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

基本信息

批准号：
7758802
负责人：
Graciela L Lorca
金额：
$ 7.25万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-02-01 至 2012-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7758802
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

项目摘要

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）：实时细菌向量是廉价和更安全疫苗开发的非常有前途的工具。但是，需要更好的诱导表达系统来规避毒性问题，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传递疫苗抗原的疗效，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。可能由FDA批准的药物触发的监管系统的发现将构成进行人类试验的巨大优势。该R03应用的目的是识别MERR，LYSR，TERT，MARR和ICLR家族中对FDA批准的小分子反应的转录调节剂。我们计划通过1）识别能够与FDA批准的小分子相互作用的基因组中的转录因子。我们将对目标的硅选择和高吞吐量技术进行合理的理性，以筛选小分子库。 2）确定使用电泳迁移率转移测定法在其假定的DNA结合位点存在下结合转录因子的能力。这些实验的目的是独立确认在高吞吐量屏幕中获得的结果，并有助于鉴定真正的粘合剂与非特异性粘合剂相比。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，旨在优化其功效的确定研究将在R01水平上证明是合理的。这种方法具有创新性，因为发现一种新蛋白质结合了已经被FDA批准的化学物质用于人类使用的化学物质，这将是进入人类试验阶段的非常重要的垫脚石。该提案完成后，我们将确定乳酸杆菌中的新调节蛋白，这些蛋白质对FDA批准的药物有很高的亲和力。其次，通过在存在天然DNA结合序列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/DNA相互作用的配体。由于为该提案选择的转录调节剂家族涉及多药耐药性的发展，因此预计所学的知识将同样适用于鉴定药物靶标，以打击由许多革兰氏阳性致病细菌引起的感染。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杆菌载体传递疫苗抗原的功效。可以通过已经批准的小分子来控制的新向量与公共卫生非常相关，因为它会影响更便宜，更安全的疫苗的开发，这些疫苗可以很容易地用于大型人群。