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The spread of antibiotic resistance in Gram-negative bacteria has sustained a continuing search for new agents with antibacterial activity against this important class of bacterial pathogen. Because the biosynthesis of lipopolysaccharide (LPS) is unique to Gram-negative bacteria and required by them for growth and virulence, attempts have been made to discover or design antibacterial agents acting at this site; however, no such agents have so far been developed. We now present definitive experimental data documenting design of the first member of the class of antibacterial compounds which specifically inhibit LPS synthesis. The target enzyme is 3-deoxy-D-manno-octulosonate cytidylytransferase (CMP-KDO synthetase), a cytoplasmic enzyme which activates 3-deoxy-D-manno-octulosonate (KDO) for incorporation into LPS. A specific inhibitor of CMP-KDO synthetase, alpha-C-(1,5-anhydro-7-amino-2,7-dideoxy-D-manno-heptopyranosyl)-carboxy late was designed using results of our studies of the purified enzyme. LPS synthesis ceased and lipid A precursor accumulated, causing growth stasis and perturbation of outer membrane structure and function, following delivery of the inhibitor to the intracellular target by a peptide carrier. Antibacterial action required an intact oligopeptide permease system and specific intracellular aminopeptidase activity to release inhibitor from the peptide prodrug.

(11,19,20,36,60DNA break to form a gate allowing subsequent DNA strand passing. Effective topoisomerase inhibitors stabilize the intermediate, termed the cleavage complex, and this stabilization in the cell triggers an unknown process causing cell death (7,30). The stabilization of the cleavage complex by topoisomerase inhibitors thus converts these enzymes into cellular poisons (28). The strategy of discovering therapeutic agents of this class is, therefore, plausible and easily justifiable because of the fact that the enzyme need not be proven to be essential to the viability of that particular cell. In principle, any cell which contains high levels of a topoisomerase is vulnerable to killing by topoisomerase inhibitors capable of stabilizing the complex.Candida albicans andAspeigilus niger are two important, life-threatening systemic fungal pathogens in individuals with impaired immunity, such as transplant recipients, patients with AIDS, and patients with cancer undergoing chemotherapy. More effective and safe antifungal agents are urgently needed for the treatment of these increasingly important opportunistic infections. Our results showed that both types of topoisomerases exist in abundance in these pathogenic fungi, indicating that the enzymes are potential targets for the discovery of fungicidal agents. By using isolated C. albicans DNA topoisomerase II and DNA breakage assays, we have further found that the responses of the fungal and the mammalian topoisomerase II to topoisomerase poisons differ in magnitude, suggesting that the discovery of specific inhibitors safe for the host is feasible. MATERIAL1 AND METHODSChemicals and reaents. Agarose (type II), Tris, EDTA, dimethyl sulfoxide, and amiloride were from Sigma. EcoRI

As an approach to discovering highly potent motilides with oral activity, novel 4"-deoxy derivatives of 8,9-anhydroerythromycin 6,9-hemiacetal were designed, synthesized, and evaluated for their gastrointestinal prokinetic activities. These compounds were orders of magnitude more potent than their 4"-hydroxy analogs in inducing smooth muscle contractions in an in vitro rabbit duodenal assay. Removal of the 12-hydroxy group, which was aimed at improving oral bioavailability, also afforded further potentiation in in vitro activity, leading to the identification of 8,9-anhydro-4"-deoxy-3'-N-desmethyl-3'-N-ethylerythromycin B 6,9-hemiacetal (ABT-229) as a potential prokinetic drug. ABT-229 was > 300,000 times more potent than erythromycin in vitro and had 39% oral bioavailability in dog compared to its 4",12-dihydroxy congener (EM-523), which was only 400 times more potent than erythromycin and had relatively low (1.4%) oral bioavailability.

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