Identification of new drug targets and resistance mechanisms in Mycobacterium tuberculosis

Identification of new drug targets is vital for the advancement of drug discovery against Mycobacterium tuberculosis , especially given the increase of resistance worldwide to first- and second-line drugs. Because traditional target-based screening has largely proven unsuccessful for antibiotic discovery, we have developed a scalable platform for target identification in M. tuberculosis that is based on whole-cell screening, coupled with whole-genome sequencing of resistant mutants and recombineering to confirm. The method yields targets paired with whole-cell active compounds, which can serve as novel scaffolds for drug development, molecular tools for validation, and/or as ligands for co-crystallization. It may also reveal other information about mechanisms of action, such as activation or efflux. Using this method, we identified resistance-linked genes for eight compounds with anti-tubercular activity. Four of the genes have previously been shown to be essential: AspS, aspartyl-tRNA synthetase, Pks13, a polyketide synthase involved in mycolic acid biosynthesis, MmpL3, a membrane transporter, and EccB3, a component of the ESX-3 type VII secretion system. AspS and Pks13 represent novel targets in protein translation and cell-wall biosynthesis. Both MmpL3 and EccB3 are involved in membrane transport. Pks13, AspS, and EccB3 represent novel candidates not targeted by existing TB drugs, and the availability of whole-cell active inhibitors greatly increases their potential for drug discovery

Mapping Spectral Spaces

How have residual marks been created, left, and remembered? How might we conceptualize these afterlives and effects of experiences, perceptions, processes, and events? Traces acquire meaning through acts of discovery, juxtaposition, re-presentation, and relocation.2 If traces are present yet not always visible, define the “thing” through a web of relations between the listener and teller yet are not animated until they come alive in particular settings, are always already known but not recognizable until they are brought out again in yet new but familiar forms, then the concept of spectral traces may provide us with a language of belonging, even as such traces speak of past loss

Compounds with whole-cell inhibition used for target identification.

<p>Compounds with whole-cell inhibition used for target identification.</p

Location of mutations in <i>Mtb</i> Pks13, mapped onto the thiolation-thioesterase domain of <i>E. coli</i> enterobactin synthase (EntF, PDB: 2roq).

<p>The mutations occur in a 2-helix lid that closes over the active site.</p

Location of mutations in AspS, based on the <i>T. thermophilus</i> model (PDB: 1efw).

<p>The peptide chains are shown as blue and gray ribbons, and the tRNA molecules are shown as atomic models. The side-chains of residues Leu515 and Thr559, corresponding to the mutations in the Mtb AspS (F526 and T570), are highlighted in green and are located in the dimer interface.</p

Location of mutations observed in MmpL3 (based on Tullius et al., 2011).

<p>Location of mutations observed in MmpL3 (based on Tullius et al., 2011).</p