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II期提案的长期目标是生产新的酮醇抗生素，具有有效的抗菌活性，可抗大花洛琳敏感性和对大花环抗性的人类病原体。在第一阶段的研究中，我们成功地开发了生产生物学过程，用于生产15-R-6-脱氧胆碱化B，这是15-R-触及霉素（R =各种化学基团）的生化前体，涉及在Escheria Colris cortring cortring cortring cortring cortring cortring cortring cortring trestry restry cortring threstry cortring thtrytry cortring the rectring requs requs requs cortring cortrant restry cortring thtry cortring threstry cortring threstry cortrant cortring thtry。在第二阶段中，该过程将通过向5R-3-羟基-2-羟基-2-甲基戊酸n-乙酰甲基半甲基半甲甲基甲酯（Thioester）喂食（15-R-3-6DEB）的大规模生产，以大规模生产所需的15-R-6-脱氧乙烯酸盐B（15-R-6DEB）（15-R-6DEB）（“ Diketide-Snac”）（“ Diketide-SnAc”）（“ Diketide-snac”）由此产生的15-R-6DEB随后将用于与另一家公司合作生产Ketolide Kosan铅。 II期研究的具体目的是：1）确定产生的聚酮化合物滴度与DEBS PKS，底物供应酶和底物之间的关系。这些数据将有助于我们设计和构建重组大肠杆菌菌株，该大肠杆菌菌株在二酮喂养的奶昔烧瓶发酵中产生> 100 mg/L的15-R-6DEB。 2）通过随机诱变的大肠杆菌菌株DEBS PK和底物供应基因的随机诱变，突变菌株在15 r-6deb滴度中的二烷二甲体喂养中的15倍增加> 10倍，摇动烧瓶发酵。预计这些突变体的改进遗传背景将增强在特定目标中创建的DEBS PKS和底物供应基因的最佳排列的性能。3）从特定的AIM 1中引入最佳代谢的DEBS PKS和底物供应基因，并从特定的大肠杆菌中从特定的AIM中产生E. Coli Recibim Bearie 2中的大肠杆菌菌株，以产生E. coli coli Bearibente> 250-l of 15-l of 15-r for 250-l of 15-r for 250-r for 250-r for 250-MG/l。 Diketide喂养，摇烧烧瓶发酵。 4）通过高细胞密度大肠杆菌培养物在2升搅拌的发酵罐中优化生理和过程参数，以> 1 g/L的最大生产> 1 g/l的生产。这将通过实现特异性目标3获得的菌株来完成。基于15-R-触及霉素A的酮底酸系列类似物具有出色的体外和体内抗菌活性，与当前临床试验中领先的酮醇相当或更好。我们打算与我们的合作伙伴合作将最佳化合物移至临床前测试。