The research described in this proposal is primarily designed to elucidate the mechanism of action of a novel cephalosporin which contains the antibacterial dipeptide Beta-c1-LA1a-Beta-C1-LAla linked to the cephem as a C10-ester. We have prepared this compound as a substrate for the Beta-lactam enzymes, with the expectation that enzymatic hydrolysis of the cephalosporin in Beta-lactamase resistant bacteria would release the antibiotic peptide. The peptide, in turn, is likely to be hydrolyzed by bacterial peptidase, and the accumulating Beta-c1-LAla should lead to inactivation of alanine racemase inside the cell. The cephalosporin peptide ester was found to be an excellent substrate for the purified Escherichia coli TEM Beta-lactamase, and Beta-C1-LA1a-Beta-C1-LAla is a product of enzymic reaction in vitro. Further, the dipeptide gives rise to inactivation of alanine racemase in E. coli. However, microbial susceptibility testing revealed that the cephem peptidyl ester is antibacterial against both lactamase producing and nonproducing bacteria. We now wish to analyze thoroughly the reactions of our compound with both types of organisms, using in vivo 13C NMR. We will look further at the ability of the penicillin-binding proteins (PBPs) to process the cephem peptide in vitro and in vivo. We suspect that the PBPs, as well as the Beta-lactamases, may be conscripted for delivery of enzyme inactivators in vivo and the research proposed is designed to explore this possibility. We further intend to prepare a cephalosporin carbamate derivative of the aminoglycoside fortimicin. We will similarly examine its reactions both with the Beta-lactamases and the PBPs. In so doing, we hope to explore whether these enzymes can be generally commandeered for the delivery of antibacterial compounds that have limited clinical utility because of adventitious cytotoxicity in the host.
