New Ketolide Antibacterial Drugs

新型酮内酯类抗菌药

• 批准号: 6645823
• 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/6645823
• 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(申请人提供)：这一第二阶段提案的长期目标是生产新的酮内酯抗生素，对人类对大环内酯类敏感和耐大环内酯类细菌病原体具有强大的抗菌活性。在第一阶段的研究中，我们成功地开发了一种生产15-R-红霉素(R=各种化学基团)的生化前体15-R-6-脱氧赤霉素内酯B的生物工艺，该工艺涉及在携带必需的PKS底物供应基因的大肠杆菌中表达6-脱氧赤霉内酯B(Debs)聚酮合成酶(PKS)基因。在第二阶段，通过将5R-3-羟基-2-甲基戊酸N-乙酰半胱胺硫酯(“二酮-SNAC”)喂入表达工程DEBS1模块2/DEBS2/DEBS3基因的大肠杆菌菌株，该工艺将进行优化，以大规模生产所需的15-R-6-脱氧赤藓内酯B(15-R-6dEB)。合成的15-R-6dEB随后将用于生产科桑与另一家公司合作发现的铅酮内酯。第二阶段研究的具体目标是：1)确定生产的聚酮滴度与底物供给酶、底物供给酶和底物的水平之间的关系。这些数据将有助于我们设计和构建一株在双酮补料摇瓶发酵中生产&gt；100 mg/L 15-R-6dEB的重组大肠杆菌。2)通过对一株含有Debs PKS和底物供应基因的大肠杆菌菌株进行随机诱变，筛选出15-R-6dEB滴度提高10倍的突变菌株。这些突变体的遗传背景的改善有望提高针对特定目的1创造的Debs PKS和底物供应基因的最佳排列的表现。3)将来自特定目的1的最优代谢工程的Debs PKS和底物供应基因从特定目的2导入大肠杆菌菌株，以创建一株重组大肠杆菌，该重组菌株在摇瓶发酵中能够生产&gt；250 mg/L的15-R-6dEB。4)优化了2升搅拌发酵罐高密度发酵生产15-R-6dEB的生理和工艺参数，使其产量达到1 g/L。这将用通过实现特定目标而获得的菌株来完成。3.几种基于15-R-红霉素A的酮内酯系列类似物在体外和体内具有优异的抗菌活性，可与当前临床试验中的主要酮内酯或FDA批准的特定用途的酮内酯相媲美或更好。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。