Molecular mechanism of antibiotic rifampicin action

抗生素利福平作用的分子机制

• 批准号: 7052765
• 负责人: IRINA ARTSIMOVITCH
• 金额: $ 18.25万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-15 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7052765
• 关键词: DNA directed RNA polymerase; analog; antibiotics; bacterial genetics; chemical binding; chemical models; drug design /synthesis /production; drug resistance; enzyme inhibitors; pharmacokinetics; radiotracer; rifamycins

DESCRIPTION (provided by applicant): Rifamycins are effective inhibitors of a wide range of bacterial RNA polymerases, earning their place as first line antibiotics in treatment of tuberculosis. However, their clinical efficiency and versatility are severely limited by the rapid rise of resistant strains. The majority of the rifamycin-resistant mutations map to the rpoB gene, encoding the subunit of RNA polymerase. Since this enzyme is both the target of and the source of resistance to rifamycins, elucidation of its interactions with rifamycins is instrumental for design of new, more potent antibiotics and expanding their use to new therapeutic targets. The structure of the rifampicin-polymerase complex has been obtained, and the mechanism of inhibition has been proposed. However, the current model fails to account for many aspects of rifamycin action, e.g., the existence ofrpoB mutants that are resistant to rifampicin yet sensitive to its derivatives, ruling out the simple loss-of-binding mechanism. A combination of genetic and biochemical approaches will be used to determine the molecular mechanism of rifamycin-resistance. First, a comprehensive mutagenesis of the E. coli rpoB gene will be used to isolate mutants resistant to different rifamycins (rifamycin SV, rifampin, rifabutin, etc), with the emphasis on the detailed analysis of mutants conferring differential resistance as the key to understanding rifamycin action. Second, RNA polymerase variants corresponding to these mutants will be purified to confirm the antibiotic resistance in a highly purified system in vitro; effective changes in transcription kinetics and thermodynamics will be determined. Third, using radio labeled rifamycin, physical parameters of rifamycins binding to different states of RNA polymerase will be quantified. Finally, single nucleotide resolved transcription assays will be used to determine the effects of various chemical modifications to basic rifamycin pharmacophore, especially those present in clinically important antibiotics rifampin and rifabutin. The data obtained as a result of this systematic analysis will be used to refine the structural model of rifamycin-RNA polymerase complex and to develop explicit kinetic and thermodynamic models of rifamycin action. Finally a set of rules for design of more potent rifamycin-like antibiotics will be formulated to achieve the long-term goal of knowledge-based creation of the new generation of antibacterial drugs.

描述（由申请人提供）：利福霉素是多种细菌RNA聚合酶的有效抑制剂，是治疗结核病的一线抗生素。然而，它们的临床效率和多功能性受到耐药菌株迅速增加的严重限制。大多数利福霉素耐药突变映射到rpoB基因，编码RNA聚合酶亚基。由于这种酶既是利福霉素耐药性的靶点，也是耐药性的来源，阐明它与利福霉素的相互作用有助于设计新的、更有效的抗生素，并将其应用于新的治疗靶点。获得了利福平-聚合酶复合物的结构，并提出了其抑制机制。然而，目前的模型未能解释利福霉素作用的许多方面，例如，存在对利福平耐药但对其衍生物敏感的rpob突变体，从而排除了简单的失去结合机制。