An in vitro screen of bacterial lipopolysaccharide biosynthetic enzymes identifies an inhibitor of ADP-heptose biosynthesis

The lipopolysaccharide (LPS)-rich outer membrane of gram-negative bacteria provides a protective barrier that insulates these organisms from the action of numerous antibiotics. Breach of the LIPS layer can therefore provide access to the cell interior to otherwise impermeant toxic molecules and can expose vulnerable binding sites for immune system components such as complement. Inhibition of LIPS biosynthesis, leading to a truncated LIPS molecule, is an alternative strategy for antibacterial drug development in which this vital cellular structure is weakened. A significant challenge for in vitro screens of small molecules for inhibition of LIPS biosynthesis is the difficulty in accessing the complex carbohydrate substrates. We have optimized an assay of the enzymes required for LIPS heptose biosynthesis that simultaneously surveys five enzyme activities by using commercially available substrates and report its use in a small-molecule screen that identifies an inhibitor of heptose synthesis.