Development of a High Throughput Screen for the Identification of Inhibitors of Peptidoglycan O-Acetyltransferases, New Potential Antibacterial Targets

Abstract

The O-acetylation of peptidoglycan occurs in many Gram-negative and most Gram-positive pathogens and this modification to the essential wall polymer controls the lytic activity of the autolysins, particularly the lytic transglycosylases, and inhibits that of the lysozymes of innate immunity systems. As such, the peptidoglycan O-acetyltransferases PatA/B and OatA are recognized as virulence factors. In this study, we present the high throughput screening of small compound libraries to identify the first known inhibitors of these enzymes. The fluorometric screening assay developed involved monitoring the respective O-acetyltransferases as esterases using 4-methylumbelliferylacetate as substrate. Pilot screens of 3921 compounds validated the usefulness of the HTS protocol. A number of potential inhibitors were identified amongst a total of 145,000 low molecular-weight compounds, some of which were common to both enzymes, while others were unique to each. After eliminating a number of false positives in secondary screens, dose response curves confirmed the apparent specificity of a benzothiazolyl-pyrazolo-pyridine as an inhibitor of Neisseria gonorrhoeae PatB, and several coumarin-based compounds as inhibitors of both this PatB and OatA from Staphylococcus aureus. The benzothiazolyl-pyrazolo-pyridine was determined to be a non-competitive inhibitor of PatB with a Ki of 126 µM. At 177 µg/mL and close to its solubility limit, this compound caused a 90% reduction in growth of N. gonorrhoeae, while growth of Escherichia coli, a bacterium that lacks PatB and, hence, does not produce O-acetylated peptidoglycan, was unaffected. These data provide preliminary proof of concept that peptidoglycan O-acetyltransferases would serve as useful antibacterial targets.

Peptidoglycan O-Acetylation as a Virulence Factor: Its Effect on Lysozyme in the Innate Immune System

“…These strategies aim to weaken the bacterium’s pathogenicity without weakening the overall fitness of the bacterium, to the point that the antibiotic is seen as a large enough threat to warrant a resistance strategy. This is done by targeting virulence factors; for example, adhesion to inhibit host colonization, or O-acetylation (reviewed in [86,87]). The potential of PG O-acetylation as an attractive and viable anti-virulence target is underscored by results of a number of animal studies involving mutant strains of pathogens with reduced PG O-acetylation levels.…”

“…Targeting PG O-acetylation as an anti-virulence strategy represents a new avenue which is now being explored and has the potential to be a longer lasting solution. Recently, we demonstrated the possibility that inhibitors of PatB and OatA with antibacterial activity can be isolated [87] and thus provided a preliminary proof of principle that is worthy of further investigation.…”

WssI from the Gram-negative bacterial cellulose synthase is an O-acetyltransferase that acts on cello-oligomers with several acetyl donor substrates

“…Data analysis was conducted using CDD Vault Analysis Software (Collaborative Drug Discovery, Inc), and hits were identified as compounds that had a Z-score −3. The hits remaining after the primary and confirmatory screening were examined further to identify possible PAINS, which could result in false-positive results ( 91 ). Further refinement of the lead candidates was also conducted by assessing the fluorescence quenching potential of lead compound hits by assaying the quenching potential of different compound concentrations (10.1 μM and/or 50.5 μM) against the fluorescence of 4-methylumbeliferone (247 μM) to reveal any potential false positives that were not actually because of Ai WssI ΔN activity.…”

High-Throughput Screening of Natural Product and Synthetic Molecule Libraries for Antibacterial Drug Discovery

“…Numerous in vitro molecular target-based HTS assays using the SML are reported to date that successfully identified inhibitors of various protein targets such as penicillin-binding protein (PBP) [ 178 ], mitogen-activated protein kinase (MKK) [ 179 ], the type III secretion system (T3SS) [ 180 ], the anthrax lethal factor (LF) [ 181 ], phosphopantetheinyl transferase (PPTase) [ 182 ], peptidoglycan O-acetyltransferase [ 183 ], telomere resolvase (ResT) [ 184 ], etc. Spicer et al screened ~292,000 compounds against M18 Aspartyl Aminopeptidase (PfM18AAP) and identified two compounds that were also active in whole cell-based assays [ 185 ].…”