Identification of New Lactobacillus Regulators Responsive to FDA-Approved Drugs

描述（由申请人提供）：活细菌载体是开发更便宜和更安全疫苗的非常有前途的工具。然而，需要能够避免毒性问题的更好的诱导型表达系统，以实现体内抗原表达的精确时间和空间控制。我们的长期目标是提高使用乳杆菌载体递送疫苗抗原的功效，并使用联邦药物管理局（FDA）批准的药物调节活载体中的抗原表达。一个监管系统的发现，可以触发一个已经被FDA批准的药物，将构成一个巨大的优势，达到人体试验。本R 03申请的目的是鉴定MerR、LysR、TetR、马尔和IclR家族中的转录调节因子，这些转录调节因子在体外对FDA批准的小分子有反应。我们计划通过以下方式实现这一目标：1）鉴定乳杆菌基因组中能够与FDA批准的小分子相互作用的转录因子。我们将进行合理的目标选择和高通量技术筛选小分子文库。2)使用电泳迁移率变动分析确定药物在存在其推定的DNA结合位点的情况下结合转录因子的能力。这些实验旨在独立确认高通量筛选中获得的结果，并帮助鉴别与非特异性结合剂相比的真正结合剂。药物-蛋白质相互作用的亲和力将通过等温滴定量热法定义。其基本原理是，一旦确定了有效的新转录因子/小分子组合，将在R 01水平证明旨在优化其疗效的确定性研究是合理的。这种方法是创新的，因为发现一种新的蛋白质，这种蛋白质结合了一种已经被FDA批准用于人类的化学物质，这将是进入人体试验阶段的一个非常重要的垫脚石。在完成这项提案后，我们将在乳酸杆菌中发现新的调节蛋白，这些蛋白对FDA批准的药物具有高亲和力。第二，通过在天然DNA结合序列存在下测试相互作用蛋白/小分子，我们将能够鉴定将破坏体内蛋白/DNA相互作用的配体。由于家族的转录调节剂选择这个建议参与多药耐药性的发展，预计所学到的将同样适用于药物靶点的识别，以打击由许多革兰氏阳性病原菌引起的感染。 公共卫生相关性：拟议的研究与公共卫生有关，因为我们的长期目标是提高使用乳酸杆菌载体递送疫苗抗原的功效。发现可以通过已经批准用于人类的小分子控制的新载体与公共卫生特别相关，因为它将影响更便宜和更安全的疫苗的开发，这些疫苗可以很容易地用于大规模人群。

DESCRIPTION (provided by applicant): Live bacterial vectors are very promising tools in the development of cheaper and safer vaccines. However, better inducible expression systems that can circumvent toxicity problems are required to enable precise temporal and spatial control of antigen expression in vivo. Our long-term goal is to increase the efficacy with which vaccine antigens can be delivered using Lactobacillus vectors and to regulate the antigen expression in the live vector using a Federal Drug Administration (FDA) approved drug. The finding of a regulatory system that could be triggered by a drug that is already approved by the FDA would constitute an enormous advantage to reach human trials. The objective of this R03 application, is to identify transcriptional regulators in the MerR, LysR, TetR, MarR, and IclR families that respond in vitro to small molecules approved by the FDA. We plan to achieve this objective by 1) Identifying transcription factors within the genomes of lactobacilli that are able to interact with FDA approved small molecules. We will perform a rational in silico selection of our targets and high throughput technology for the screening of small molecule libraries. 2) Determining the ability of the drug to bind the transcription factor in presence of its putative DNA binding site using electrophoretic mobility shift assays. These experiments are aimed to independently confirm the results obtained in the high throughput screen and help in the identification of true binders in contrast to unspecific binders. The affinity of the drug-protein interaction will be defined by isothermal titration calorimetry. The rationale is that, once efficacious new transcription factor/small molecule combos are identified, definitive studies designed to optimize their efficacy will be justified at the R01 level. The approach is innovative because the discovery of a new protein that binds a chemical that has already been approved by the FDA for its use in humans will be a very important stepping stone in reaching the human trial phase. After completion of this proposal we will have identified new regulatory proteins in lactobacilli that respond with high affinity to FDA approved drugs. Second, by testing the interaction protein/small molecule in the presence of their native DNA binding sequence we will be able to have identified ligands that will disrupt the protein/DNA interaction in vivo. Since the families of transcriptional regulators selected for this proposal are involved in the development of multidrug resistance it is anticipated that what is learned will be equally applicable to the identification of drug targets to combat infections caused by many gram-positive pathogenic bacteria. Public Health Relevance: The proposed research is relevant to the public health because our long-term goal is to increase the efficacy with which vaccine antigens can be delivered using Lactobacillus vectors. The finding of new vectors that can be controlled by already-approved small molecules for its use in humans is exceptionally relevant to public health since it will impact in the development of cheaper and safer vaccines that could be easily administer to large populations.