Potent, novel in vitro inhibitors of the Pseudomonas aeruginosa deacetylase LpxC.

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.

A slow, tight-binding inhibitor of the zinc-dependent deacetylase LpxC of lipid A biosynthesis with antibiotic activity comparable to ciprofloxacin.

The zinc-dependent enzyme LpxC catalyzes the deacetylation of UDP-3-O-acyl-GlcNAc, the first committed step of lipid A biosynthesis. Lipid A is an essential component of the outer membranes of most Gram-negative bacteria, including Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, making LpxC an attractive target for antibiotic design. The inhibition of LpxC by a novel N-aroyl-l-threonine hydroxamic acid (CHIR-090) from a recent patent application (International Patent WO 2004/062601 A2 to Chiron and the University of Washington) is reported here. CHIR-090 possesses remarkable antibiotic activity against both E. coli and P. aeruginosa, comparable to that of ciprofloxacin. The biological activity of CHIR-090 is explained by its inhibition of diverse LpxC orthologues at low nanomolar concentrations, including that of Aquifex aeolicus, for which structural information is available. The inhibition of A. aeolicus LpxC by CHIR-090 occurs in two steps. The first step is rapid and reversible, with a K(i) of 1.0-1.7 nM, depending upon the method of assay. The second step involves the conversion of the EI complex with a half-life of about a minute to a tightly bound form. The second step is functionally irreversible but does not result in the covalent modification of the enzyme, as judged by electrospray ionization mass spectrometry. CHIR-090 is the first example of a slow, tight-binding inhibitor for LpxC and may be the prototype for a new generation of LpxC inhibitors with therapeutic applicability.

19F NMR studies of tryptophan/serum albumin binding.

19F NMR provides direct measures of the Trp binding avidity of 'fatty acid free' bovine serum albumin when D- and L-6-fluorotryptophan are used as the probes. Both a high and low affinity binding site are present. The addition of octanoate either displaces the ligand from both sites or greatly decreases the affinity such that little binding occurs at 2 mM levels. In the case of L-6-fluorotryptophan separate signals are observed for the high and low affinity binding sites and titrations with competing ligands can be used to establish the relative affinities of ligands at the high affinity site. Binding at this site appears to be hydrophobic and shape specific with L-Phe being a very poor ligand (K(D)[L-Phe]/K(D)[L-Trp]=800) while both GHKalphaNal and GHKW displace L-6-fluorotryptophan from this site. In tripeptides of the general formula GHK[ epsilon NH(CH(2))(n)(CO)W], affinity increases with tether length and binding at the low affinity site is restored. This NMR assay appears well-suited for the discovery of selective binding agents in this and other biorecognition phenomena.