Profiling the Activity of Mycobacterium Tuberculosis Inhibitors Against the Trans-Translation Pathway

Resistance in tuberculosis infections is becoming a widespread problem and the demand for new anti-tubercular drugs is increasing. The TB Alliance has discovered compounds that are known to kill Mycobacterium tuberculosis (MTB) but the mechanism by which they operate still remains unknown. Using a luciferase-based screen we discovered that several of the compounds inhibit trans-translation. This process, which is used by bacteria to rescue non-stop ribosomes and release the nascent polypeptide for degradation, is required for the survival of MTB cells. Ribosomes stall once they reach the 3’ end of a non-stop mRNA, trans-translation uses tmRNA and small protein B (SmpB), interact to form a ribonucleoprotein complex which recognizes this ribosome and enters the empty A-site. While rescuing the ribosome at the end of the mRNA, the polypeptide is tagged for degradation [1]. A select set of compounds including a carbazole (KKL-896), tetrahydroquinazoline (KKL-539), and triazole (KKL-1005) showed superior activity in preliminary testing. To begin optimization of new anti-TB therapies, minimum inhibitory concentration and minimum bactericidal concentration assays were used to confirm these three compounds demonstrated broad-spectrum bacterial inhibition. Broad-spectrum inhibition includes the inhibition of growth of both Gram-positive and Gram-negative bacterial species. Secondary screens, including a mCherry reporter assay and an in vitro translation assay, ruled out inhibition of translation and confirmed that the compounds are specific to trans-translation. Through the use of intracellular photo-affinity labeling and click chemistry, attempts were made to identify the molecular target of these compounds. Identification of these targets permits further testing to define the mechanism of action that can be used in structural optimization, leading to the development of improved treatment regimens for MTB infections.

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Work Title Profiling the Activity of Mycobacterium Tuberculosis Inhibitors Against the Trans-Translation Pathway
Access
Penn State
Creators
  1. Alexandria E. Lewis
Keyword
  1. Biochemistry and Molecular Biology
  2. Mycobacterium Tuberculosis
  3. TB Alliance
License Attribution-NonCommercial-NoDerivs 3.0 United States
Work Type Thesis
Acknowledgments
  1. Kenneth Keiler - research mentor
  2. Sara Ades - thesis reader
Publisher
  1. Eberly College of Science
  2. Department of Biochemistry and Molecular Biology
Publication Date Spring 2016
Subject
  1. Biochemistry and Molecular Biology
Language
  1. English
Deposited June 22, 2016

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