Biophysical and Mechanistic Determinants for Cancer Cell Import of Hydrocarbon-Stapled Peptides

癌细胞输入碳氢化合物肽的生物物理和机制决定因素

基本信息

批准号：
9178990
负责人：
Loren David Walensky
金额：
$ 19.03万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-02 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9178990
关键词：
Address Affinity Algorithms Antineoplastic Agents BCL1 Oncogene BCL2 gene BCL2L11 gene Biological Biology Biostatistical Methods CRISPR screen CRISPR/Cas technology Candidate Disease Gene Cell physiology Cells Chemicals Circular Dichroism Complex Computing Methodologies Data Set Disease EZH2 gene Environment Event Exhibits Foundations Gene Deletion Glean Goals High Pressure Liquid Chromatography Human Hydrocarbons KRAS2 gene Laboratories Libraries Malignant Neoplasms Measurement Measures Mediating Metabolic Methods Modality Molecular Nutrient Oncogenic Pathologic Pathway interactions Penetrance Peptide Hydrolases Peptide Library Peptides Pharmaceutical Preparations Pinocytosis Population Principal Component Analysis Property Protein Family Proteins Research Resistance Scanning Screening for cancer Shapes Signal Transduction Signaling Protein Son of Sevenless Proteins Specificity Statistical Data Interpretation Statistical Methods Targeted Research Therapeutic Time Translating Vesicle Transport Pathway base biophysical properties cancer cell cancer therapy crosslink cytotoxicity design drug development experience genome-wide genome-wide analysis insight interdisciplinary approach novel therapeutics peptide drug peptide structure prototype small molecule therapeutic target tool uptake validation studies

项目摘要

PROJECT SUMMARY Deregulated intracellular protein interactions mediate a complex network of pathologic signaling events that drive human cancer. The development of drugs to modulate the large, flat, and complex interfaces of oncogenic signaling proteins remains a formidable challenge and has inspired alternative approaches to traditional small molecule discovery. Although the very peptide structures that define the molecular handshakes between proteins are ideally suited to modulate such signaling events, structured peptides in the context of a protein typically lose their bioactive shape when isolated from the whole. We previously developed hydrocarbon-stapled peptides that recapitulate the natural shape of -helical interaction motifs by insertion of chemical crosslinks. Stapled peptides are structurally-stable, protease-resistant, and retain the capacity to engage their biological targets with natural potency and specificity. Unexpectedly, select stapled -helical peptides are also cell- permeable, opening the door to an entirely new modality for dissecting and potentially drugging pathologic protein interactions in cancer. However, despite a decade of progress in the creation of new research tools to investigate and target disease-causing proteins, the true promise of structured peptides as a novel therapeutic platform for cancer has been hampered by our very limited understanding of two fundamental questions: (1) What biophysical properties dictate whether a stapled peptide will be taken up by a cancer cell? (2) What is the explicit molecular mechanism of cellular import and intracellular release for cell-permeable stapled peptides? The answers to these questions not only carry the potential to transform these research tools and prototype therapeutics into an arsenal of bona fide cancer drugs, but will also provide critical new insight into the essential cellular process of vesicle transport. For example, certain cancer cells enforce metabolic immortality by upregulating protein nutrient uptake via the pinocytosis import pathway – a mechanism that could be leveraged to achieve a therapeutic window for structured peptide-based cancer drugs. Here, I propose a two-pronged research plan that harnesses our deep experience with stapled peptide design and application, but diverges from our traditional protein targeting research to instead focus on the why and how distinct stapled peptides access the intracellular environment. To achieve our goals, we will (1) employ biostatistical and computational methods to glean what biophysical parameters confer cellular penetrance among our libraries of stapled peptides, and (2) perform a genome-wide CRISPR-based screen to identify and then vet those cellular components that alternatively impair and enhance the import pathway for stapled peptides. By integrating our laboratory’s foundation in the chemical biology of oncogenic protein interactions with the proposed biostatistical, CRISPR screening, and cancer cell import validation studies, we hope to break new ground in our understanding of just how stapled peptides and their uptake mechanisms can be harnessed for therapeutic benefit in cancer.

项目概要细胞内蛋白质相互作用失调介导病理信号事件的复杂网络，开发调节大而平坦且复杂的致癌界面的药物。信号蛋白仍然是一个艰巨的挑战，并激发了传统小信号蛋白的替代方法分子发现。虽然正是肽结构定义了分子之间的握手。蛋白质非常适合调节此类信号事件，蛋白质背景下的结构化肽当从整体中分离出来时，通常会失去其生物活性形状。通过插入化学交联来重现α-螺旋相互作用基序的自然形状的肽。钉合肽结构稳定，具有蛋白酶抗性，并保留与其生物结合的能力出乎意料的是，选定的钉合α-螺旋肽也具有细胞特异性。可渗透，为解剖和潜在药物病理学的全新模式打开了大门然而，尽管在创建新的研究工具方面取得了进展，但癌症中的蛋白质相互作用。研究并靶向致病蛋白质，这是结构化肽作为新型治疗方法的真正前景我们对两个基本问题的理解非常有限，这阻碍了癌症平台的发展：(1) 哪些生物物理特性决定了钉合肽是否会被癌细胞吸收？（2）什么是？细胞渗透性钉合肽的细胞输入和细胞内释放的明确分子机制？这些问题的答案不仅有可能改变这些研究工具和原型治疗方法进入真正的癌症药物库，但也将为基本的治疗提供重要的新见解例如，某些癌细胞通过囊泡运输来实现代谢永生。通过胞饮作用输入途径上调蛋白质营养吸收——一种可以利用的机制为了实现基于结构化肽的癌症药物的治疗窗口，我提出了两个方面的建议。研究计划利用了我们在钉合肽设计和应用方面的丰富经验，但存在分歧从我们传统的蛋白质靶向研究转向关注钉合肽的原因和差异为了实现我们的目标，我们将（1）采用生物统计和计算。收集哪些生物物理参数赋予我们的装订文库中的细胞外显率的方法肽，(2) 进行基于 CRISPR 的全基因组筛选，以识别并审查这些细胞通过整合我们的成分，可以损害或增强装订肽的进口途径。实验室在致癌蛋白相互作用的化学生物学方面的基础与所提出的生物统计学， CRISPR筛选和癌细胞导入验证研究，我们希望在我们的理解上取得新的突破如何利用钉合肽及其摄取机制来治疗癌症。