Understanding B-Lactam Resistance in Acinetobacter baumannii

描述(由申请人提供)：鲍曼不动杆菌对内酰胺类抗生素的耐药性是当代抗菌化疗的最大挑战之一。β-内酰胺酶的产生是这种表型的特殊原因。外排泵的重要性日益凸显，这也是“阻力的武器”。此次R01更新的目标是通过研究AmpC头孢菌素酶、ADC-7(不动杆菌衍生头孢菌素酶)和鲍曼不动杆菌的外排泵，开始采用多学科和综合的方法来深入了解导致β-内酰胺类抗生素耐药的分子细节。我们的工作解决了成功的最重要的障碍-内酰胺疗法。为了解决这个重要的问题，我们将分析含有不同R1和R2侧链的特定硼酸抑制剂(BaI)如何使ADC-7失活。我们的方法将需要进行动力学和生物物理(等温量热法、蛋白质热稳定性和圆二色谱)测量，以补充结构表征。在一起，我们将获得对失活机制的独特见解，以及定义有效抑制剂的能量要求，并了解内酰胺酶专一性的演变。一旦BAI的特性和每个BAI/ADC-7复合体的结构被确定，我们还将通过对与BAI相互作用的氨基酸残基进行定点突变来检验我们的结论，并测试这些变化对敏感性、动力学、蛋白质稳定性和结构的影响。这一综合方法将揭示哪些氨基酸位置是：(A)对BAI和内酰胺化合物的结合很重要；以及(B)对酶结构、稳定性和/或功能至关重要。我们的努力将为新奇百货的开发和优化提供合理的依据。结合这些研究，我们将确定迄今尚未确定的外排泵在内酰胺类/BAI耐药中的作用。利用AdeABC和AdeIJK的“基因敲除”(adeABC/adeIJK)，我们将探索“二次”外排泵和转运体在β-内酰胺耐药中的作用。在两个影响内酰胺类耐药性的领域实现这些目标，将有助于我们设计出更有效的对抗鲍曼不动杆菌的内酰胺类抗生素。此外，我们的工作是理解为什么鲍曼不动杆菌的外排系统对抗生素构成了强大的障碍的“第一步”。整合这些知识将提供关于内酰胺类耐药性的必要见解，这将有助于药物化学家和内科医生面对这一严重的传染病威胁。

DESCRIPTION (provided by applicant): ¿-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. The production of ¿-lactamases is singularly responsible for this phenotype. Added to this "arsenal of resistance" is the emerging importance of efflux pumps. The goal of this R01 renewal is to embark upon a multidisciplinary and integrative approach to gain insight into the molecular details that result in ¿-lactam resistance by studying the AmpC cephalosporinase, ADC-7 (Acinetobacter derived cephalosporinase), and the efflux pumps of A. baumannii. Our work addresses the most important barriers to successful ¿-lactam therapy. To address this significant problem, we will analyze how specific boronic acid inhibitors (BAIs) that contain different R1 and R2 side chains inactivate ADC-7. Our approach will entail kinetic and biophysical (isothermal calorimetry, protein thermal stability, and circular dichroism) measurements that will complement structural characterization. Together, we will gain unique insights into the mechanism of inactivation, the energetic requirements that define a potent inhibitor, and understand the evolution of ¿-lactamase specificity. Once the BAIs are characterized and the structures of each BAI/ADC-7 complex are determined, we will also test our conclusions by performing site-directed mutagenesis of amino acid residues that interact with the BAI and test the impact of these changes on susceptibility, kinetics, protein stability, and structure. This comprehensive approach will reveal which amino acid positions are: (a) important for binding of BAI and ¿-lactam compounds; and (b) critical for enzyme structure, stability, and/or function. Our efforts will provide a rational basis for the development and optimization of novel BAIs. In concert with these investigations, we will define the role of heretofore uncharacterized efflux pumps in ¿-lactam/BAI resistance. Using "knockouts" of AdeABC and AdeIJK (adeABC/adeIJK), we will explore the role of "secondary" efflux pumps and transporters in ¿-lactam resistance. Achieving these aims in two areas affecting ¿-lactam resistance will help us design more potent ¿-lactams against A. baumannii. Additionally, our work is a "first step" to understanding why the efflux systems of A. baumannii present a formidable barrier to antibiotics. Integrating this knowledge will provide the necessary insights regarding ¿-lactam resistance that will assist medicinal chemists and physicians faced with this serious infectious disease threat.