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

肺炎克雷伯菌中碳青霉烯酶的持续挑战：KPC-2

• 批准号: 8441988
• 负责人: ROBERT A. BONOMO
• 金额: --
• 依托单位: LOUIS STOKES CLEVELAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8441988
• 关键词: Active Sites; Address; American; Amino Acid Sequence; Amino Acids; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Drug Resistance; Biochemical; Carbapenems; Catalysis; Cessation of life; Clinical; Complex; Emerging Communicable Diseases; Future; Genetic; Gram-Negative Bacterial Infections; Healthcare; Hot Spot; Hydrolysis; Imipenem; Infection; Kinetics; Klebsiella; Klebsiella pneumonia bacterium; Knowledge; Laboratories; Lactamase; Lactams; Meropenem; Microbiology; Morbidity - disease rate; Mutagenesis; Mutation; Nosocomial Infections; Pattern; Pharmaceutical Preparations; Phenotype; Physicians; Positioning Attribute; Predisposition; Proteins; Research; Resistance; Role; Sentinel; Site; Source; Structure-Activity Relationship; System; Therapeutic Agents; Treatment Protocols; Variant; antimicrobial; beta-Lactamase; beta-Lactams; carbapenem resistance; carbapenemase; combat; design; doripenem; inhibitor/antagonist; mortality; novel; pathogen; public health relevance; tertiary care

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.

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与医疗保健相关的发病率和死亡率的重要来源，碳青霉烯酶（即碳青霉烯水解β-内酰胺酶）可能会灭活所有 ¿ -内酰胺严重限制了可用于治疗这些细菌感染的抗菌药物，目前，克雷伯菌中出现了两种主要的碳青霉烯酶，它们对我们的抗生素武器库构成了主要威胁：肺炎克雷伯菌碳青霉烯酶（KPC-2）和新德里金属酶。 -内酰胺酶 (NDM-1 MBL) 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、多尼培南和 ¿ - 内酰胺酶抑制剂，几乎在美国每个主要的三级护理机构中都有使用，以便设计未来 ¿ - 对抗内酰胺 对于这些迫在眉睫的威胁，我们需要了解拥有 KPC 和 NDM-1 的菌株的底物特征和敏感性模式将如何因“热点”中的替换而发生变化。 -内酰胺酶（即，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入口）。这些挑战构成了本文将要解决的主要研究方向。我们将在此优点提案中提出的研究问题是：1）如果 KPC-2 中出现 &-loop 中的替换，它们是否会通过代表新的表型而构成重大的临床威胁，从而呈现出更大的效果2) NDM-1 MBL 的独特结构特征如何影响 ¿ - 内酰胺酶水解 ¿为了回答第一个问题，我们的计划是确定 KPC-2 中 R164、E166、N170 和 D179 氨基酸位置的 &-环替换的影响，并确定这些突变如何改变微生物表型、蛋白质稳定性和为了解决第二个问题，我们将使用诱变以逐步的方式去除氨基酸位置 163 处的“独特的附加序列”。 NDM-1 中的 166 (163-166) 并评估这些缺失对抗性（表型）、标准水解的影响 ¿ -内酰胺和碳青霉烯底物以及蛋白质稳定性 接下来，我们将使用诱变来替换（位点饱和）并删除 NDM-1 中活性位点入口处的氨基酸 F70 (70)，并评估其活性。通过关注肺炎克雷伯菌的碳青霉烯酶，我们将提供新疗法的关键知识。我们的实验室正在积极确定其氨基酸序列要求。 KPC-2 和这个新项目将成为进一步探索 &-loop 的作用和 NDM-1 生化研究中的一项重要任务，实现我们的目标将促进药物化学家所需的一般理解。设计有效的治疗剂作为这些“多功能”的底物和抑制剂 ¿ -内酰胺酶并帮助我们设计抗生素治疗方案，帮助医生应对这种严重的和新出现的传染病威胁。