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、更安全疫苗的非常有前途的工具。然而，需要更好的诱导表达系统来规避毒性问题，从而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酸菌载体递送疫苗抗原的效力，并使用美国联邦药物管理局（FDA）批准的药物调节抗原在活载体中的表达。发现一种已经被FDA批准的药物可以触发的监管系统，将对进入人体试验构成巨大的优势。此次R03申请的目的是鉴定MerR、LysR、TetR、MarR和IclR家族中对FDA批准的小分子有体外反应的转录调节因子。我们计划通过1)鉴定乳酸菌基因组中能够与FDA批准的小分子相互作用的转录因子来实现这一目标。我们将对我们的目标和高通量技术进行合理的硅选择，以筛选小分子文库。2)利用电泳迁移位移法测定药物在假定DNA结合位点存在的情况下与转录因子结合的能力。这些实验旨在独立确认高通量筛选中获得的结果，并有助于鉴别真正的结合物与非特异性结合物。药物-蛋白质相互作用的亲和力将通过等温滴定量热法来确定。其基本原理是，一旦确定了有效的新转录因子/小分子组合，设计用于优化其疗效的明确研究将在R01水平上得到证明。这种方法是创新的，因为发现一种新的蛋白质可以结合一种已经被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。在完成该提案后，我们将在乳酸菌中确定新的调节蛋白，这些蛋白对FDA批准的药物具有高亲和力。其次，通过测试蛋白质/小分子在其天然DNA结合序列存在下的相互作用，我们将能够确定在体内破坏蛋白质/DNA相互作用的配体。由于本提案选择的转录调节因子家族涉及多药耐药的发展，预计所学到的知识将同样适用于识别药物靶点，以对抗许多革兰氏阳性致病菌引起的感染。

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.