Dual Targeting Antibacterial Peptide Inhibitor of Early Lipid A Biosynthesis

Abstract

UDP-3-O-(R-3-hydroxyacyl)GlcN N-acyltransferase (LpxD) has been shown to be essential to survival of lipid A producing Gram-negative bacteria. In this study, LpxD-binding peptides 12 amino acids in length were identified from a phage-bound random peptide library screen. Three peptides displayed antibacterial activity when expressed intracellularly, one of which (RJPXD33) represented 15% of the total hits. RJPXD33 binds to E. coli LpxD with a Kd of 6 μM and is competitive with R-3-hydroxymyristoyl-ACP binding. RJPXD33 can be C-terminally fused in vivo with thioredoxin or N-terminally modified in vitro with beta-alanyl-fluorescein and maintain LpxD binding. The latter was used to develop an LpxD fluorescent binding assay used to evaluate unlabeled ligands, and is amenable to small molecule library screening. Furthermore, RJPXD33 also binds to and inhibits E. coli UDP-N-acetylglucosamine acyltransferase (LpxA) with a Kd of 20 μM, unearthing the possibility for the development of small molecule, dual-binding LpxA/LpxD inhibitors as novel antimicrobials.

Structural Basis for the Recognition of Peptide RJPXD33 by Acyltransferases in Lipid A Biosynthesis

“…When the assay was performed with EcLpxA, the observed K d for FITC-RJPXD33⌬6 was 12 Ϯ 1 M (Table 2). This value was in close proximity to the previously published value (K d ϭ 17 Ϯ 1.6 M) for the full-length FITC-RJPXD33 (13). This suggested that the six terminal amino acids are not making extensive binding contacts to LpxA, in agreement with the crystal structure.…”

“…by phage display against immobilized LpxD (13). Although no EcLpxD-RJPXD33 structure is available, this current work suggests that RJPXD33 would overlap with the acyl-PPan arm of acyl-ACP, thereby inhibiting acyl-ACP from binding to LpxD.…”

“…The fluorescence polarization assay was performed as previously described (13). In 384-well black Costar plates, LpxA or His 6 -LpxD were serially diluted while holding the fluorescent peptide at 20 nM in a final volume of 50 l in 20 mM HEPES pH 8.0 (0.01% DMSO).…”

“…Recently, through a phage display screen against LpxD, an inhibitor (RJPXD33; TNLYMLPKWDIP-NH 2 ) was identified that demonstrated affinity and inhibitory activity for both LpxA and LpxD (13). Although peptides display susceptibility to proteases and poor bioavailability, they have become a coveted tool in chemists' and biologists' repertoires because of the ease of synthesis and functional utility.…”

“…Herein, we have utilized x-ray crystallography and peptide photoaffinity probes to provide insights into the binding interactions between the LpxA acyltransferase and RJPXD33 (13). Furthermore, acyltransferase binding studies using truncations of RJPXD33 were undertaken to provide biochemical verification of the structural data obtained.…”