probes to evaluate the efficiency of cellular internalization, de-esterification, functional engagement, and catalytic degradation of K-Ras G12D. The chimeric probes will be obtained through N-terminus conjugation of small-molecules to R11.1.6. Carboxyl groups in the resulting chimeras will be esterified with a tuned diazo compound, which should enable the chimera to enter the cytosol of human cells. The consequences of the chimeric probes will be assessed in relevant cell lines. The work proposed in this fellowship will be performed in the Raines laboratory at MIT and will provide me with world-class training in chemical biology. The training under this project also includes plans for applicant’s career development through a course in cancer biology, a teaching certification program, and attending scientific conferences. Overall, I anticipate that the proposed work in this fellowship will help further the mission of the NCI Ras initiative to explore new therapeutic approaches for Ras- driven cancers.