A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier

Abstract

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K. BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K. Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA Stability

“…Overall, our results highlight OM biogenesis as an excellent target for novel antibiotic discovery. Thanatin joins the increasing list of molecules that disrupt the assembly of the OM with diverse mechanisms (Hart et al, 2019;Imai et al, 2019;Lehman and Grabowicz, 2019;Psonis et al, 2019). Based on their mechanisms, these compounds could be employed not only to fight multidrug resistant pathogens but also in combination with existing antibiotics not sufficiently effective.…”

Identification of Genes Required for Resistance to Peptidomimetic Antibiotics by Transposon Sequencing

“…Recent research has identified novel antibiotics that target essential OM proteins (Ghequire et al, 2018;Storek et al, 2018a;Hart et al, 2019;Imai et al, 2019;Luther et al, 2019;Sousa, 2019). While these compounds show great promise for clinical applications, it is unclear how fast resistance against this new class of antibiotic may evolve and what the underlying mechanisms of resistance are.…”

Identification of a Compound That Inhibits the Growth of Gram-Negative Bacteria by Blocking BamA–BamD Interaction

“…Two subunits in this complex, BamA and BamD, are essential for bacteria growth. In recent years, a wild variety of compounds targeting BamA or BamD have been identified to impair the OM and show promising antibacterial activity (Hagan et al, 2015;Hart et al, 2019b;Imai et al, 2019). In particular, darobactin, a newly reported large molecule shows anti-bacteria activity both in vitro and in vivo by targeting the lateral gate conformation of BamA, which is outside the OM (Imai et al, 2019).…”

“…In recent years, a collection of compounds that disrupts OM structure by targeting BamA or BamD have been identified, which present with promising anti-bacterial potential (Hagan et al, 2015;Hart et al, 2019b;Imai et al, 2019). These results give us implications that BAM complex is an effective and attractive target for developing novel antibiotics.…”