Potent, Novel in Vitro Inhibitors of thePseudomonasaeruginosaDeacetylase LpxC

Abstract

Deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by LpxC is the first committed step in the Pseudomonas aeruginosa biosynthetic pathway to lipid A; homologous enzymes are found widely among Gram-negative bacteria. As an essential enzyme for which no inhibitors have yet been reported, the P. aeruginosa LpxC represents a highly attractive target for a novel antibacterial drug. We synthesized several focused small-molecule libraries, each composed of a variable aromatic ring, one of four heterocyclic/spacer moieties, and a hydroxamic acid and evaluated the LpxC inhibition of these compounds against purified P. aeruginosa enzyme. To ensure that the in vitro assay would be as physiologically relevant as possible, we synthesized a tritiated form of the specific P. aeruginosa glycolipid substrate and measured directly the enzymatically released acetate. Several of our novel compounds, predominantly those having fluorinated substituents on the aromatic ring and an oxazoline as the heterocyclic moiety, demonstrated in vitro IC(50) values less than 1 microM. We now report the synthesis and in vitro evaluation of these P. aeruginosa LpxC inhibitors.

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

“…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.…”

“…Therefore, targeting the hydrophobic tunnel exclusively yields inhibitor leads of simpler design and higher affinity. Given the increasing occurrence of antibiotic resistance in current day therapy, and given that LpxC is validated as a target for the design of potent antibacterials (20)(21)(22)(23)(24)(25), we advance that the next step in this program, the structure-based design of broad spectrum LpxC inhibitors targeting the hydrophobic tunnel in the LpxC active site, may represent a new and effective strategy for the successful treatment of Gram-negative sepsis. …”

“…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.…”

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

“…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.…”

Mild and Efficient Synthesis of 2‐Indole‐2′‐oxazolines at Room Temperature—A Simple Access to Novel IndOX Ligands

“…A racemic version corresponding to this structure has been reported earlier. [7] The oxazoline ring chirality has been utilized in ligand design for only two decades. [8 -10] IndOX compounds are not yet tested as ligands, but recent studies of pyrrolyl oxazolines (PyrOX) in metal-mediated asymmetric reactions [11] can also be seen as an indication of IndOX-ligand activity.…”