Molecular mechanism of antibiotic rifampicin action

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，耐药菌株的迅速崛起严重限制了它们的临床效率和通用性。大多数利福霉素耐药突变映射到编码RNA聚合酶亚单位的rpoB基因。由于该酶既是利福霉素的靶点，也是其耐药的来源，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，并将其扩大到新的治疗靶点。得到了利福平-聚合酶复合体的结构，并提出了其抑制机理。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，排除了简单的结合丢失机制。 将结合遗传和生化方法来确定利福霉素耐药的分子机制。首先，对大肠杆菌rpoB基因进行综合诱变，筛选出对不同利福霉素(利福霉素SV、利福平、利福平等)具有不同抗性的突变株，重点分析具有不同抗性的突变株，以此作为了解利福霉素作用的关键。其次，与这些突变体相对应的RNA聚合酶变异体将被提纯，以确认体外高纯度系统中的抗生素耐药性；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，将量化与不同状态的RNA聚合酶结合的利福霉素的物理参数。最后，单核苷酸分辨转录分析将用于确定对基本利福霉素药效团的各种化学修饰的效果，特别是那些存在于临床重要抗生素利福平和利福布汀中的化学修饰。这一系统分析得到的数据将用于完善利福霉素-RNA聚合酶复合体的结构模型，并开发利福霉素作用的显式动力学和热力学模型。最后，将制定一套设计更有效的利福霉素类抗生素的规则，以实现基于知识创造新一代抗菌药物的长期目标。

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.