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

 描述（由申请人提供）：多重耐药病原微生物的迅速出现对公共卫生构成了重大威胁，对发现新的抗菌剂提出了不断增长的需求。大环内酯类抗生素，如14元大环内酯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疗法。然而，由于这些药物的临床过度使用，大环内酯类耐药机制迅速出现。相比之下，16元大环内酯类药物已经证明了克服影响14元和15元大环内酯类药物的耐药机制的能力。事实上，一些选择的16元大环内酯类已成功地用于美国以外的细菌感染的临床治疗。尽管它们的潜力已被证明，但是，16元大环内酯类药物在开发新的人类抗菌药物方面仍然有待开发。泰乐菌素生物合成途径产生一系列16元大环内酯类，其中兽用治疗药物泰乐菌素最为人所知。在这项SBIR提案中，Alluvium Biosciences提出对泰乐菌素生物合成途径进行基因工程改造，以生产新型16元大环内酯化合物，用于新的抗生素药物发现。在这项工作中，一种基因工程链霉菌 将通过异源细胞色素P450单加氧酶mycG的基因组整合来产生草莓属菌株。该细胞色素P450是原生于负责在灰红小单孢菌中产生16元大环内酯的霉素家族的霉素生物合成途径。先前已经确定，在麦司那霉素的生物合成期间，MycG活性将区域和立体特异性羟基和/或环氧化物官能团安装到麦司那霉素核心支架上，从而产生单氧化和二氧化的生物活性化合物。已经确定，这种氧化功能性对于霉素生物活性至关重要。根据初步工作，冲积预计工程S。Fracheniculum菌株将能够产生杂合的基于泰乐菌素的类似物，其特征在于氧化模式类似于在大环内酯类的霉素家族中观察到的氧化模式。由于已知区域和立体特异性氧化官能团可以影响大环内酯化合物的生物活性，Alluvium假设新的16元大环内酯将显示针对大环内酯抗性细菌病原体的效力。因此，将在该初始I期研究中对一系列细菌菌株（包括显示大环内酯类耐药的菌株）的抗菌活性进行体外评价。如果这一假设得到支持，证明有希望的活性的化合物将进入II期研发，其中Alluvium将继续进行药物化学工作，以优化药理学特性并建立一种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.