Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase

Abstract

Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery.

Interrupting Biosynthesis of O Antigen or the Lipopolysaccharide Core Produces Morphological Defects in Escherichia coli by Sequestering Undecaprenyl Phosphate

“…The present results, especially when combined with previous observations (29,30), strongly suggest that the accumulation of any Und-PP-linked intermediate interferes with PG synthesis. This possibility, which we consider highly probable, has significant implications for past and future experiments that explore the physiological relevance of any glycan polymer whose synthesis requires a Und-P intermediate.…”

“…For example, some Corynebacterium glutamicum mutants accumulate lethal amounts of arabinogalactan and lipoarabinomannan intermediates, but the cells survive if the pool of decaprenyl phosphate is increased (5). Similarly, several Staphylococcus aureus mutants produce lethal WTA intermediates, but an increase in the pool of Und-P restores cell growth (29). Lastly, an increase in the amount of Und-P reverses the morphological and membrane defects caused by dead-end Und-PP-linked ECA intermediates in Escherichia coli (30).…”

Antiinfectives targeting enzymes and the proton motive force

“…Rv3378c is of interest because it is a target for antivirulence-based therapeutics for TB. As one example of a UPPS uncoupler-inhibitor, we reported in recent work that the fertility drug clomiphene also had activity against S. aureus and that a major target was UPPS (71). What is interesting about the clomiphene (36) structure is that it is remarkably similar to the structure of tamoxifen (20), which itself has antiinfective activity.…”

Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

“…Recent in silico, in vitro, and in vivo approaches have identified inhibitors of UppS (13)(14)(15), including a method that used clustered regularly interspersed short palindromic repeat (CRISPR) interference (CRISPRi) to identify drug targets (16). We demonstrated previously that a ribosome-binding-site (RBS) mutation that decreased the expression of UppS led to vancomycin resistance and activation of the M -dependent cell envelope stress response (17).…”