Identification of New Lactobacillus Regulators Responsive to FDA-Approved Drugs

描述(由申请人提供)：活细菌载体在开发更便宜和更安全的疫苗方面是非常有前途的工具。然而，需要更好的可诱导表达系统来规避毒性问题，以实现对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运送疫苗抗原的效率，并使用联邦药品管理局(FDA)批准的药物来调节活载体中的抗原表达。发现一种可能由FDA已经批准的药物触发的监管系统，将构成进入人体试验的巨大优势。此R03应用程序的目标是确定MerR、LysR、TetR、MARR和ICLR家族中对FDA批准的小分子产生体外反应的转录调节因子。我们计划通过1)在乳酸菌基因组中识别能够与FDA批准的小分子相互作用的转录因子来实现这一目标。我们将合理选择我们的靶标和高通量技术来筛选小分子文库。2)用凝胶迁移率改变分析确定药物与转录因子结合的能力，以确定存在其假定的DNA结合部位。这些实验的目的是独立确认在高通量筛选中获得的结果，并帮助鉴定真正的粘结剂，而不是非特异性粘结剂。药物-蛋白质相互作用的亲和力将通过等温滴定量热法确定。其基本原理是，一旦鉴定出有效的新转录因子/小分子组合，旨在优化其有效性的决定性研究将在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。在这项提案完成后，我们将在乳酸菌中鉴定出与FDA批准的药物具有高亲和力的新调节蛋白。其次，通过在存在天然DNA结合序列的情况下测试相互作用的蛋白质/小分子，我们将能够识别出将在体内扰乱蛋白质/DNA相互作用的配体。由于为这项建议选择的转录调控因子家族参与了多重耐药的发展，预计所学到的将同样适用于确定药物靶标，以对抗许多革兰氏阳性病原菌引起的感染。 公共卫生相关性：拟议的研究与公共健康相关，因为我们的长期目标是提高使用乳杆菌载体运送疫苗抗原的有效性。发现可以由已经批准的小分子控制的新载体用于人类，这与公共卫生格外相关，因为这将影响到更便宜、更安全的疫苗的开发，这些疫苗可以很容易地在大量人群中使用。

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.