Identification of New Lactobacillus Regulators Responsive to FDA-Approved Drugs

鉴定对 FDA 批准的药物有反应的新型乳酸菌调节剂

• 批准号: 7588490
• 负责人: Graciela L Lorca
• 金额: $ 7.33万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7588490
• 关键词: Adjuvant; Adopted; Affinity; Agreement; Animal Model; Antibiotic Resistance; Antigens; Aromatic Compounds; Bacillus (bacterium); Bacteria; Binding; Binding Sites; Calculi; Calorimetry; Chemicals; Clostridium; Collaborations; Computer Simulation; DNA; DNA Binding; DNA-Protein Interaction; Data; Development; Drug Delivery Systems; Electrophoretic Mobility Shift Assay; Environment; Family; Food Industry; Gastrointestinal tract structure; Gene Expression; Gene Targeting; Genome; Genus staphylococcus; Goals; Grant; Health Food; Human; Immune response; In Vitro; Infection; Innovative Therapy; Lactobacillus; Lactose; Learning; Life; Ligands; Listeria; Molecular Conformation; Multi-Drug Resistance; Multidrug Resistance Induction; Outcome; Pharmaceutical Preparations; Phase; Population; Production; Protein Binding; Proteins; Public Health; Publications; Recombinants; Regulation; Research; Research Design; Research Personnel; Research Project Grants; Resources; Screening Result; Screening procedure; Solutions; Streptococcus; System; Technology; Testing; Titrations; Toxic effect; Training; Vaccine Antigen; Vaccines; Work; antimicrobial drug; bacterial vector; base; combat; design; efflux pump; functional genomics; genetic regulatory protein; genome sequencing; high throughput screening; high throughput technology; in vivo; innovation; meetings; pathogenic bacteria; promoter; protein purification; public health relevance; research study; small molecule; small molecule libraries; tool; transcription factor; vector

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.

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，需要能够规避毒性问题的更好的诱导表达系统，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。发现一个可以由 FDA 批准的药物触发的监管系统将为进行人体试验带来巨大的优势。该 R03 应用的目的是鉴定 MerR、LysR、TetR、MarR 和 IclR 家族中的转录调节因子，这些调节因子在体外对 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) 鉴定乳酸杆菌基因组内能够与 FDA 批准的小分子相互作用的转录因子。我们将对目标进行合理的计算机选择，并采用高通量技术来筛选小分子文库。 2) 使用电泳迁移率变动分析确定药物在其推定的 DNA 结合位点存在的情况下结合转录因子的能力。这些实验旨在独立确认在高通量筛选中获得的结果，并帮助识别与非特异性结合物相比的真正结合物。药物-蛋白质相互作用的亲和力将通过等温滴定量热法来定义。理由是，一旦确定了有效的新转录因子/小分子组合，旨在优化其功效的明确研究将在 R01 水平上得到证实。该方法具有创新性，因为发现一种与 FDA 已批准用于人体的化学物质结合的新蛋白质将成为进入人体试验阶段的非常重要的垫脚石。完成该提案后，我们将在乳酸杆菌中鉴定出新的调节蛋白，这些蛋白对 FDA 批准的药物具有高亲和力。其次，通过在天然 DNA 结合序列存在的情况下测试相互作用蛋白/小分子，我们将能够鉴定出会破坏体内蛋白质/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性的发展，因此预计所学到的知识将同样适用于识别药物靶标，以对抗许多革兰氏阳性病原菌引起的感染。 公共健康相关性：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传递疫苗抗原的功效。发现可以由已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、更安全的疫苗的开发，这些疫苗可以轻松地用于大量人群。