New Ketolide Antibacterial Drugs

新型酮内酯类抗菌药

• 批准号: 6742436
• 负责人: CHARLES R HUTCHINSON
• 金额: $ 37.5万
• 依托单位: KOSAN BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-15 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6742436
• 关键词: Escherichia coli; Streptomyces; antibacterial agents; bacteria infection mechanism; bacteriolysis; biotechnology; biotherapeutic agent; biotransformation; cholinesterases; coenzyme A; combinatorial chemistry; drug design /synthesis /production; drug discovery /isolation; drug resistance; enzyme activity; enzyme biosynthesis; erythromycin; macrolide antibiotics; microorganism disease chemotherapy; microorganism immunology; molecular genetics; polyketide synthase; polymerase chain reaction; recombinant proteins

DESCRIPTION (provided by applicant): The long-term goal of this Phase II proposal is the production of new ketolide antibiotics with potent antibacterial activity against macrolide-susceptible and macrolide-resistant bacterial pathogens of humans. In Phase I research, we successfully developed a biological process for production of 15-R-6-deoxyerythronolide B, the biochemical precursor of 15-R-erythromycins (R=various chemical groups), that involves expression of the 6-deoxyerythronolide B (DEBS) polyketide synthase (PKS) genes in an Escherichia coli strain carrying the requisite PKS substrate supply genes. In Phase II this process will be optimized for large-scale production of the desired 15-R-6-deoxyerythronolide B (15-R-6dEB) by feeding the 5R-3-hydroxy-2-methylpentanoic acid N-acetylcysteamine thioester ("diketide-SNAC") to an E. coli strain expressing the engineered DEBS1 module 2/DEBS2/DEBS3 genes. The resulting 15-R-6dEB will be used subsequently to produce a lead ketolide Kosan has discovered in partnership with another company. The specific aims for the Phase II research are: 1) to determine the relationship between the titer of polyketide produced and the level of DEBS PKS, substrate supply enzymes and substrates. These data will help us design and construct a recombinant E. coli strain that produces >100 mg/L of 15-R-6dEB in a diketide-fed, shake flask fermentation. 2) To isolate, by random mutagenesis of an E. coli strain bearing DEBS PKS and substrate supply genes, mutant strains with a >10-fold increase in 15-R-6dEB titer in a diketide-fed, shake flask fermentation. The improved genetic background of these mutants is expected to enhance the performance of the optimum arrangement of the DEBS PKS and substrate supply genes created in Specific Aim 1. 3) To introduce the optimal metabolically engineered DEBS PKS and substrate supply genes from Specific Aim 1 into the E. coli strain from Specific Aim 2 to create an E. coli recombinant strain that produces >250 mg/L of 15-R-6dEB in a diketide-fed, shake flask fermentation. 4) To optimize the physiological and process parameters for maximum production of 15-R-6dEB at >1 g/L by high cell density E. coli cultures in a 2 liter stirred fermentor. This will be done with a strain obtained through achievement of Specific Aim 3. Several of the ketolide series of analogs based on 15-R-erythromycin A have excellent in vitro and in vivo antibacterial activity, comparable to or better than the leading ketolides in current clinical trials or approved by the FDA for specific uses. We intend to move the best compound to pre-clinical testing in collaboration with our partner.

描述（由申请方提供）：本II期提案的长期目标是生产对人类大环内酯类敏感和耐药细菌病原体具有强效抗菌活性的新型酮内酯类抗生素。在I期研究中，我们成功地开发了一种生产15-R-6-脱氧红霉素B（15-R-红霉素的生化前体，R=各种化学基团）的生物学方法，该方法涉及在携带必需的PKS底物供应基因的大肠杆菌菌株中表达6-脱氧红霉素B（DEBS）聚酮合酶（PKS）基因。在阶段II中，通过将5 R-3-羟基-2-甲基戊酸N-乙酰基半胱胺硫B（“二酮-SNAC”）进料至E.表达工程化DEBS 1模块2/DEBS 2/DEBS 3基因的大肠杆菌菌株。由此产生的15-R-6dEB随后将用于生产Kosan与另一家公司合作发现的铅酮内酯。II期研究的具体目标是：1）确定所产生的聚酮化合物的滴度与DEBS PKS、底物供应酶和底物水平之间的关系。这些数据将有助于我们设计和构建重组大肠杆菌。大肠杆菌菌株，其在双酮化合物进料的摇瓶发酵中产生>100 mg/L的15-R-6dEB。2)通过随机诱变，分离出一株E.携带DEBS PKS和底物供应基因的大肠杆菌菌株，在双酮化合物进料的摇瓶发酵中15-R-6dEB滴度增加>10倍的突变菌株。预期这些突变体的遗传背景的改善将增强在特定目的1中创建的DEBS PKS和底物供应基因的最佳排列的性能。3)目的：将代谢工程优化的DEBS PKS基因和底物供应基因从特异性目的基因1导入大肠杆菌中。coli菌株，以产生E.大肠杆菌重组菌株，其在双酮化合物进料的摇瓶发酵中产生>250 mg/L的15-R-6dEB。4)为优化高密度E.大肠杆菌培养物在2升搅拌发酵罐中。这将使用通过实现特定目标3获得的菌株进行。 基于15-R-红霉素A的几种酮内酯系列类似物具有优异的体外和体内抗菌活性，与目前临床试验中或FDA批准用于特定用途的主要酮内酯相当或更好。我们打算与我们的合作伙伴合作，将最好的化合物转移到临床前测试。