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In vitro characterization of wild-type and rifamycin-resistant Mycobacterium tuberculosis RNA polymerases

机译：野生型和耐利福霉素结核分枝杆菌RNA聚合酶的体外表征

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摘要

Rifampin (a semi-synthetic rifamycin derivative) is one of the first-line antituberculosis drugs. Rifampin inhibits transcription by binding to the beta-subunit of the RNA polymerase (RNAP). The majority of the clinically-relevant Mycobacterium tuberculosis (MTB) rifamycin-resistant (RifR) mutations result from amino acid substitutions of one of the following three residues: betaAsp435, betaHis445, and betaSer450 in a highly conserved 27 amino acid region of the RNAP beta-subunit.;The core subunits of wild-type and RifR mutants (Asp435Val, His445Tyr, Ser450Leu) of MTB RNAP were overexpressed in and purified from E. coli. The rifamycins were all found to bind tightly (IC50) to the wild-type MTB and E. coli RNAPs, whereas dramatic (~102-105 fold) losses of affinity for rifamycins were observed for the RifR mutants from both bacteria. Additional studies with efflux pump-deficient E. coli (EC2880) confirm that the differential sensitivity of MTB and E. coli to rifamycin antibiotic activity is due to rifamycin efflux from E. coli, rather than any differences in the target RNAPs. The activities of C-8 modified rifamycins are consistent with X-ray crystal structures that show Ser450 acting as a hydrogen bond donor to the C-8 hydroxyl of rifamycins and that rifamycin resistance in the Ser450 mutants is likely due to loss of this hydrogen bond and loss of affinity. A series of novel benzoxazinorifamycin analogues displayed superior affinity toward wild-type and RifR mutants of the MTB RNAP than rifampin and rifalazil (RifR mutants, but not WT), but the IC50 values were still in the 10-6 M (muM) range with the RifR MTB RNAPs.;Rifampin exhibits significant drug-drug interactions via potent induction of cytochrome P450 3A4 (CYP3A4). Selected commercially available rifamycins and our synthetic analogues were screened in the human pregnane X receptor (hPXR) activation assay to determine their extents of hPXR activation as an indicator of potential for CYP3A4 induction. One of our analogues exhibited very low (similar to rifalazil) activation of hPXR, while the others did show significant activation and some cytotoxicity.;The results of these studies have provided encouraging evidence that rifamycins with improved activity against RifR MTB RNAP and lower drug-drug interaction liabilities can be developed.
机译：利福平（一种半合成的利福霉素衍生物）是一线抗结核药物之一。利福平通过与RNA聚合酶（RNAP）的β亚基结合来抑制转录。大多数与临床相关的结核分枝杆菌（MTB）利福霉素抗性（RifR）突变是由以下三个残基之一的氨基酸取代引起的：RNAA beta的高度保守的27个氨基酸区域中的betaAsp435，betaHis445和betaSer450 MTB RNAP的野生型和RifR突变体（Asp435Val，His445Tyr，Ser450Leu）的核心亚基在大肠杆菌中过表达并从大肠杆菌中纯化。利福霉素均被发现与野生型MTB和大肠杆菌RNAP紧密结合（IC50），而两种细菌的​​RifR突变体均对利福霉素发生了显着（〜102-105倍）的亲和力丧失。对缺乏流出泵的大肠杆菌（EC2880）的其他研究证实，MTB和大肠杆菌对利福霉素抗生素活性的差异敏感性是由于大肠杆菌的利福霉素流出引起的，而不是靶标RNAP的任何差异。 C-8修饰的利福霉素的活性与X射线晶体结构一致，X射线晶体结构显示Ser450充当利福霉素C-8羟基的氢键供体，并且Ser450突变体中的利福霉素耐药性可能是由于该氢键的丢失和失去亲和力。一系列新颖的苯并恶嗪利福霉素类似物对MTB RNAP的野生型和RifR突变体表现出比利福平和利福拉齐（RifR突变体，但不是WT）更高的亲和力，但IC50值仍在10-6 M（muM）范围内。利福平通过有效诱导细胞色素P450 3A4（CYP3A4）表现出显着的药物相互作用。在人的孕烷X受体（hPXR）激活试验中筛选了选定的市售利福霉素和我们的合成类似物，以确定其hPXR激活的程度，作为CYP3A4诱导潜力的指标。我们的一种类似物对hPXR的激活非常低（类似于利福拉齐），而其他类似物的确具有显着的激活作用和一定的细胞毒性。这些研究结果提供了令人鼓舞的证据，表明利福霉素对RifR MTB RNAP的活性增强且药物-可以开发药物相互作用的负债。

著录项

  • 作者

    Atwal, Sumandeep Kaur.;

  • 作者单位

    University of Michigan.;

  • 授予单位 University of Michigan.;
  • 学科 Biochemistry.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 194 p.
  • 总页数 194
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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