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（由申请人提供）：基础研究将导致医学方面的新方法，这是NIH的核心思想，我们认为我们已经找到了一种。由于该赠款的较早资助，我们发现了一种肽，即（1）靶向体内酸性组织，包括肿瘤，（2）可以将极性分子传递到细胞中，并在细胞质中释放它们，并且（3）给出了（3）有机会更好地了解肽如何在整个膜上插入。现在，我们计划探索这一发现的基本和应用方面。我们称之为“ phlip”（对于pH（低）插入肽）的肽是在水溶液中溶于无组织单体的肽，它作为非结构化的单体与双层或膜的表面结合，并插入双层，并插入反式的单体。 - 板螺旋螺旋（TM）降低时。我们已经建立了肽插入的基本能量和动力学。我们已经表明，小鼠中标记的Phlip靶标和图像肿瘤的标记版本小至1 mm，并且成像准确地识别了肿瘤边界。我们还确定，通过二硫化物连接到Phlip插入的大型极性货物分子（M〜1000 DA，log P〜 -2）在低pH值以低pH值的培养肿瘤细胞的细胞肿瘤中释放。通过继续我们的基础研究，我们希望构架该技术在诊所中的使用。我们将研究将水溶性肽插入膜中的过程和序列要求，找到靶向肿瘤和其他酸性组织的改进方法，并开发出扩展的方式将极性分子递送到细胞中，从。将成像剂靶向具有PHLIP的肿瘤可以有助于诊断或充当手术指南，并且提供治疗剂可以帮助治疗。使用生物物理，生化和生物学方法，我们将寻求以下问题的答案：1。插入过程中双层和肽的动力学中间体，能量和结构是什么？ 2。哪个序列特征允许水溶性肽自发插入以形成TM？ 3。脂质在TM插入中扮演什么角色？ 4。可以使用phlip在体内图像货物交付吗？ 5。可以输送到细胞的极性分子的范围是多少？ 公共卫生相关性：与公共卫生相关，我们使用了一种基于肿瘤的酸度来靶向肿瘤的新方法，该肽使用小肽插入组织中细胞的膜上的小肽。肽在肿瘤中优先积累，这是由于其代谢而酸性的，我们可以在肽上放置标签，以对肿瘤进行成像以诊断或通过显示其边界的位置来帮助外科医生去除肿瘤。肽也可用于将药物输送到肿瘤细胞中，因此可以改善治疗。