Molecular mechanism of antibiotic rifampicin action

描述（由申请人提供）：利福霉素是广泛的细菌RNA聚合酶的有效抑制剂，在结核病治疗中作为一线抗生素获得了一席之地。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定位于rpoB基因，编码RNA聚合酶的亚基。由于这种酶既是利福霉素的靶标又是其耐药性的来源，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应用扩展到新的治疗靶标。已获得的利福平-聚合酶复合物的结构，并已提出的抑制机制。然而，目前的模型未能解释利福霉素作用的许多方面，例如，rpoB突变体的存在对利福平有抗性，但对其衍生物敏感，排除了简单的结合丧失机制。 遗传学和生物化学方法的组合将用于确定利福霉素耐药的分子机制。首先，对E.大肠杆菌rpoB基因将用于分离对不同利福霉素（rifamycin SV、rifampin、rifablide等）的抗性突变体，重点是对赋予差异抗性的突变体的详细分析，这是理解利福霉素作用的关键。其次，将纯化对应于这些突变体的RNA聚合酶变体，以在体外高度纯化的系统中确认抗生素抗性;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，利福霉素与不同状态的RNA聚合酶结合的物理参数将被定量。最后，将使用单核苷酸解析转录测定来确定各种化学修饰对基本利福霉素药效团的影响，特别是那些存在于临床上重要的抗生素利福平和利福霉素中的化学修饰。作为系统分析的结果所获得的数据将被用来完善利福霉素-RNA聚合酶复合物的结构模型，并开发明确的利福霉素作用的动力学和热力学模型。最后，一套规则的设计更有效的利福霉素类抗生素将制定实现知识为基础的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.