Screening for Antibacterial Inhibitors of the UDP-3-O-(R-3-Hydroxymyristoyl)-N-Acetylglucosamine Deacetylase (LpxC) Using a High-Throughput Mass Spectrometry Assay

A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)-N- acetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby gener- ates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z' factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coli with reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 mg/mL). The results show that mass spectrometry-based screening is a valuable high-throughput screening tool for detecting inhibitors of enzy- matic targets involving difficult to detect reactions. (Journal of Biomolecular Screening XXXX:xx-xx)