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Research Themes Drug discovery

Microbial Pathogenesis: Targeting Drug Resistance in Mycobacterium tuberculosis

SBKB [doi:10.1038/sbkb.2012.184]
Featured Article - February 2014
Short description: Whole-cell screening for antituberculosis compounds combined with identification of resistance-linked mutations provide novel scaffolds for drug development.

Resistance mutations to compound 4 in the M. tuberculosis aspartyl-tRNA synthetase mapped onto the crystal structure of the AspRS (tRNA:synthetase) complex from T. thermophilus (PDB 1EFW). The resistance mutations map to the dimer interface. Figure courtesy of James Sacchettini.

As the development of drug resistance continues to be a threat to public health, new methods are sought for the rapid identification of novel targets. This is particularly the case for tuberculosis, where the prevalence of drug-resistant strains is rising rapidly. Current antibiotics target a limited number of cellular processes, and efforts in the past two decades to develop new drugs based on pre-specified targets have yielded limited returns.

To identify new drugs effective against the pathogen Mycobacterium tuberculosis, Sacchettini and colleagues (PSI MTBI) have developed a workflow that avoids target bias by combining high-throughput, whole-cell screening with whole- genome sequencing of resistant strains. In addition to eliminating target bias, this platform allows for the rapid identification of a specific target of the lead compound, an essential step for subsequent medicinal chemistry. First, a library of compounds was screened for general inhibition of cell growth, followed by in vitro selection of mutations that confer resistance. To pinpoint the gene product responsible for drug sensitivity, polymorphisms potentially responsible for selected resistance are identified through deep sequencing and confirmed as the basis for resistance by reintroducing single mutations in a clean genetic background, via a phage recombinase.

To demonstrate the utility of this protocol, eight compounds with antitubercular activity were selected from a number of previous whole-cell screens. Several resistant strains were then isolated and sequenced, with the responsible mutations identified. In four cases, the genes associated with the resistant mutations were known to be essential, and thus likely to be the direct targets of the drugs: an ESX-3 type VII secretion system component (EccB3), the aspartyl-tRNA synthetase (AspS), membrane transporter MmpL3 and a polyketide synthase (Pks13). In all but one case, re-engineering the isolated mutations into a wild-type background conferred resistance to the original drug, confirming that the targets had been correctly identified. While MmpL3 had previously been shown to be the target of several small molecules, EccB3, AspS and Pks13 are not targeted by currently available drugs and therefore represent novel target leads. In the case of AspS and Pks13, structural homology modeling also suggested mechanisms of action.

In addition to providing an efficient approach to the identification of novel drug targets in any microorganism, the described protocol can also be used to rapidly build on the data generated by previous whole-cell screens.

Stéphane Larochelle

References

  1. T.R. Ioerger et al. Identification of new drug targets and resistance mechanisms in Mycobacterium tuberculosis.
    PLoS One. 8, e75245 (2013). doi:10.1371/journal.pone.0075245

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