An innovative metabolic engineering strategy for the discovery of novel macrolide antibiotics

 描述(申请人提供)：耐多药病原微生物的迅速出现对公众健康构成了重大威胁，对发现新的抗菌剂的需求日益增长。大环内酯类抗生素，如14元大环内酯类红霉素和第二代类似物克拉霉素和阿奇霉素，是临床上用于治疗呼吸道感染的一线药物。然而，由于这些药物在临床上的过度使用，大环内酯类药物的耐药机制迅速出现。相反，16元大环内酯显示了克服影响14元和15元大环内酯的抗性机制的能力。事实上，一些精选的16元大环内酯已经成功地用于美国以外的细菌感染的临床治疗。然而，尽管16元大环内酯类化合物的潜力已被证明，但在开发新的人类抗菌剂方面仍未得到充分开发。泰乐菌素的生物合成途径产生一系列16元大环内酯类化合物，其中最著名的是兽医治疗泰乐菌素。在这项SBIR建议中，Alluvium Biosciences建议对泰乐菌素的生物合成途径进行基因工程，以生产新的16元大环内酯化合物，用于新的抗生素药物开发。在这项努力中，一种遗传工程链霉菌 Frdiae菌株将通过异源细胞色素P450单加氧酶mycG的基因组整合而产生。这种细胞色素P450是天然的霉素素生物合成途径，该途径负责在灰色小单孢菌中产生16元大环内酯类的霉素素家族。已有研究表明，在霉素的生物合成过程中，MycG活性会将区域和立体特异的羟基和/或环氧化物官能团安装到Mycinamicin核心支架上，从而产生单氧化和双氧化的生物活性化合物。已经证实，这种氧化功能对霉素的生物活性至关重要。在前期工作的基础上，Alluvium公司预计，工程菌株将能够产生以泰乐菌素为基础的杂交类似物，其氧化模式与大环内酯类抗生素中观察到的霉素家族相似。众所周知，区域和立体特异的氧化官能团可以影响大环内酯类化合物的生物活性，Alluvium假设新的16元大环内酯类化合物将显示出对大环内酯类耐药细菌病原体的效力。因此，对一系列细菌菌株的体外抗菌活性评估，包括那些表现出大环内酯类耐药性的菌株，将在这项初步的第一阶段研究中进行。如果这一假设得到支持，显示出有前景的活性的化合物将进入第二阶段的研发，在这一阶段，Alluvium将进行药物化学努力，以优化药理特性，并建立一个领先的大环内酯类抗生素候选药物。

 DESCRIPTION (provided by applicant): The rapid emergence of multi-drug resistant pathogenic microorganisms represents a major threat to public health, placing an ever-increasing demand for the discovery of new antibacterial agents. Macrolide antibiotics, such as the 14-membered macrolide erythromycin and second-generation analogs clarithromycin and azithromycin, are among the first line therapies clinically employed to treat respiratory tract infections. However, as a consequence of the clinical overuse of these agents, macrolide resistance mechanisms have rapidly emerged. In contrast, 16-membered macrolides have demonstrated the capability of overcoming resistance mechanisms that affect 14- and 15-membered macrolides. Indeed, a few select 16- membered macrolides have been successfully employed in the clinical treatment of bacterial infections outside of the United States. Despite their demonstrated potential, however, 16-membered macrolides still remain underexplored in the development of new human antibacterial agents. The tylosin biosynthetic pathway produces a series of 16-membered macrolides, of which the veterinary therapeutic tylosin is best known. In this SBIR proposal, Alluvium Biosciences proposes to genetically engineer the tylosin biosynthetic pathway to enable the production of novel 16-membered macrolide compounds for application in new antibiotic drug discovery. In this effort, a genetically engineered Streptomyces fradiae strain will be generated via genomic integration of the heterologous cytochrome P450 monooxygenase, mycG. This cytochrome P450 is native to the mycinamicin biosynthetic pathway that is responsible for the production of the mycinamicin family of 16- membered macrolides in Micromonospora griseorubida. It has previously been established that during the biosynthesis of the mycinamicins, MycG activity installs a regio- and stereospecific hydroxyl and/or epoxide functionality onto the mycinamicin core scaffold, resulting in both mono and di-oxidized bioactive compounds. It is established that this oxidative functionality is critical for mycinamicin bioactivity. Based on preliminary work, Alluvium expects that the engineered S. fradiae strain will be capable of producing hybrid tylosin-based analogs featuring an oxidation pattern similar to that observed in the mycinamicin family of macrolides. As it is known that the regio- and stereospecific oxidative functionalities can influence the bioactivity of macrolide compounds, Alluvium hypothesizes that the novel 16-membered macrolides will display potency against macrolide resistant bacterial pathogens. Accordingly, in vitro evaluation of antibacterial activities against a series of bacterial strains, including those that display macrolide resistance will be performed within this initial Phase I study. If the hypothesis is supported, compounds demonstrating promising activity will proceed to Phase II R&D wherein Alluvium will pursue medicinal chemistry efforts in order to optimize pharmacological properties and establish a lead macrolide antibiotic candidate.