Discovery of a Small Molecule that Blocks Wall Teichoic Acid Biosynthesis in Staphylococcus aureus

Abstract

Both Gram-positive and Gram-negative bacteria contain bactoprenol-dependent biosynthetic pathways expressing non-essential cell surface polysaccharides that function as virulence factors. Although these polymers are not required for bacterial viability in vitro, genes in many of the biosynthetic pathways are conditionally essential: they cannot be deleted except in strains incapable of initiating polymer synthesis. We report a cell-based, pathway-specific strategy to screen for small molecule inhibitors of conditionally essential enzymes. The screen identifies molecules that prevent the growth of a wildtype bacterial strain but do not affect the growth of a mutant strain incapable of initiating polymer synthesis. We have applied this approach to discover inhibitors of wall teichoic acid (WTA) biosynthesis in Staphylococcus aureus. WTAs are anionic cell surface polysaccharides required for host colonization that have been suggested as targets for new antimicrobials. We have identified a small molecule, 7-chloro-N,N-diethyl-3-(phenylsulfonyl)-[1,2,3]triazolo[1,5-a]quinolin-5-amine (1835F03), that inhibits the growth of a panel of S. aureus strains (MIC = 1–3 μg mL−1), including clinical methicillin-resistant S. aureus (MRSA) isolates. Using a combination of biochemistry and genetics, we have identified the molecular target as TarG, the transmembrane component of the ABC transporter that exports WTAs to the cell surface. We also show that preventing the completion of WTA biosynthesis once it has been initiated triggers growth arrest. The discovery of 1835F03 validates our chemical genetics strategy for identifying inhibitors of conditionally essential enzymes, and the strategy should be applicable to many other bactoprenol-dependent biosynthetic pathways in the pursuit of novel antibacterials and probes of bacterial stress response.

New Structural Templates for Clinically Validated and Novel Targets in Antimicrobial Drug Research and Development

“…Adapted from (Sewell and Brown 2014) The first reported late-stage WTA biosynthesis inhibitor is targocil 21 (Scheme 12), which is a selective TarG inhibitor identified by the high-throughput screening (HTS) of a library of 55000 small molecules against wild-type S. aureus and a tarO-deletion mutant (Lee et al 2010;Swoboda et al 2010). Targocil has been proven to induce significant cell wall stress (Campbell et al 2012) and displayed a submicromolar MIC against MRSA.…”

In Vitro Antimicrobial Activity of Wall Teichoic Acid Biosynthesis Inhibitors against Staphylococcus aureus Isolates

“…The WTA biosynthesis pathway constitutes a novel target for small-molecule inhibitors, and two inhibitory compounds have recently been reported (23,36). WTAs are phosphaterich, highly anionic polymers that are covalently linked to the peptidoglycan cell wall of most Gram-positive pathogens.…”

“…This mixed gene dispensability pattern implies that blocking lateacting WTA biosynthetic enzymes after flux into the pathway has been initiated is deleterious to bacterial growth, likely through the accumulation of toxic intermediates (or the depletion of the peptidoglycan carrier lipid bactoprenol). A cellbased, pathway-specific high-throughput screen exploiting this mixed gene dispensability was used to identify the first small molecule to target WTA biosynthesis, 1835F03 (36) (Fig. 1).…”

“…1). The target of 1835F03 was identified to be tarG, the transmem-brane component of the ABC transporter that exports WTA to the cell surface (36). This compound inhibits the growth of select S. aureus strains, including MRSA (36).…”

“…The target of 1835F03 was identified to be tarG, the transmem-brane component of the ABC transporter that exports WTA to the cell surface (36). This compound inhibits the growth of select S. aureus strains, including MRSA (36). A structureactivity relationship study of 1835F03 led to the discovery of an analog, targocil, that is 10-fold more potent (23) (Fig.…”