Antibacterial Activities and Characterization of Novel Inhibitors of LpxC

122

The UDP-3- O -( R -3-hydroxyacyl)- N -acetylglucosamine deacetylase LpxC is an essential enzyme of lipid A biosynthesis in Gram-negative bacteria and a promising antibiotic target. CHIR-090, the most potent LpxC inhibitor discovered to date, displays two-step time-dependent inhibition and kills a wide range of Gram-negative pathogens as effectively as ciprofloxacin or tobramycin. In this study, we report the solution structure of the LpxC–CHIR-090 complex. CHIR-090 exploits conserved features of LpxC that are critical for catalysis, including the hydrophobic passage and essential active-site residues. CHIR-090 is adjacent to, but does not occupy, the UDP-binding pocket of LpxC, suggesting that a fragment-based approach may facilitate further optimization of LpxC inhibitors. Additionally, we identified key residues in the Insert II hydrophobic passage that modulate time-dependent inhibition and CHIR-090 resistance. CHIR-090 shares a similar, although previously unrecognized, chemical scaffold with other small-molecule antibiotics such as L-161,240 targeting LpxC, and provides a template for understanding the binding mode of these inhibitors. Consistent with this model, we provide evidence that L-161,240 also occupies the hydrophobic passage.

Section: Methodsmentioning

confidence: 99%

“…CHIR-090's exploitation of highly conserved LpxC features may partially explain the extremely low incidence of spontaneous resistance compared with L-161,240 and BB-78485 (5,8).…”

Section: Chir-090mentioning

confidence: 99%

Structure of the deacetylase LpxC bound to the antibiotic CHIR-090: Time-dependent inhibition and specificity in ligand binding

Barb

Jiang

Raetz

et al. 2007

122

“…To be useful against the LpxC enzymes from a broad range of Gramnegative bacteria, an inhibitor should target interactions with conserved features and surfaces in the enzyme active site. The primary determinant of LpxC-inhibitor recognition is zinc coordination, and some of the most potent inhibitors known to date exploit a hydroxamate functionality for this task (20)(21)(22)(23)(24)(25). Secondary determinants of recognition are conserved surfaces in the enzyme active site, perhaps the most important of which is the hydrophobic tunnel.…”

Section: Resultsmentioning

confidence: 99%

“…Recent mutagenesis studies (19) with LpxC enzymes from Escherichia coli and Aquifex aeolicus indicate residues important for catalysis and zinc binding. To date, inhibitors developed against LpxC enzymes from different Gram-negative bacteria contain hydroxamate or phosphonate zinc-binding motifs and some exhibit potent antibacterial properties (20)(21)(22)(23)(24)(25). LpxC is thus validated as a target for the development of antibacterial agents selective against Gram-negative bacteria.…”

mentioning

confidence: 99%

Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis

Whittington

Rusche

Shin

et al. 2003

153

227

The outer leaflet of the outer membrane of the Gram-negative bacterium serves as a permeability barrier and is composed of lipopolysaccharide, also known as endotoxin. The membrane anchor of lipopolysaccharide is lipid A, the biosynthesis of which is essential for cell viability. The first committed step in lipid A biosynthesis is catalyzed by UDP-(3-O-(R-3-hydroxymyristoyl))-Nacetylglucosamine deacetylase (LpxC), a zinc-dependent deacetylase. Here we report the crystal structure of LpxC from Aquifex aeolicus, which reveals a new ␣؉␤ fold reflecting primordial gene duplication and fusion, as well as a new zinc-binding motif. The catalytic zinc ion resides at the base of an active-site cleft and adjacent to a hydrophobic tunnel occupied by a fatty acid. This tunnel accounts for the specificity of LpxC toward substrates and inhibitors bearing appropriately positioned 3-O-fatty acid substituents. Notably, simple inhibitors designed to target interactions in the hydrophobic tunnel bind with micromolar affinity, thereby representing a step toward the structure-based design of a potent, broad-spectrum antibacterial drug.

“…Many pathogens, such as strains of Pseudomonas aeruginosa in cystic fibrosis patients (29,30), now are resistant to commercially available antibiotics. Therefore it is necessary to develop new antibacterial agents that target previously unexploited systems, such as the enzymes that assemble the lipid A component of lipopolysaccharide (2,8,9,(31)(32)(33)(34)(35). The cytosolic acyltransferase LpxA catalyzes the first step of the lipid A pathway ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide

Williams

Immormino

Gewirth

et al. 2006

UDP-GlcNAc acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein (ACP) to the glucosamine 3-OH group of UDP-GlcNAc. LpxA is essential for the growth of Escherichia coli and related Gram-negative bacteria. The crystal structure of the E. coli LpxA homotrimer, determined previously at 2.6 Å in the absence of substrates or inhibitors, revealed that LpxA contains an unusual, left-handed parallel ␤-helix fold. We now present the crystal structure at 1.8 Å resolution of E. coli LpxA in a complex with a pentadecapeptide, peptide 920. Three peptides, each of which adopts a ␤-hairpin conformation, are bound per LpxA trimer. The peptides are located at the interfaces of adjacent subunits in the vicinity of the three active sites. Each peptide interacts with residues from both adjacent subunits. Peptide 920 is a potent inhibitor of E. coli LpxA (Ki ‫؍‬ 50 nM). It is competitive with respect to acyl-ACP but not UDP-GlcNAc. The compact ␤-turn structure of peptide 920 bound to LpxA may open previously uncharacterized approaches to the rational design of LpxA inhibitors with antibiotic activity.antibiotic ͉ crystal structure ͉ Escherichia coli ͉ inhibitor ͉ outer membrane L ipopolysaccharide constitutes the outer monolayer of the outer membrane in Gram-negative bacteria (1-3). Lipopolysaccharide maintains the integrity of the outer membrane, which functions as a barrier to molecules Ͼ500 Da (4). Lipid A (endotoxin) is the hydrophobic moiety that anchors lipopolysaccharide into the outer membrane (1, 2). It is a potent activator of the human innate immune system via Toll-like receptor 4 (TLR4) (5-7). The minimal lipopolysaccharide required for growth usually consists of two 3-deoxy-D-manno-octulosonic acid (Kdo) residues attached to lipid A ( Fig. 1; ref. 2). Because lipid A biosynthetic enzymes have no mammalian counterparts, they are attractive targets for the design of new antibiotics (10, 11).Kdo 2 -lipid A is synthesized by a conserved pathway consisting of nine enzymes (1, 2). LpxA catalyzes the first step (Fig. 1), the thermodynamically unfavorable 3-O-acylation of UDP-GlcNAc (K eq ϭ 0.01) (12-14). The crystal structure of Escherichia coli LpxA, previously determined at 2.6 Å (15), revealed that the enzyme is a homotrimer. It adopts a distinctive, left-handed parallel ␤-helix fold ( Fig. 2 A and B), which is specified by the presence of 24 complete and six partial hexad repeats (15). Three contiguous hexad repeats (18-aa residues) fold into one coil of the ␤-helix (Fig. 2 A and B). Many other bacterial acyl and acetyl transferases later were shown to possess this fold (17)(18)(19)(20)(21)(22).Structures of LpxA have not been solved in the presence of substrates or inhibitors. Mutagenesis suggests that the active site is located in a large cleft between adjacent subunits (8, 23). This region contains several conserved basic residues (K76, H122, H125, H144, H160, and R204) (23). H125 (Fig. 2 C and D) may be the catalytic ...