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

用于等位基因特异性阻断 K-Ras G12D 信号传导的分子探针

基本信息

批准号：
10474345
负责人：
Aniekan Matthew Okon
金额：
$ 7.17万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10474345
关键词：
Affinity Alleles Amino Acids Antibodies Arginine Binding Binding Proteins Biological Biology Biophysics Biotin Cancer Biology Cancer Model Cell Line Cell Proliferation Cell model Cells Chemicals Chimera organism Cytosol Development Educational process of instructing Ensure Esterification Esters Evaluation Event FRAP1 gene Fellowship Fluorescent Dyes Generations Goals Growth and Development function Guanosine Triphosphate Phosphohydrolases Human Hydrolysis Impairment In Vitro KRAS2 gene Laboratories Lesion Ligands Link Lysine MEKs Malignant Neoplasms Mammalian Cell Mission Modification Molecular Probes Mutation N-Dimethylacetamide N N-terminal Nature Oncogenic Pathway interactions Pharmaceutical Preparations Pharmacology Polyethylene Glycols Positioning Attribute Protein Isoforms Proteins Proteolysis Proto-Oncogene Proteins c-akt Rain Signal Transduction Site Technology Testing Therapeutic Intervention Training Ubiquitination Variant Work antagonist anticancer research base cancer cell career development cell growth certificate program design diazo compound in vitro Assay mutant novel therapeutic intervention novel therapeutics overexpression pancreatic cancer cells ras Proteins small molecule symposium targeted treatment theories therapy development thermostability tumor tumor growth tumor progression ubiquitin-protein ligase

项目摘要

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.

项目概要/摘要已知 G12、G13 或 Q61 处的单个氨基酸激活突变会损害 GTP 酶的内在活性 K-Ras 导致其组成性激活，以驱动肿瘤的发展和生长，并进行了测试和抑制。除了小分子外，K-Ras 信号传导的研究还为癌症的治疗干预提供了一种有吸引力的策略。 K-Ras G12C 的共价修饰剂，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物的棘手性质，该研究尚未成功。抗体和设计器已证明 K-Ras 突变体的离线、高亲和力拮抗作用特别是，7-kDa 蛋白 R11.1.6 最近被证明可以拮抗 K-Ras G12D。然而，当相对于细胞中 K-Ras G12D 的量过度表达时，信号传导却没有受到抑制。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胜过 K-Ras G12D-Raf 相互作用的高局部有效摩尔浓度，在这种情况下。理论上，可以通过向含有 K-Ras G12D 的细胞外源施用 R11.1.6 来克服这一问题。此外，通过 K-Ras G12D 降解进行抑制也可以避免对高细胞内我将检验外源递送 R11.1.6 或 R11.1.6 衍生的假设。嵌合降解剂可以绕过化学计量瓶颈，产生 K-Ras 的有效拮抗作用为了测试这些假设，我将制备 R11.1.6 的蛋白质-小分子嵌合体。探针评估细胞内化、脱酯化、功能参与和的效率 K-Ras G12D 的催化降解将通过 N 端缀合获得嵌合探针。所得嵌合体中的小分子R11.1.6将被调整的重氮基酯化。化合物，它应该能够使嵌合体进入人类细胞的细胞质。嵌合探针将在相关细胞系中进行评估。本研究金中提出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一流的化学生物学培训。该项目还包括申请人通过癌症生物学课程、教学等职业发展计划总体而言，我预计这方面的拟议工作。奖学金将有助于进一步实现 NCI Ras 计划的使命，探索 Ras 的新治疗方法驱动癌症。