The Continuing Challenge of Carbapenemases in K. pneumoniae: KPC-2 & NDM-1

描述（由申请人提供）： 碳青霉烯类耐药革兰氏阴性病原体的出现和传播是医疗保健相关发病率和死亡率的重要来源。碳青霉烯酶（即， 碳青霉烯水解酶- 内酰胺酶），这些酶可能会严重限制可用于治疗这些细菌感染的抗菌剂。目前，在克雷伯氏菌中出现了两种主要的碳青霉烯酶，并且是对我们的抗生素设备的主要威胁：肺炎克雷伯氏菌碳青霉烯酶（KPC-2）和新德里金属--内酰胺酶（NDM-1 MBL）。KPC是一种A类碳青霉烯酶，可水解亚胺培南、厄他培南、美罗培南、多利培南和β-内酰胺酶抑制剂，在美国几乎所有主要的三级医疗机构中都有发现。为了设计未来的内酰胺类药物， 对于这些迫在眉睫的威胁，我们需要了解具有KPC和NDM-1的菌株的底物谱和敏感性模式将如何由于β-内酰胺酶中“热点”的取代而改变（即，α-环，KPC-2中的氨基酸残基164至179，以及NDM-1的环区域和活性位点的入口）。这些挑战框架的研究方向，将在这里解决。 我们将在此Merit提案中提出的主要研究问题是： 1)如果在KPC-2中出现了&环的替换，它们是否会对临床造成重大威胁， 代表新的表型，将提出更大的挑战，临床医生？ 2)NDM-1 MBL的独特结构特征如何影响？-内酰胺酶 水解内酰胺类？ 为了回答第一个问题，我们的计划是确定在KPC-2中氨基酸位置R164、E166、N170和D179处的α-环中的取代的效果，并确定这些突变如何改变微生物表型、蛋白质稳定性和催化机制。 为了解决第二个问题，我们将使用诱变以逐步方式去除NDM-1中氨基酸位置163至166（163-166）处的“独特附加序列”，并评估这些缺失对耐药性（表型）、标准内酰胺和碳青霉烯底物的水解以及蛋白质稳定性的影响。接下来，我们将使用诱变来替换（位点饱和）并缺失NDM-1中的F70（70），即活性位点入口处的氨基酸，并评估该变体的活性及其在蛋白质稳定性和催化中的作用。 通过对克雷伯氏菌碳青霉烯酶的研究，我们将提供导致新疗法的关键知识。我们的实验室正在积极确定KPC-2的氨基酸序列要求，这个新项目将在进一步探索β-loop的作用和NDM-1的生化研究中脱颖而出。实现我们的目标将促进药物化学家设计有效的治疗剂所需的一般理解，这些治疗剂将作为底物和抑制剂对抗这些“多功能”的内酰胺酶，并帮助我们设计抗生素治疗方案，以帮助医生面对这种严重的和新出现的传染病威胁。

DESCRIPTION (provided by applicant): The emergence and dissemination of carbapenem-resistant Gram-negative pathogens is a significant source of healthcare-associated morbidity and mortality. Carbapenemases (i.e., carbapenem-hydrolyzing ¿-lactamases) that potentially inactivate all ¿-lactams severely limit the antimicrobials available to treat infections with these bacteria. Presently, two major carbapenemases are emerging in Klebsiella and are a major threat to our antibiotic armamentarium: the Klebsiella pneumoniae carbapenemase (KPC-2), and the New Delhi Metallo-¿-lactamase (NDM-1 MBL). KPC is a class A carbapenemase that hydrolyzes imipenem, ertapenem, meropenem, doripenem, and ¿- lactamase inhibitors and is found in almost every major tertiary care facility in the US. In order to design future ¿-lactams to combat these imminent threats, we need to understand how the substrate profile and susceptibility patterns of strains that possess KPC and NDM-1 will change as a result of substitutions in "hot spots" in the ¿-lactamase (i.e., &-loop, amino acid residues 164 to 179 in KPC-2, and the loop regions and entrance to the active site of NDM-1). These challenges frame the research direction that will be addressed herein. The major research questions we will ask in this Merit Proposal are: 1) If substitutions in the &-loop arise in KPC-2, will they pose a significant clincal threat by representing novel phenotypes that will present even greater challenges to the clinician? 2) How do unique structural features of NDM-1 MBL impact the ability of the ¿-lactamase to hydrolyze ¿-lactams? To answer the first question our plan is to determine the effect of substitutions in the &-loop at amino acid positions R164, E166, N170, and D179 in KPC-2 and ascertain how these mutations alter microbiological phenotype, protein stability and catalytic mechanism. To address the second question we will use mutagenesis to remove, in a stepwise manner, the "distinctive additional sequence" at amino acid positions 163 to 166 ( 163-166) in NDM-1 and assess the impact of these deletions on resistance (phenotype), hydrolysis of standard ¿-lactam and carbapenem substrates, and protein stability. Next, we will use mutagenesis to replace (site-saturation) and to delete F70 ( 70) in NDM-1, an amino acid at the entrance of the active site and assess the activity of this variant and its role in protein stability and catalysis. By focusing on the carbapenemases of K. pneumoniae we will provide the critical knowledge that leads to novel therapies. Our laboratory is actively defining the amino acid sequence requirements of KPC-2 and this new project will stand out as an important undertaking in the further exploration of the role of the &-loop and in the biochemical study of NDM-1. Achieving our objectives will advance the general understanding needed for the medicinal chemist to design effective therapeutic agents that will act as substrates and inhibitors against these "versatile" ¿-lactamases and help us devise antibiotic treatment regimens that will assist physicians faced with this serious and emerging infectious disease threats.