Antibacterial agents that target lipid A biosynthesis in Gram-negative bacteria -: Inhibition of diverse UDP-3-O-(R-3-hydroxymyristoyl)-N-acetyglucosamine deacetylases by substrate analogs containing zinc binding motifs

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A, a unique amphiphilic molecule found in the outer membranes of virtually all Gramnegative bacteria. Since lipid A biosynthesis is required for bacterial growth, inhibitors of LpxC have potential utility as antibiotics. The enzymes of lipid A biosynthesis, including LpxC, are encoded by single copy genes in all sequenced Gram-negative genomes, We have now cloned, overexpressed, and purified LpxC from the hyperthermophile Aquifex aeolicus. This heat-stable LpxC variant (the most divergent of all known LpxCs) displays 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 phenyloxazoline-based hydroxamate that inhibits E, coli LpxC with K-i of similar to 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 inhibit both E, coli and A. aeolicus LpxC at similar concentrations. We have also identified a phosphinate-containing substrate analog that inhibits both E. coli and A. aeolicus LpxC, suggesting that the LpxC reaction proceeds by a mechanism similar to that described for other zinc metalloamidases, like carboxypeptidase A and thermolysin, The differences between the phenyloxazoline and the substrate-based LpxC inhibitors might be exploited for developing novel antibiotics targeted either against some or all Gram-negative strains. We suggest that LpxC inhibitors with antibacterial activity be termed "deacetylins."