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

项目总结/摘要 已知G12、G13或Q61处的单个氨基酸激活突变会损害GT3的固有活性。 K-Ras，其导致其组成性激活以驱动肿瘤发展和生长。因此，抑制 K-Ras信号转导为癌症的治疗干预提供了一种有吸引力的策略。除了小分子 K-Ras G12 C的共价修饰剂，致力于开发K-Ras突变体的可逆小分子拮抗剂 由于K-Ras蛋白对可逆小分子结合剂的难处理性质， 相反，已经用抗体和设计者证明了K-Ras突变体的高亲和力拮抗作用。 结合蛋白特别地，最近显示7-kDa蛋白R11.1.6拮抗K-Ras G12 D 当相对于细胞中K-Ras G12 D的量过表达时，K-Ras G12 D的信号传导。然而， 在更相关的癌症模型中观察到，其中R11.1.6的细胞内浓度不足以 竞争胜过K-Ras G12 D-Raf相互作用的高局部有效摩尔浓度。这种化学计量瓶颈， 理论上，可以通过向携带K-Ras G12 D的细胞外源施用R11.1.6来克服。 此外，通过降解K-Ras G12 D的抑制也可以避免对高细胞内表达的需要。 R11.1.6的浓度。我将检验外源性递送R11.1.6或R11.1.6衍生物的假设， 嵌合降解剂可以绕过化学计量瓶颈，产生K-Ras的有效拮抗作用 G12 D-Raf相互作用。为了验证这些假设，我将制备R11.1.6的蛋白质-小分子嵌合体， 用于评估细胞内化、去酯化、功能接合和 K-Ras G12 D的催化降解。嵌合探针将通过N-末端缀合 小分子至R11.1.6。由此产生的嵌合体中的羧基将被调谐的重氮基酯化， 化合物，这应该使嵌合体进入人类细胞的胞质溶胶。的后果 将在相关细胞系中评估嵌合探针。本奖学金中提出的工作将在 麻省理工学院的雷恩斯实验室，并将为我提供世界一流的化学生物学培训。培训根据 该项目还包括申请人的职业发展计划，通过癌症生物学课程， 认证计划，并参加科学会议。总的来说，我预计这方面的拟议工作 奖学金将有助于进一步NCI Ras倡议的使命，探索Ras的新治疗方法， 驱动癌症。

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.