Mechanism and Uses of Transmembrane Helix Insertion by Soluble Peptides

描述(由申请人提供)：基础研究将导致医学上的新方法，这是NIH的一个中心思想，我们相信我们已经找到了一个。由于这笔赠款的早期资助，我们发现了一种多肽，它(1)靶向体内的酸性组织，包括肿瘤，(2)可以将极性分子输送到细胞中，在细胞质中释放它们，以及(3)提供了更好地了解多肽如何跨膜插入的机会。我们现在计划探索这一发现的基本和应用两个方面。这种多肽，我们称之为“PHLIP”(低pH插入肽)，在水溶液中以非结构单体的形式溶解，以非结构单体的形式结合到双层或膜的表面，并在pH降低时以跨膜螺旋(TM)的形式插入双层。我们已经建立了多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靶标和图像在小鼠体内的肿瘤小到1毫米，成像准确地识别肿瘤边界。我们还证实了在低pH条件下，大的极性货物分子(M~1000 Da，logP~-2)通过二硫键连接到PHLIP的插入端，通过膜转运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，我们希望将这项技术框架用于临床。我们将研究将水溶性多肽插入膜的过程和序列要求，找到针对肿瘤和其他酸性组织的改进方法，并开发扩展的方法将极性分子输送到细胞中，通过二硫键或酯裂解将它们释放到细胞质中。靶向PHLIP肿瘤的显像剂可以帮助诊断或作为手术指南，而提供治疗药物可以帮助治疗。利用生物物理、生化和生物学方法，我们将寻求以下问题的答案：1.双层和多肽在插入过程中的动力学中间体、能量学和结构是什么？2.哪些序列特征允许水溶性多肽自发插入形成TM？3.脂质在TM插入中起什么作用(S)？4.PHLIP能否用于体内货物递送的成像？5.可传递到细胞的极性分子的范围是多少？ 与公共健康相关：与公共健康相关我们发现了一种根据肿瘤的酸性来定位肿瘤的新方法，这种方法使用一种小肽，这种小肽可以插入到组织中的细胞膜上。多肽优先积聚在肿瘤中，由于其新陈代谢的结果是酸性的，我们可以在多肽上贴上标签，以对肿瘤进行成像诊断，或通过显示其边界来帮助外科医生切除肿瘤。这种多肽还可以用来将药物输送到肿瘤细胞中，因此改进治疗方法是可能的。

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.