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.

项目摘要/摘要已知在G12，G13或Q61处的单氨基酸激活突变会损害固有的GTPase活性 K-RAS导致其组成型激活以驱动肿瘤的发育和生长。因此，抑制作用 K-Ras信号的含量为癌症的治疗干预提供了有吸引力的策略。除了小分子 K-Ras G12C的共价修饰符，努力发展可逆的小分子拮抗剂K-RAS突变体由于K-RAS蛋白对可逆的小分子粘合剂的棘手性质，因此没有成功。相反，抗体和设计师已经证明了K-RAS突变体的高亲和力拮抗剂结合蛋白。特别是，最近显示了7-kDa蛋白R11.1.6与K-Ras G12D拮抗相对于细胞中K-RAS G12D的量过表达时的信号传导。但是，没有抑制是在更相关的癌症模型中观察到，R11.1.6的细胞内浓度不足胜任K-RAS G12D – RAF相互作用的高局部有效摩尔度。这样的化学计量瓶颈理论可以通过外源给具有K-Ras G12d的细胞的外源给予R11.1.6来克服。此外，通过降解k-ras G12D抑制也可能规避高细胞内的需求 R11.1.6的浓度。我将测试外源交付R11.1.6或R11.1.6衍生的假设嵌合降解器可以绕过化学计量瓶颈，以产生K-Ras的有效拮抗作用 G12D -RAF相互作用。为了检验这些假设，我将准备R11.1.6的蛋白质 - 小分子嵌合体评估细胞内在化，去酯化，功能参与和的问题 K-Ras G12d的催化降解。嵌合问题将通过N末端结合获得小分子至R11.1.6。所得嵌合体中的羧基将用调谐的重氮进行酯化化合物，这应该使嵌合体进入人类细胞的细胞质。后果嵌合问题将在相关的细胞系中进行评估。该奖学金中提出的工作将在麻省理工学院的Raines实验室将为我提供化学生物学的世界一流培训。下面的培训该项目还包括通过癌症生物学课程的申请人职业发展的计划，教学认证计划，并参加科学会议。总的来说，我预计拟议的工作奖学金将有助于进一步探索NCI RAS倡议的使命，以探索RAS-的新治疗方法 - 驱动的癌症。