Novel Chemical Scaffolds for Inhibition of Rifamycin-Resistant RNA Polymerase Discovered from High-Throughput Screening

Scharf, Nathan T.; Molodtsov, Vadim; Kontos, Arrin; Murakami, Katsuhiko; Garcia, George A.

doi:10.1177/1087057116679994

Cited by 1 publication

(1 citation statement)

References 29 publications

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“…An in vitro plasmid based transcription assay using a Malachite Green Aptamer (MGA) gene (Scharf et al, 2016) that was previously developed for high-throughput screening was modified for these studies. Individual modified pTZ18U vectors containing the E. coli rrnB P1 (266 to 15) and MTB rrnA P3 (255 to 115) ribosomal RNA promoters followed by 4 consecutive repeats of DNA encoding the MGA and three consecutive repeats of the synB artificial terminator sequence were prepared, pMGA4-Ec-rrnB1-SynBx3 and pMGA4-Mt-rrnA3-SynBx3 respectively.…”

Section: In Vitro Transcription Assaysmentioning

confidence: 99%

Structural basis for rifamycin resistance of bacterial RNA polymerase by the three most clinically important RpoB mutations found in Mycobacterium tuberculosis

Molodtsov

Scharf

Stefan

et al. 2017

Molecular Microbiology

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105

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SummarySince 1967, Rifamaycin (RIF) has been used as a first line antibiotic treatment for tuberculosis (TB), and it remains the cornerstone of current short-term TB treatment. Increased occurrence of RIF-resistant (RIF R ) TB, ~41% of which results from the RpoB S531L mutation in RNA polymerase (RNAP), has become a growing problem worldwide. In this study, we determined the X-ray crystal structures of the Escherichia coli RNAPs containing the most clinically important S531L mutation and two other frequently observed RIF R mutants, RpoB D516V and RpoB H526Y. The structures reveal that the S531L mutation imparts subtle if any structural or functional impact on RNAP in the absence of RIF. However, upon Rifampin (RMP) binding, the S531L mutant exhibits a disordering of the RIF binding interface, which effectively reduces the RIF affinity. In contrast, the H526Y mutation reshapes the RIF binding pocket, generating significant steric conflicts that essentially prevent RIF binding. While the D516V mutant does not exhibit any such gross structural changes, certainly the electrostatic surface of the RIF binding pocket is dramatically changed, likely resulting in the decreased affinity for RIF. Analysis of interactions of RMP with three common RIF R mutant RNAPs suggests that modifications to RMP may recover its efficacy against RIF R TB.

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Section: In Vitro Transcription Assaysmentioning

confidence: 99%