Molecular mechanism of antibiotic rifampicin action

抗生素利福平作用的分子机制

• 批准号: 6911366
• 负责人: IRINA ARTSIMOVITCH
• 金额: $ 20.94万
• 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/6911366
• 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，在结核病治疗中赢得了一线抗生素的地位。然而，它们的临床效率和多功能性因耐药菌株的迅速增加而受到严重限制。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的靶标，也是利福霉素耐药性的来源，因此阐明其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用途扩大到新的治疗靶点。获得了利福平-聚合酶复合物的结构，并提出了抑制机制。然而，当前的模型未能解释利福霉素作用的许多方面，例如，对利福平具有抗性但对其衍生物敏感的rpoB突变体的存在，排除了简单的结合丧失机制。 将结合遗传和生化方法来确定利福霉素耐药性的分子机制。首先，对大肠杆菌rpoB基因进行全面诱变，分离出对不同利福霉素（利福霉素SV、利福平、利福布丁等）耐药的突变体，重点是对具有差异耐药性的突变体进行详细分析，这是了解利福霉素作用的关键。其次，将纯化与这些突变体相对应的RNA聚合酶变体，以在体外高度纯化的系统中确认抗生素耐药性；将确定转录动力学和热力学的有效变化。第三，使用放射性标记的利福霉素，对与不同状态的RNA聚合酶结合的利福霉素的物理参数进行定量。最后，单核苷酸解析转录测定将用于确定各种化学修饰对基本利福霉素药效团的影响，特别是临床上重要的抗生素利福平和利福布丁中存在的化学修饰。通过系统分析获得的数据将用于完善利福霉素-RNA聚合酶复合物的结构模型，并开发利福霉素作用的明确动力学和热力学模型。最终将制定一套更有效的利福霉素类抗生素的设计规则，以实现基于知识创造新一代抗菌药物的长期目标。