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

RIG：开发一种全酶法来生成肽模拟物

• 批准号: 1021547
• 负责人: Anthony Bell
• 金额: $ 19.85万
• 依托单位: University of Southern Mississippi
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1021547&HistoricalAwards=false
• 关键词: RIG; Toward; Development; Enzymatic; Method

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.

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型化合物库。 这些文库将由具有增强的体内应用能力的肽组成。肽由于其高靶结合特异性、效力和低毒性而理想地适合于体内研究（即生物分子成像和基因递送）。因此，肽提供了一种有效的手段来研究细胞途径的特定方面，而不破坏关键的细胞功能。尽管它们具有固有的特异性和效力，但肽具有低稳定性和降低的膜渗透性-这些因素限制了它们的生物稳定性和用于体内研究的实用性。为了克服这些限制，将使用重组表达系统（“PURE”系统）来合成由非标准或“非天然”氨基酸结构单元组成的肽。使用非天然氨基酸的前提是基于被称为非核糖体肽（NRP）的一类有效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能力。然而，参与NRP生产的合成途径是复杂的，并且尚未证明适合于筛选文库的新功能。与传统的表达系统不同，PURE系统提供了对蛋白质/肽合成所需的每个反应组分的精确控制。这允许标准L-氨基酸被多种非天然氨基酸潜在取代以产生NRP样肽。有效的体外分析（即腺苷酸化、核酸酶保护等）已经鉴定出可用于确定定量标准（结合亲和常数），所述定量标准建立底物翻译的阈值。有了这些标准，将合成由L-和非天然氨基酸组成的各种肽库。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大大提高肽诊断和试剂的开发和经济表达，这将影响整个化学生物学领域。更广泛的影响该项目还将为南密西西比大学（USM）的学生提供基础研究的教育培训和经验。作为一个代表性不足的少数群体的成员，主要研究者敏锐地意识到需要提高少数群体在科学中的曝光和参与程度。在USM有来自代表性不足的少数群体，PI可以参与研究和导师的学生的显着池。今年夏天，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加该研究项目。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在STEM相关领域进入大学的代表性不足的少数民族的数量。为了通过外展活动提高公众的科学素养水平，PI将与eMerging Careers Institute（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了探索职业选择和在就业市场竞争的途径。ECI正在密西西比开发一个类似的项目，PI将与ECI合作开发其科学重点。PI认为，这些活动将是提高代表性不足和服务不足群体参与的有效手段，有助于造福科学和整个社会。