Discovery of B. pseudomallei therapeutics for Biodefense

发现类鼻疽杆菌用于生物防御的疗法

• 批准号: 7285226
• 负责人: Donald T Moir
• 金额: $ 117.01万
• 依托单位: MICROBIOTIX, INC
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7285226
• 关键词: Acute; Aerosols; Anabolism; Animal Model; Animals; Anti-Bacterial Agents; Antibiotic Therapy; Antibiotics; Bacteria; Biological; Biological Assay; Bioterrorism; Burkholderia pseudomallei; Categories; Cell Wall; Cells; Centers for Disease Control and Prevention (U.S.); Ciprofloxacin; Class; Containment; Development; Dose; Drug Delivery Systems; Drug Design; Drug Kinetics; Engineering; Environment; Enzymes; Exhibits; Exposure to; Facility Construction Funding Category; Genes; Goals; Gram-Negative Bacteria; Growth; Human; Human Resources; Impairment; In Vitro; Infection; Isopropyl Thiogalactoside; Lead; Licensing; Luciferases; Mammalian Cell; Maximum Tolerated Dose; Melioidosis; Metabolic; Modeling; Molecular Profiling; Mus; Operon; Organism; Orthologous Gene; Pharmaceutical Preparations; Pharmacology; Phase; Phase I Clinical Trials; Play; Population; Property; Protein Secretion; Proteins; Pseudomonas aeruginosa; Regulation; Relative (related person); Reporter; Research; Research Personnel; Risk; Role; Safety; Score; Screening procedure; Serum; Specificity; Structure; Testing; Therapeutic; Therapeutic Agents; Toxic effect; Toxicology; Transgenic Organisms; Vaccines; Validation; Virulent; Work; biodefense; cell growth; cytotoxic; drug discovery; high throughput screening; inhibitor/antagonist; innovation; mouse model; novel; promoter; resistance mechanism; response; small molecule libraries; vitamin biosynthesis

DESCRIPTION (provided by applicant): Burkholderia pseudomallei is a bioterrorist threat. With the best current therapies, lethality is typically as high as 40%. The overall goal of this proposal is the development of new drugs against this organism. In Phase I, the high sequence similarity between B. pseudomallei and its less virulent relative Pseudomonas aeruginosa was exploited to build innovative screens for rapid, safe discovery of effective therapeutic agents. Three key discoveries of the Phase I research established the feasibility of this approach: (a) cross-species complementation is efficient enough between these two species to permit construction of P. aeruginosa deletions surviving by virtue of expression of the 6. pseudomallei ortholog in almost 50% of cases attempted; (b) lac regulation is adequate to deplete targets to the point of limiting cell growth in about 33% of cases tested; (c) expression profiling by microarray hybridization revealed genes specifically up-regulated for 4 of 5 depleted target strains tested. Promoters from target depletion-responsive genes were fused to the P. luminescens luxCDABE operon to generate luciferase reporter assays in P. aeruginosa for inhibition of B. pseudomallei targets. Construction and implementation of a model whole cell luciferase reporter screen for inhibitors of GyrA established the feasibility of such screens for antibacterial drug discovery. In Phase II, three new reporter assays for inhibitors of validated B. pseudomallei targets will be used to screen two diverse chemical libraries representing over 150,000 compounds. Confirmed hits will be subjected to a panel of secondary assays for validation as target specific, potent on B. pseudomallei cells, and non- cytotoxic to mammalian cells in culture. Validated hits will be prioritized by potency and drug-like features and optimized by rational drug design aided by structural information for several inhibitor-target co-crystals. Lead compounds will be tested in mice to determine the maximum tolerated dose, pharmacokinetic properties, and efficacy in a B. pseudomallei infection mouse model. In addition, utilizing the expertise gained in Phase I studies, P. aeruginosa whole cell reporter assays will be developed and screened for inhibitors of at least three additional B. pseudomallei targets. In Phase III, a potent, safe, orally active B. pseudomallei inhibitor will be advanced into IND enabling toxicology and safety pharmacology studies and file an IND.

描述（由申请人提供）：Burkholderia pseudomallei是一种生物恐怖主义威胁。使用当前最佳疗法，致死性通常高达40％。该提案的总体目标是针对这种生物体开发新药。在第一阶段中，假杆菌与其毒性较低的相对假单胞菌的高序列相似性被利用以构建创新的筛选，以快速，安全地发现有效的治疗剂。第一阶段研究的三个关键发现确定了这种方法的可行性：（a）这两个物种之间的跨物种互补是有效的，可以允许建造铜绿假单胞菌的缺失，从而在几乎50％的企图的病例中表达了6个。 （b）在大约33％的测试病例中，LAC调节足以耗尽靶标的限制细胞生长； （c）通过微阵列杂交进行表达分析，揭示了针对5个耗尽靶标的菌株中4个特异性上调的基因。将来自靶耗尽响应基因的启动子融合到P. luminescens luxcdabe操纵子上，以在铜绿假单胞菌中生成荧光素酶报告基因测定，以抑制假单胞菌芽孢杆菌的靶标。 Gyra抑制剂的全细胞荧光素酶报告基筛查模型的构建和实施确立了此类筛查对抗菌药物发现的可行性。在第二阶段中，将使用三个新的记者分析，用于验证的B.假单胞菌靶标的抑制剂，用于筛选两个代表150,000种化合物的不同化学文库。确认的命中将接受一组二级测定面板，以验证靶向特异性，对假芽孢杆菌细胞有效，并且在培养中对哺乳动物细胞非细胞毒性。经过验证的命中将通过效力和类似药物的特征来优先考虑，并通过结构信息提供了几种抑制剂 - 靶标的共晶体的合理药物设计。铅化合物将在小鼠中进行测试，以确定假芽孢杆菌感染小鼠模型中最大耐受剂量，药代动力学特性和功效。此外，利用在I期研究中获得的专业知识，将开发并筛选铜绿假单胞菌全细胞报告基因测定法，以筛选至少三个额外的B. pseudomallei靶标的抑制剂。在第三阶段中，有效，安全，活跃的B.假单胞菌抑制剂将被提高到IND促进毒理学和安全药理学研究，并提交IND。