Mechanism and Uses of Transmembrane Helix Insertion by Soluble Peptides

可溶性肽跨膜螺旋插入的机制和用途

• 批准号: 8106730
• 负责人: Oleg A Andreev
• 金额: $ 51.3万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-15 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8106730
• 关键词: Acidity; Appearance; Basic Science; Binding; Biochemical; Biological; Biological Assay; C-terminal; Cell membrane; Cells; Chemicals; Cholesterol; Clinic; Collaborations; Coupled; Cultured Cells; Cultured Tumor Cells; Cytoplasm; Diagnosis; Disulfides; Environment; Escherichia coli; Esters; Event; Fluorescence; Funding; Genetic Screening; Genetic Variation; Goals; Grant; Human; Image; In Vitro; Informatics; Ions; Kinetics; Label; Laboratories; Lead; Link; Lipid Bilayers; Lipids; Location; Medicine; Membrane; Metabolism; Methods; Molecular Weight; Mus; Operative Surgical Procedures; Pathway interactions; Peptides; Pharmaceutical Preparations; Play; Process; Property; Public Health; Publications; Publishing; Reporting; Rhodamine; Role; Signal Transduction; Solutions; Sphingomyelins; Structure; Surface; Surgeon; Technical Expertise; Technology; Testing; Therapeutic; Thermodynamics; Tissues; United States National Institutes of Health; Variant; Vesicle; Water; Work; aqueous; base; cell growth; improved; in vivo; insight; monomer; neoplastic cell; novel strategies; tumor; whole body imaging

DESCRIPTION (provided by applicant): It is a central idea of the NIH that basic research will lead to new approaches in medicine, and we believe that we have found one. As a result of earlier funding of this grant, we have discovered a peptide that (1) targets acidic tissues in vivo, including tumors, (2) can deliver polar molecules into cells, releasing them in the cytoplasm, and (3) gives an opportunity to better understand how peptides can insert across membranes. We now plan to explore both the basic and applied aspects of this discovery. The peptide, which we call "pHLIP" (for pH (Low) Insertion Peptide) is soluble as an unstructured monomer in aqueous solution, binds as an unstructured monomer to the surface of a bilayer or membrane, and inserts across the bilayer as a trans-membrane helix (TM) when the pH is lowered. We have established the basic energetics and kinetics of peptide insertion. We have shown that a labeled version of pHLIP targets and images tumors as small as 1 mm in mice, and that the imaging accurately identifies tumor borders. We have also established that large, polar cargo molecules (M ~ 1000 Da, log P ~ -2) attached to the inserting end of pHLIP by a disulfide are delivered across membranes and released in the cytoplasms of cultured tumor cells at low pH. By continuing our basic research we hope to frame the technology for use in the clinic. We will study the process of and sequence requirements for insertion of water soluble peptides into membranes, find improved ways to target tumors and other acidic tissues, and develop expanded ways to deliver polar molecules into cells, releasing them into the cytoplasm by disulfide or ester cleavage. Targeting imaging agents to tumors with pHLIP could aid in diagnosis or act as a guide for surgery, and delivering therapeutics could assist in treatment. Using biophysical, biochemical, and biological approaches, we will seek answers to the following questions: 1. What are the kinetic intermediates, energetics and structures of the bilayer and peptide during insertion? 2. Which sequence features allow a water-soluble peptide to insert spontaneously to form a TM? 3. What role(s) do lipids play in TM insertion? 4. Can pHLIP be used to image cargo delivery in vivo? 5. What is the range of polar molecules that can be delivered to cells? PUBLIC HEALTH RELEVANCE: Relevance to Public Health We have discovered a new way to target tumors based on their acidity, using a small peptide that inserts across the membranes of the cells in the tissue. The peptide accumulates preferentially in the tumor, which is acidic as a result of its metabolism, and we can put a label on the peptide to image the tumor for diagnosis or to assist a surgeon in removing it by showing where its boundaries are. The peptide can also be used to deliver drugs into the cells of the tumor, so improved therapy might be possible.

描述（申请人提供）：NIH的中心思想是基础研究将导致医学的新方法，我们相信我们已经找到了一个。由于这项资助的早期资助，我们发现了一种肽，它（1）靶向体内酸性组织，包括肿瘤，（2）可以将极性分子递送到细胞中，将它们释放到细胞质中，（3）提供了一个更好地了解肽如何跨膜插入的机会。我们现在计划探索这一发现的基础和应用方面。我们称之为“pHLIP”的肽（pH（低）插入肽）作为非结构化单体可溶于水溶液中，作为非结构化单体结合到双层或膜的表面，并且当pH降低时作为跨膜螺旋（TM）插入穿过双层。我们已经建立了肽插入的基本能量学和动力学。我们已经证明，标记版本的pHLIP靶向和成像小至1 mm的小鼠肿瘤，并且成像准确地识别肿瘤边界。我们还确定了通过二硫键连接到pHLIP插入端的大极性货物分子（M ~ 1000 Da，log P ~-2）在低pH下穿过膜并释放到培养的肿瘤细胞的胞浆中。通过继续我们的基础研究，我们希望将该技术用于临床。我们将研究水溶性肽插入细胞膜的过程和序列要求，找到靶向肿瘤和其他酸性组织的改进方法，并开发将极性分子递送到细胞中的扩展方法，通过二硫键或酯裂解将其释放到细胞质中。用pHLIP将成像剂靶向肿瘤可以帮助诊断或作为手术指南，并且递送治疗剂可以帮助治疗。利用生物物理学、生物化学和生物学方法，我们将寻求以下问题的答案：1。在插入过程中，双层和肽的动力学中间体、能量学和结构是什么？2.哪些序列特征允许水溶性肽自发插入形成TM？3.脂质在TM插入中起什么作用？4. pHLIP能否用于体内货物运输成像？5.可以传递到细胞的极性分子的范围是什么？ 公共卫生相关性：与公共卫生的相关性我们发现了一种基于酸性靶向肿瘤的新方法，使用一种小肽插入组织中细胞的膜。这种肽优先在肿瘤中积累，由于它的代谢，肿瘤是酸性的，我们可以在肽上贴上标签，对肿瘤进行成像诊断，或者通过显示肿瘤的边界来帮助外科医生切除肿瘤。这种肽还可以用于将药物输送到肿瘤细胞中，因此改善治疗可能是可能的。