Targeting Lsr2/DNA Complexation for Dysregulation of Gene Expression in Mycobacterium tuberculosis
The identification of new drug targets and mechanisms of action against tuberculosis has become urgent, as the emergence of drug-resistant strains has made many treatments ineffective. Lsr2 is a protein encoded by the Mycobacterium tuberculosis genome that acts as a global repressor of transcription by binding AT-rich promoter regions of the mycobacterial DNA. Inhibiting this function could potentially dysregulate a wide array of genes, as Lsr2 binds about 20% of the Mycobacterium tuberculosis genome. Among them are key genes involved in mycobacterial virulence, cell wall biosynthesis or antibiotics-inducible genes that confer multi-drug tolerance. The structure of the DNA-binding C-terminal domain has been solved by nuclear magnetic resonance while the dimerization N-terminal domain was solved using X-ray crystallography, but there is no crystal structure of the full-length protein to this day.The first part of this work describes the development of a high-throughput assay that enabled the identification of Zafirlukast as an inhibitor of the Lsr2/DNA complexation. This ability was shown in vitro using a fluorescence polarization assay. Zafirlukast also exhibits growth inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis on a Kirby Bauer assay, but shows no activity against Escherichia coli which does not encode the Lsr2 protein. Transcription levels of genes shown to be bound by Lsr2 have been monitored in vivo after applying the drug to a culture of Mycobacterium tuberculosis and the results suggest that Zafirlukast promotes dysregulation of mycobacterial transcription. This drug is already commercially available and is commonly used as a treatment for asthma. As its mechanism of action appears to be distinct from current drugs against tuberculosis, it offers a potential new lead for the treatment of this disease.The second part of this work describes different attempts made to gain structural knowledge about Lsr2 particularly in the context of an Lsr2/DNA complex. Different fusion proteins consisting of Lsr2 and lysozyme sequences were engineered for this purpose. Oligonucleotides were also designed to try to obtain crystals of the Lsr2/DNA complex.
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