Understanding ceftazidime resistance in SHV B-Lactamases

了解 SHV B 内酰胺酶中的头孢他啶耐药性

• 批准号: 7561658
• 负责人: ROBERT A. BONOMO
• 金额: $ 27万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7561658
• 关键词: Active Sites; Affinity; Amino Acid Substitution; Amino Acids; Binding; Boronic Acids; Carboxylic Acids; Catalysis; Cefotaxime; Ceftazidime; Ceftriaxone; Cephalosporins; Charge; Drug Delivery Systems; Enzymes; Escherichia coli; Ethers; Evaluation; Evolution; Generations; Genetic; Goals; Gram-Negative Bacteria; Hydrolysis; Kinetics; Klebsiella pneumonia bacterium; Lactamase; Minor; Modeling; Multi-Drug Resistance; MutS DNA mismatch-binding protein; Mutagenesis; Mutation; Oximes; Penicillin Resistance; Phenotype; Predisposition; Production; Property; Proteins; Research Personnel; Resistance; Rotation; Side; Site; Structure; Substrate Interaction; Water; Work; analog; antimicrobial; base; beta-Lactam Resistance; beta-Lactamase; beta-Lactams; beta-lactamase SHV-2; beta-lactamase SHV-3; chemotherapy; inhibitor/antagonist; insight; mortality; mutant; novel; pathogen; programs

DESCRIPTION (provided by applicant): Multi-drug resistant Gram negative bacteria (MDR-GNB) present a significant threat to successful antimicrobial chemotherapy. Among MDR-GNB, extended spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae resistant to ceftazidime are associated with increased mortality. The atomic structure determination of the SHV-2 beta-lactamase, the first ESBL described, revealed the active site of the enzyme is widened to accommodate the side chain of ceftazidime. Despite this insight, our understanding is still incomplete. The broad, long-term objectives of this proposal are: 1) To determine the atomic structures of boronic acid transition-state analogue inhibitors that have the R1 side chains of ceftazidime and cefotaxime in the SHV-1, -2 and -5 beta-lactamases. This will elucidate the critical interactions that define the ability of ESBLs to hydrolyze advanced generation cephalosporins. 2) Using strains of E. coli deficient in the principal DNA mismatch repair protein, mutS, select blaSHV mutants that stabilize and enhance expression of SHV beta-lactamase. 3) To determine if mutator phenotypes exist in K. pneumoniae; assess the relationship between ESBL enzymes, mutator phenotypes, and mutS expression. 4) To develop a model for ESBL evolution using blaSHV in a mutS deficient strain of E. coli. Accomplishing these goals will achieve an unprecedented understanding of the protein and substrate interactions and genetic properties responsible for the ESBL phenotype. This work will also serve as a paradigm for the prediction of novel ESBL phenotypes in SHV enzymes, form a basis for the evaluation of novel beta-lactams, and show how enzyme drug targets influence substrate affinity and catalysis.

描述（由申请人提供）：多药耐药革兰氏阴性细菌（MDR-GNB）对成功的抗菌化疗构成了重大威胁。在MDR-GNB中，产生肺炎克雷伯氏菌对头孢烷具有抗药性的延伸光谱β-内酰胺酶（ESBL）与死亡率升高有关。 SHV-2β-内酰胺酶的原子结构测定是第一个ESBL所描述的，揭示了该酶的活性位点被扩大以容纳头孢氮夹的侧链。尽管有这种见解，但我们的理解仍然不完整。该提议的广泛，长期目标是： 1）确定硼酸过渡态类似物抑制剂的原子结构，这些抑制剂在SHV-1，-2和-5β-内酰胺酶中具有头孢济和头孢氨酸的R1侧链。这将阐明定义ESBL水解晚期头孢菌素的能力的关键相互作用。 2）使用缺乏主DNA不匹配修复蛋白的大肠杆菌菌株，MUTS，选择稳定和增强SHVβ-内酰胺酶表达的BLASHV突变体。 3）确定K.肺炎中是否存在突变体表型；评估ESBL酶，突变表型和MUTS表达之间的关系。 4）在缺乏大肠杆菌菌株中使用BlashV开发ESBL进化的模型。 实现这些目标将对蛋白质和底物相互作用以及负责ESBL表型的遗传特性实现前所未有的理解。这项工作还将作为SHV酶中新型ESBL表型预测的范式，构成了评估新型β-内酰胺的基础，并展示酶药物靶标如何影响底物亲和力和催化。