New Ketolide Antibacterial Drugs

新型酮内酯类抗菌药

• 批准号: 6906470
• 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/6906470
• 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（由申请人提供）：该二期提案的长期目标是生产新型酮内酯类抗生素，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。在第一阶段研究中，我们成功开发了一种生产 15-R-6-deoxyerythronolide B 的生物工艺，15-R-6-deoxyerythronolide B 是 15-R-红霉素（R=各种化学基团）的生化前体，其中涉及 6-deoxyerythronolide B 的表达（ DEBS) 大肠杆菌菌株中的聚酮合酶 (PKS) 基因携带必需的 PKS 底物供应基因。在第二阶段，该工艺将通过喂入 5R-3-羟基-2-甲基戊酸 N-乙酰半胱胺硫酯（“ diketide-SNAC”）转化为表达工程化 DEBS1 模块 2/DEBS2/DEBS3 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酮内酯铅。 II期研究的具体目标是：1）确定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。这些数据将帮助我们设计和构建重组大肠杆菌菌株，该菌株在双酮化合物补料的摇瓶发酵中产生 >100 mg/L 的 15-R-6dEB。 2) 通过对带有 DEBS PKS 和底物供应基因的大肠杆菌菌株进行随机诱变，分离出在双酮化合物补料的摇瓶发酵中 15-R-6dEB 滴度增加 >10 倍的突变菌株。这些突变体遗传背景的改善预计将增强特定目标 1 中创建的 DEBS PKS 和底物供应基因的最佳排列的性能。 3) 将特定目标 1 中的最佳代谢工程 DEBS PKS 和底物供应基因引入来自 Specific Aim 2 的大肠杆菌菌株，用于创建大肠杆菌重组菌株，该菌株在二酮化合物补料的摇瓶发酵中产生 >250 mg/L 的 15-R-6dEB。 4) 优化生理和工艺参数，以在 2 升搅拌发酵罐中通过高细胞密度大肠杆菌培养物以 >1 g/L 最大产量生产 15-R-6dEB。这将通过实现特定目标 3 获得的菌株来完成。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，与目前领先的酮内酯相当或更好。临床试验或 FDA 批准用于特定用途。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。