Abstract

The natural product lysobactin is a cyclic peptide with greater antibiotic activity than vancomycin against Gram-positive aerobic and anaerobic bacteria. Although the potency of orally administered lysobactin has not been reported, lysobactin hydrolysis in the digestive tract following oral administration would be difficult to predict because of the cyclic structure and presence of beta-hydroxy and D-amino acids. Therefore, lysobactin served as a model compound for study using an in vitro assay to predict if hydrolysis in the digestive tract might severely limit its bioavailability. The in vitro immobilized digestive enzyme assay consisted of a gastric digester containing immobilized pepsin, and an intestinal digester consisting of immobilized trypsin, chymotrypsin, and mucosal peptidases. In order to determine the sites of lysobactin hydrolysis by each enzyme, lysobactin was incubated separately with immobilized pepsin, trypsin, chymotrypsin, or mucosal peptidases. Lysobactin was also incubated with a mixture of intestinal enzymes. The hydrolysis products of lysobactin in each incubation mixture were rapidly identified using reversed-phase HPLC, continuous-flow FAB/LC-MS, and tandem MS. Lysobactin was essentially stable at low pH and was not hydrolyzed by immobilized pepsin. Above pH 7, nonenzymatic hydrolysis of the lactone moiety occurred followed by proteolysis by all intestinal enzymes investigated. Lysobactin was not directly hydrolyzed by immobilized pepsin, chymotrypsin, trypsin, or mucosal peptidases. Hydrolysis rates of lysobactin at pH 7.5 with active or inactivated chymotrypsin were measured and were compared with the rate of chymotrypsin hydrolysis of the open-chain form following hydrolysis of the lactone.(ABSTRACT TRUNCATED AT 250 WORDS)