Mechanism and Uses of Transmembrane Helix Insertion by Soluble Peptides

描述（由申请人提供）：基础研究将为医学带来新方法，这是NIH的核心理念，我们相信我们已经找到了一个。由于这项拨款的早期资助，我们已经发现了一种肽，它(1)针对体内的酸性组织，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，(3)为更好地了解肽如何跨膜插入提供了机会。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”（低pH插入肽）的肽在水溶液中是可溶的非结构化单体，它以非结构化单体的形式结合在双分子层或膜的表面，并在pH降低时以跨膜螺旋（TM）的形式插入双分子层。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毫米的小鼠肿瘤，并且成像准确地识别肿瘤边界。我们还确定，通过二硫化物附着在phillip插入端的大极性货物分子（M ~ 1000 Da, log P ~ -2）在低ph下通过膜传递并释放到培养的肿瘤细胞的细胞质中。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，寻找针对肿瘤和其他酸性组织的改进方法，并开发将极性分子传递到细胞中的扩展方法，通过二硫或酯裂解将其释放到细胞质中。针对肿瘤的显像剂可以帮助诊断或作为手术指导，并且提供治疗方法可以帮助治疗。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。在插入过程中，双分子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. philips能否用于活体货物运输成像？5. 可以输送到细胞的极性分子的范围是多少？

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.