Combating Fosfomycin Resistance in Methicillin-Resistant Staphylococcus aureus

项目总结 细菌感染的治疗因多药耐药(MDR)的传播而受到影响，并广泛 耐药(XDR)病原体。对抗耐药细菌和新出现的细菌的新战略迫在眉睫 需要限制抗菌素耐药性的进一步发展。将目前批准的抗生素与 抗性中和剂是一个很有前途的方向。我们研究的总体目标是开发有效的 一线抗菌策略来自经批准的、安全的和广谱的抗生素，因此较新的抗菌药物， 以及最终选择的药物，可以保留下来，以最大限度地减少新出现的耐药性。 磷霉素对革兰氏阴性和革兰氏阳性病原菌都有效，是一种很有前途的 开发一线代理商的候选人。对磷霉素的原发耐药性源于磷霉素的修饰 邻位氧络合物(VOC)超家族的酶。VOC酶的主要作用是解毒 内源化合物和异源化合物。编码VOC磷霉素抗性酶的基因已被 在几乎所有最耐药的革兰氏阴性和革兰氏阳性病原体中发现 分枝杆菌。FosB是耐甲氧西林金黄色葡萄球菌的主要磷霉素修饰酶 (MRSA)。它将杆菌硫醇(BSH)共价连接到磷霉素上，使抗生素失活。FosB基因敲除株 耐甲氧西林金黄色葡萄球菌对磷霉素的敏感性显著增加，表明该酶受到抑制 作为一种潜在的治疗策略。我们假设结合BSH结合的FosB的X射线结构数据 通过基于结构的虚拟抑制剂筛选，将识别可以作为FosB抑制剂的小分子和 降低磷霉素在耐甲氧西林金黄色葡萄球菌中的MIC值，从而使磷霉素成为治疗耐甲氧西林金黄色葡萄球菌的有效药物。在AIM 1，我们将使用基于结构的虚拟筛选来寻找抑制FosB和FosB的新的小分子支架 评价它们与磷霉素联合使用时的动力学和协同效应。此外, 我们将用新化合物来确定FosB的晶体结构，以指导未来的药物化学 接近了。在目标2中，我们将确定FosB与BSH络合的新结构。到目前为止，还没有 VOC磷霉素抗性酶相对于其天然硫醇的结构特征，以及 FosB与BSH结合的结构将对我们理解VOC的机制产生革命性的影响。 催化磷霉素耐药。BSH结合的结构随后将被用作其他 基于结构的虚拟筛选。这项研究将让本科生参与有意义的项目， 让他们接触各种各样的技术，并帮助培养他们的批判性思维、研究技能和兴趣 在生物医学职业生涯中。

PROJECT SUMMARY The treatment of bacterial infections is compromised by the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) pathogens. New strategies to combat resistant as well as emerging bacteria are urgently needed to limit further development of antimicrobial resistance. Combining currently approved antibiotics with resistance neutralizing agents is a promising direction. The overall goal of our research is to develop effective front-line antimicrobial strategies from an approved, safe, and broad-spectrum antibiotic so newer antimicrobials, as well as final-option drugs, can be held in reserve to minimize emerging resistance. Fosfomycin is effective against both Gram-negative and Gram-positive pathogens and represents a promising candidate for developing a front-line agent. Primary resistance to fosfomycin arises from fosfomycin-modifying enzymes of the Vicinal Oxygen Chelate (VOC) superfamily. The primary role of VOC enzymes is to detoxify endogenous and xenobiotic compounds. Genes encoding VOC fosfomycin resistance enzymes have been identified in almost all of the most drug-resistant Gram-negative and Gram-positive pathogens and in Mycobacteria. FosB is the principal fosfomycin-modifying enzyme of methicillin-resistant Staphylococcus aureus (MRSA). It covalently attaches bacillithiol (BSH) to fosfomycin, inactivating the antibiotic. FosB knockout strains of MRSA demonstrate significantly increased susceptibility to fosfomycin, identifying suppression of the enzyme as a potential therapeutic strategy. We hypothesize that combining X-ray structural data of BSH-bound FosB with structure-based virtual inhibitor screening will identify small molecules that can serve as FosB inhibitors and lower the MIC of fosfomycin in MRSA, thereby making fosfomycin an effective treatment against MRSA. In Aim 1, we will use structure-based virtual screening to identify new small molecule scaffolds that inhibit FosB and evaluate them with respect to kinetics and synergistic effectiveness when combined with fosfomycin. In addition, we will determine crystal structures of FosB with the new compounds to guide future medicinal chemistry approaches. In Aim 2, we will determine a novel structure of FosB complexed with BSH. To date, none of the VOC fosfomycin resistance enzymes have been structurally characterized with respect to their native thiol, and a structure of FosB with BSH bound will be transformative to our understanding of the mechanism of VOC- catalyzed fosfomycin resistance. The BSH-bound structure will then be used as a starting model for additional structure-based virtual screening. This research will involve undergraduate students in meaningful projects that expose them to a wide range of techniques and help develop their critical thinking, research skills, and interest in biomedical careers.