Structural and Biological Basis of Small Molecule Inhibition of Escherichia coli LpxD Acyltransferase Essential for Lipopolysaccharide Biosynthesis

LpxD, acyl-ACP-dependent -acyltransferase, is the third enzyme of lipid A biosynthesis in Gram-negative bacteria. A recent probe-based screen identified several compounds, including 6359-0284 (compound ), that inhibit the enzymatic activity of ( ) LpxD. Here, we use these inhibitors to chemically validate LpxD as an attractive antibacterial target. We first found that compound was oxidized in solution to the more stable aromatized tetrahydro-pyrazolo-quinolinone compound . From the strain deficient in efflux, we isolated a mutant that was less susceptible to compound and had an missense mutation (Gly268Cys), supporting the cellular on-target activity. Using surface plasma resonance, we showed direct binding to LpxD for compound and other reported LpxD inhibitors . Furthermore, we determined eight cocrystal structures of LpxD/inhibitor complexes. These costructures pinpointed the 4'-phosphopantetheine binding site as the common ligand binding hotspot, where hydrogen bonds to Gly269 and/or Gly287 were important for inhibitor binding. In addition, the LpxD/compound costructure rationalized the reduced activity of compound in the LpxD mutant. Moreover, we obtained the LpxD structure in complex with a previously reported LpxA/LpxD dual targeting peptide inhibitor, RJPXD33, providing structural rationale for the unique dual targeting properties of this peptide. Given that the active site residues of LpxD are conserved in multidrug resistant Enterobacteriaceae, this work paves the way for future LpxD drug discovery efforts combating these Gram-negative pathogens.