Understanding B-Lactam Resistance in Acinetobacter baumannii

描述（由申请方提供）：鲍曼不动杆菌的<$-内酰胺耐药性是当代抗微生物化疗面临的最大挑战之一。这种表型的产生主要是由于β-内酰胺酶的产生。除了这个“抵抗武器库”外，外排泵的重要性也在不断显现。此次R 01更新的目标是采用多学科和综合方法，通过研究AmpC头孢菌素酶、ADC-7（不动杆菌衍生的头孢菌素酶）和A.鲍曼不动杆菌。我们的工作解决了成功的内酰胺治疗的最重要的障碍。 为了解决这个重要问题，我们将分析含有不同R1和R2侧链的特定硼酸抑制剂（BAI）如何抑制ADC-7。我们的方法将需要动力学和生物物理（等温量热法，蛋白质热稳定性和圆二色性）测量，这将补充结构表征。我们将共同获得对灭活机制的独特见解，定义有效抑制剂的能量需求，并了解<$-内酰胺酶特异性的演变。一旦对BAI进行了表征并确定了每个BAI/ADC-7复合物的结构，我们还将通过对与BAI相互作用的氨基酸残基进行定点诱变来测试我们的结论，并测试这些变化对易感性、动力学、蛋白质稳定性和结构的影响。这种全面的方法将揭示哪些氨基酸位置：（a）对BAI和？-内酰胺化合物的结合很重要;（B）对酶的结构、稳定性和/或功能至关重要。我们的工作将为新型BAI的开发和优化提供合理的依据。与这些研究相一致，我们将定义迄今为止未表征的外排泵在？-内酰胺/BAI耐药性中的作用。使用AdeABC和AdeIJK的“敲除”（adeABC/adeIJK），我们将探索“次级”外排泵和转运蛋白在内酰胺耐药性中的作用。在影响<$-内酰胺耐药性的两个领域实现这些目标将有助于我们设计出更有效的<$-内酰胺类抗生素。鲍曼不动杆菌。此外，我们的工作是理解为什么A.鲍曼不动杆菌是抗生素的一个可怕的屏障。整合这些知识将提供有关内酰胺耐药性的必要见解，这将有助于药物化学家和医生面对这种严重的传染病威胁。

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.