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

项目摘要/摘要 已知G12、G13或Q61处的单一氨基酸激活突变会削弱GTPase的内在活性 K-RAS导致其结构性激活，从而驱动肿瘤的发展和生长。因此，抑制 K-RAS信号通路的研究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 G12C共价修饰剂，努力开发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体的顽固性，一直没有成功。 相反，K-RAS突变体的高亲和力拮抗作用已被抗体和设计者证明 结合蛋白。特别是，7-kDa蛋白R11.1.6最近被证明能拮抗K-RAS G12D 当信号相对于细胞中K-RAS G12D的量过表达时。然而，没有抑制作用。 在更相关的癌症模型中观察到R11.1.6的细胞内浓度不足以 胜过K-RAS G12D-Raf相互作用的高局域有效摩尔浓度。这样的化学计量瓶颈，在 理论上，可以通过外源R11.1.6给含有K-RAS G12D的细胞来克服。 此外，通过降解K-RAS G12D来抑制也可以绕过高细胞内的需要 R11.1.6的浓度。我将测试从外部提供R11.1.6或R11.1.6派生版本的假设 嵌合降解物可以绕过化学计量瓶颈产生有效的K-RAS拮抗作用 G12D-Raf相互作用。为了验证这些假设，我将准备R11.1.6的蛋白质-小分子嵌合体AS 评估细胞内化、去酯化、功能参与和 K-RAS G12D的催化降解嵌合探针将通过N-末端接合获得 小分子至R11.1.6.得到的嵌合体中的羧基将与调谐的重氮化酯化。 化合物，这应该使嵌合体进入人类细胞的胞浆。这场战争的后果 嵌合探针将在相关细胞系中进行评估。该奖学金中提议的工作将在 麻省理工学院的雷恩斯实验室将为我提供世界级的化学生物学培训。培训计划下的培训 该项目还包括申请者的职业发展计划，通过癌症生物学课程，教学 认证计划，并参加科学会议。总体而言，我预计在这方面拟议的工作 奖学金将有助于促进NCI RAS倡议的使命，探索RAS的新治疗方法- 导致癌症。

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.