Drug-resistance in M. tuberculosis & the characterisation of a new anti-tuberculosis drug-candidate

University dissertation from Stockholm : Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology

Abstract: The occurrence of multidrug-resistant (MDR) tuberculosis (TB) has been increasing at alarming levels globally, and the spread of extensively drug-resistant (XDR) M. tuberculosis is threatening the control of tuberculosis. Understanding the molecular mechanisms behind resistance is an important tool in minimising and preventing its spread, as faster diagnostic methods can de developed and treatment guidelines be optimised. There is also an urgent need to find and develop effective drugs that simplify and shorten the existing treatment regimen, as well as being effective against all forms of TB. The current thesis presents the characterisation of in vitro-resistance to rifampicin (RIF), as well as the pre-clinical characterisation of a promising new anti-TB drug candidate. In Paper I we investigated RIF-resistance mutations within the rpoB gene of in vitro-selected resistant mutants. The array and frequency of mutations, as well as the resistance-levels found in these mutants were similar to those reported for RIF-resistant clinical isolates. Furthermore, we saw that mutants of the Beijing genotype, a family of strains known to be spreading globally and commonly associated with MDR, did not portray a different span of resistance mutations or resistance-level. Papers II-IV present the pre-clinical characterisation of R207910. R207910 is a new anti-TB drug candidate identified for its high Mycobacterial specificity. This Diarylquinoline, a new class of compounds, was shown to have a strong inhibitory effect on both drug-susceptible and MDR M. tuberculosis (Minimum Inhibitory Concentration 0.03?g/ml). In combination with the standard combinatorial TB-treatment regimen, R207910 further had an equal, if not stronger, bactericidal activity in mice than the standard regimen alone. In vitro studies showed that resistance occurs through mutations in the bacilli s ATP synthase indicating that the compound targets a unique site; the bacilli s energy-producing ATP synthase. Resistance to R207910 occurred at a relatively slow rate (approx 10-8 mutations per cell generation) and this spontaneous acquisition of resistance was prevented at an R207910-concentration of 3 mg/ml, a level deemed attainable within humans without causing adverse effects. R207910 shows the potential of shortening and simplifying the treatment of both DS and MDR-TB. Furthermore, having characterised the dynamics of resistance development to the compound before it reaches clinical use, treatment doses and guidelines can be established that will cure TB-patients as well as prolong, and hopefully prevent, the emergence of clinical R207910-resistance.

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