Antibacterial Agents That Target Lipid A Biosynthesis in Gram-negative Bacteria

Abstract

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the bio- synthesis of lipid A, a unique amphiphilic molecule found in the outer membranes of virtually all Gram- negative bacteria. Since lipid A biosynthesis is required for bacterial growth, inhibitors of LpxC have potential utility as antibiotics. The enzymes of lipid A biosynthe- sis, including LpxC, are encoded by single copy genes in all sequenced Gram-negative genomes. We have now cloned, overexpressed, and purified LpxC from the hy- perthermophile Aquifex aeolicus. This heat-stable LpxC variant (the most divergent of all known LpxCs) dis- plays 32% identity and 51% similarity over 277 amino acid residues out of the 305 in Escherichia coli LpxC. Although A. aeolicus LpxC deacetylates the substrate UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine at a rate comparable with E. coli LpxC, a phenyloxazo- line-based hydroxamate that inhibits E. coli LpxC with Ki of ?50 nM (Onishi, H. R., Pelak, B. A., Gerckens, L. S., Silver, L. L., Kahan, F. M., Chen, M. H., Patchett, A. A., Galloway, S. M., Hyland, S. A., Anderson, M. S., and Raetz, C. R. H. (1996) Science 274, 980–982) does not inhibit A. aeolicus LpxC. To determine whether or not broad-spectrum deacetylase inhibitors can be found, we have designed a new class of hydroxamate-containing inhibitors of LpxC, starting with the structure of the physiological substrate. Several of these compounds in- hibit both E. coli and A. aeolicus LpxC at similar con- centrations. We have also identified a phosphinate-con- taining substrate analog that inhibits both E. coli and A. aeolicus LpxC, suggesting that the LpxC reaction pro- ceeds by a mechanism similar to that described for other zinc metalloamidases, like carboxypeptidase A and ther- molysin. The differences between the phenyloxazoline and the substrate-based LpxC inhibitors might be ex- ploited for developing novel antibiotics targeted either against some or all Gram-negative strains. We suggest that LpxC inhibitors with antibacterial activity be termed “deacetylins.”