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）：多重耐药革兰氏阴性菌（MDR-GNB）对成功的抗菌化疗构成重大威胁。在 MDR-GNB 中，产生对头孢他啶耐药的肺炎克雷伯菌的超广谱 β-内酰胺酶 (ESBL) 与死亡率增加相关。 SHV-2 β-内酰胺酶（第一个描述的 ESBL）的原子结构测定表明，该酶的活性位点被加宽以适应头孢他啶的侧链。尽管有这样的见解，我们的理解仍然不完整。该提案的广泛、长期目标是： 1) 确定SHV-1、-2和-5 β-内酰胺酶中具有头孢他啶和头孢噻肟R1侧链的硼酸过渡态类似物抑制剂的原子结构。这将阐明决定 ESBL 水解新一代头孢菌素能力的关键相互作用。 2) 使用主要 DNA 错配修复蛋白 mutS 缺陷的大肠杆菌菌株，选择稳定和增强 SHV β-内酰胺酶表达的 blaSHV 突变体。 3) 确定肺炎克雷伯菌是否存在突变表型；评估 ESBL 酶、突变表型和 mutS 表达之间的关系。 4) 在 mutS 缺陷型大肠杆菌菌株中使用 blaSHV 开发 ESBL 进化模型。 实现这些目标将对蛋白质和底物相互作用以及导致 ESBL 表型的遗传特性产生前所未有的了解。这项工作还将作为预测 SHV 酶中新型 ESBL 表型的范例，为评估新型 β-内酰胺奠定基础，并展示酶药物靶点如何影响底物亲和力和催化作用。