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可能包含具有重要生物活性的特定分子的新化合物库。这些文库将由具有增强的体内应用能力的肽组成。多肽由于其高靶向结合特异性、效力和低毒性，非常适合于体内研究（即生物分子成像和基因传递）。因此，多肽提供了一种有效的方法来研究细胞通路的特定方面，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，但其稳定性低，膜通透性降低，这些因素限制了其生物稳定性和体内研究的实用性。为了克服这些限制，重组表达系统（“PURE”系统）将用于合成由非标准或“非天然”氨基酸构建块组成的肽。使用非天然氨基酸的前提是基于一类被称为非核糖体肽（nrp）的强效天然产物的化学成分。nrp含有多种非天然残基，使这些生物分子具有增强的功能能力。然而，NRP产生的合成途径是复杂的，并没有被证明可以筛选新的功能库。与传统的表达系统不同，PURE系统可以精确控制蛋白质/肽合成所需的每个反应组分。这允许用各种非天然氨基酸替代标准的l -氨基酸来生成nrp样肽。有效的体外测定（即腺苷酸化，核酸酶保护等）已被确定，可用于确定定量标准（结合亲和常数），建立底物翻译的阈值。有了这些标准，由L-氨基酸和非天然氨基酸组成的多种肽库将被合成。该研究项目融合了成熟的和新兴的细胞表达技术；该翻译系统的智力和科学优点可以极大地促进多肽诊断和试剂的发展和经济表达，这将影响化学生物学的一般领域。更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。作为一个代表性不足的少数群体的成员，首席研究员敏锐地意识到需要增加少数群体在科学中的曝光和参与水平。在南加州大学，有大量来自未被充分代表的少数群体的学生，PI可以参与研究和指导。今年夏天，一个参加USM本科生研究经验（REU）计划的代表性不足的本科生将参与研究项目。主要研究者目前也指导来自密西西比研究生教育联盟（AGEM）的学生。AGEM的一个主要目标是增加未被充分代表的少数民族进入STEM相关领域的学术界的人数。为了通过外联活动提高公众的科学素养水平，PI将与新兴职业研究所（ECI）合作，通过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探索职业选择的途径，并在就业市场上竞争。ECI正在密西西比州开发一个类似的项目，PI将与ECI合作发展其科学重点。PI认为，这些活动将是增加代表性不足和服务不足的群体的参与的有效手段，以帮助造福科学和整个社会。

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.