Pathway-Directed Screen for Inhibitors of the Bacterial Cell Elongation Machinery

Abstract

New antibiotics are needed to combat the growing problem of resistant bacterial infections. An attractive avenue toward the discovery of such next-generation therapies is to identify novel inhibitors of clinically validated targets, like cell wall biogenesis. We have therefore developed a pathway-directed whole-cell screen for small molecules that block the activity of the Rod system of Escherichia coli. This conserved multiprotein complex is required for cell elongation and the morphogenesis of rod-shaped bacteria. It is composed of cell wall synthases and membrane proteins of unknown function that are organized by filaments of the actin-like MreB protein. Our screen takes advantage of the conditional essentiality of the Rod system and the ability of the beta-lactam mecillinam (also known as amdinocillin) to cause a toxic malfunctioning of the machinery. Rod system inhibitors can therefore be identified as molecules that promote growth in the presence of mecillinam under conditions permissive for the growth of Rod– cells. A screen of ∼690,000 compounds identified 1,300 compounds that were active against E. coli. Pathway-directed screening of a majority of this subset of compounds for Rod inhibitors successfully identified eight analogs of the MreB antagonist A22. Further characterization of the A22 analogs identified showed that their antibiotic activity under conditions where the Rod system is essential was strongly correlated with their ability to suppress mecillinam toxicity. This result combined with those from additional biological studies reinforce the notion that A22-like molecules are relatively specific for MreB and suggest that the lipoprotein transport factor LolA is unlikely to be a physiologically relevant target as previously proposed.

Countering Gram-Negative Antibiotic Resistance: Recent Progress in Disrupting the Outer Membrane with Novel Therapeutics

“…MAC13243 and A22 were suggested to act on LolA to permeabilize the OM. However, recent work from the Bernhardt group proposed an alternate explanation that could account for the resistance against A22, MAC13243, and related compounds conferred by LolA overproduction [132]. When LolA is produced in excess, the Rcs cell envelope stress response is activated, and LolA overproduction is only protective against the compounds when the Rcs response is intact.…”

“…When LolA is produced in excess, the Rcs cell envelope stress response is activated, and LolA overproduction is only protective against the compounds when the Rcs response is intact. The Bernhardt group illustrated that inactivating RcsB, the transcriptional regulator of the Rcs system, abrogates the protective effect of LolA overproduction [132]. LolA and LolB are also potentially interesting targets for novel therapeutics.…”

Status of Targeting MreB for the Development of Antibiotics

“…It is believed that CBR-4830 inhibits MreB by competing with ATP for binding to the nucleotide binding pocket (Robertson et al, 2007). It has also been reported that CBR-4830 inhibits the growth of wild-type E. coli and induces spherical cells but may have significant off-target activity (Buss et al, 2019). Sceptrin ( Figure 2G) is a natural product isolated from a marine sponge, Agelas confera (Walker et al, 1981;Cipres et al, 2010), and seems to have affinity for MreB (Rodríguez et al, 2008).…”

“…Like MAC13234, compound A is also believed to target MreB in the A22-like fashion by hydrolyzing to produce MP265. A more recent study (Buss et al, 2019) adopted a pathwaydirected whole-cell screening technique to identify compounds 1 and 4 ( Figures 2D,E) as active against E. coli by targeting MreB. Compounds 1 and 4 are also believed to inhibit MreB by hydrolyzing to produce A22 analogs.…”

A Staphylococcus aureus clpX Mutant Used as a Unique Screening Tool to Identify Cell Wall Synthesis Inhibitors that Reverse β-Lactam Resistance in MRSA

“…In addition, whole cell screens for growth inhibition typically generate large numbers of active compounds, many of which have non-specific activities (Silver, 2011). Therefore, including a counter-screen that facilitates exclusion of non-specific inhibitors and allows identification of compounds targeting specific pathways early in the screening workflow can speed up the screening process tremendously (French et al, 2017;Buss et al, 2018).…”