Disrupting Dogma: Investigating LPS Biosynthesis Inhibition as an Alternative Mechanism of Action of Aminoglycoside Antibiotics

项目摘要 随着大量革兰氏阴性菌对抗生素的耐药性，鉴定 新的抑制剂和现有的抑制剂的优化是必要的，以便能够有效地治疗 疾病。我们实验室和其他实验室最近的研究工作表明，氨基糖苷类化合物具有最低限度的 对蛋白质合成的影响，事实上，它们有效地与大肠杆菌中的肝素转移酶I(HEPI)结合。这 是一项重要的发现，因为它可能会使这类抗生素被戏剧性地重新设计成更好的 副作用较少的药物，因为HEPI和核糖体结合部位的大小非常不同，而且它们 在电荷上有很大的差异(HEPI带正电，而核糖体带负电)。这 提案将推进重新设计氨基糖苷类抗生素的努力，以增强HEPI的结合并减少 与其他细胞靶点结合，可能导致耳毒性和肾毒性等副作用。 我们的研究将解决三个假设：(1)氨基糖苷类药物与其他细胞靶标结合 包括庚基转移酶在内的核糖体，(2)了解肝素与氨基糖苷的相互作用 将能够进行结构修改和杀菌活性的优化，以及(3)计算方法 可以加强氨基糖苷类药物的重新设计。到目前为止，重新设计氨基糖苷类药物以更有效地结合 核糖体未能产生更有效的药物，这可能是因为其作用机制涉及 其他酶，如HEPI。这项工作承诺加强药物发现工作，同时也为 我实验室的学生和卫斯理两门高级生物化学课程的学生在21世纪的药物发现 方法：研究方法。

Project Summary With numerous Gram-negative bacterial species demonstrating antimicrobial drug resistance, the identification of new inhibitors and the optimization of existing inhibitors is necessary to enable an effective treatment of illnesses. Recent research efforts in our lab and others have demonstrated that aminoglycosides have minimal impact on protein synthesis and in fact they potently bind to heptosytransferase I (HepI) in Escherichia coli. This is an important finding, because it may allow for this class of antibiotics to be dramatically redesigned to be better drugs with fewer side effects, because the HepI and ribosome binding sites have very different sizes and they have dramatic differences in charges (HepI is positively charged, while the ribosome is negatively charged). This proposal will advance efforts to redesign aminoglycoside antibiotics to enhance HepI binding and to reduce binding to other cellular targets that can lead to side effects like oto- and nephrotoxicity. Our investigation will address three hypotheses: (1) that aminoglycosides bind to other cellular targets beyond the ribosome including heptosyltransferase enzymes, (2) understanding the HepI-aminoglycoside interactions will enable structural modification and optimization of bactericidal activity, and (3) that computational methods can enhance aminoglycoside redesign. To date, efforts to redesign aminoglycosides for more potent binding to the ribosome has failed to lead to more potent drugs, and this is likely because the mechanism of action involves other enzymes like HepI. This work promises to enhance drug discovery efforts while also providing training for students in my lab and in two upper-level biochemistry courses at Wesleyan in 21st century drug discovery methods.