RIG: Toward the Development of an All Enzymatic Method to Generate Peptidomimetics

该项目的长期目标是开发一种独特的方法来生成新化合物的库，这些化合物可能包含具有重要生物活性的特定分子。这些文库将被组成或具有增强能力的多肽，用于体内应用。多肽具有较高的靶标结合特异性、效价和低毒性，非常适合于体内研究(如生物分子成像和基因传递)。因此，多肽提供了一种有效的手段来研究细胞途径的特定方面，而不会破坏关键的细胞功能。尽管多肽具有固有的特异性和效力，但它们的稳定性较低，膜透性降低--这些因素限制了它们的生物稳定性和在体内研究中的应用。为了克服这些限制，重组表达系统(“纯”系统)将被用来合成由非标准或“非天然”氨基酸组成的多肽。使用非天然氨基酸的前提是基于一类被称为非核糖体肽(NRPs)的天然产物的化学组成。NRP含有各种非天然残基，这些残基使这些生物分子具有增强的功能。然而，参与NRP产生的合成途径是复杂的，并且还没有被证明适合于为新的功能筛选文库。与传统的表达系统不同，纯系统提供了对蛋白质/肽合成所需的每个反应成分的精确控制。这使得标准的L氨基酸有可能被各种非天然氨基酸取代，从而产生核糖核酸样肽。高效的体外检测(如腺化、核酸酶保护等)已确定可用于确定建立底物翻译阈值的定量标准(结合亲和力常数)。有了这些标准，就可以合成由L和非天然氨基酸组成的不同的多肽文库。该研究项目融合了成熟的和新兴的细胞表达技术；这种翻译系统的智力和科学价值可以极大地促进多肽诊断和试剂的开发和经济表达，这些将影响整个化学生物学领域。广泛的影响该项目还将为南密西西比大学(USM)的学生提供教育培训和基础研究经验。作为代表不足的少数群体的一员，首席调查员敏锐地意识到有必要增加少数群体在科学方面的接触和参与程度。在密歇根州立大学，有一大批学生来自代表不足的少数群体，PI可以让他们参与研究和指导。今年夏天，参加密歇根州立大学本科生研究体验(REU)计划的一名人数不足的本科生将参加该研究项目。这位首席调查员目前还指导密西西比州研究生教育联盟(AGEM)的学生。AGEM的一个主要目标是增加在STEM相关领域进入学术界的人数不足的少数群体。为了通过外联活动提高公众的科学素养水平，该协会将与新兴职业研究所(ECI)合作，通过一个旨在影响青年和年轻人的在线出版物传播科学信息。ECI为阿拉米达县的高中和大专青年提供了探索职业选择和在就业市场上竞争的途径。ECI正在密西西比州开发一个类似的项目，PI将与ECI合作开发其科学重点。国际和平协会认为，这些活动将是增加代表不足和服务不足群体的参与的有效手段，有助于造福于整个科学和社会。

The long-term goal of this project is to develop a unique method to generate libraries of novel compounds that are likely to contain specific molecules that have important biological activities. These libraries will be composed or peptides with enhanced capabilities for in vivo applications. Peptides are ideally suited for in vivo studies (i.e. biomolecular imaging and gene delivery) due to their high target binding specificity, potency and low toxicity. As a result, peptides provide an effective means to investigate specific aspects of cellular pathways without disrupting critical cellular functions. Despite their inherent specificity and potency, peptides suffer from low stability and reduced membrane permeability -- factors that limit their biological stability and utility for in vivo studies. To overcome these limitations, a recombinant expression system (the "PURE" system) will be used to synthesize peptides composed of nonstandard or 'unnatural' amino acid building blocks. The premise for using unnatural amino acids is based upon the chemical composition of a potent class of natural products known as nonribosomal peptides (NRPs). NRPs contain a variety of unnatural residues that equip these biomolecules with enhanced functional capabilities. However, the synthetic pathways involved in NRP production are complex, and have not proven amenable to screening libraries for novel functionalities. Unlike conventional expression systems, the PURE system affords precise control over each of the reaction components required for protein/peptide synthesis. This allows potential substitution of the standard L-amino acids by a variety of unnatural amino acids to generate NRP-like peptides. Efficient in vitro assays (i.e. adenylation, nuclease protection, etc.) have been identified that can be used to determine quantitative criteria (binding affinity constants) that establish a threshold for translation of a substrate. With these criteria in hand, diverse peptide libraries composed of L- and unnatural amino acids will be synthesized. The research project merges well established and emerging cell expression techniques; the intellectual and scientific merits of this translation system could greatly enhance the development and economic expression of peptide diagnostics and reagents that will impact the field of chemical biology in general.Broader ImpactsThe project will also offer educational training and experience in basic research to the students of The University of Southern Mississippi (USM). As a member of an underrepresented minority group, the principle investigator is keenly aware of the need to increase the level of exposure and participation of minority groups in science. At USM there is a significant pool of students from underrepresented minority groups that the PI can involve in research and mentor. This summer an underrepresented undergraduate student participating in the Research Experience for Undergraduate (REU) program at USM will participate in the research project. The principle investigator also currently mentors students from the Alliance for Graduate Education in Mississippi (AGEM). A primary goal of AGEM is to increase the number of underrepresented minorities entering academe in STEM related fields. To raise the level of scientific literacy of the public through outreach activities, the PI will partner with the eMerging Careers Institute (ECI) to disseminate scientific information through an online publication designed to impact youth and young adults. ECI provides high school and junior college youth of Alameda County with pathways to explore career options, and compete in the job market. ECI is developing a similar program in Mississippi and the PI will work with ECI on the development of its scientific focus. The PI believes that these activities will be an effective means to increase the participation of underrepresented and underserved groups to help benefit science and society as a whole.