Bacterial infections, a major cause of death and morbidity prior to the development of modern antibiotics during the first half of this century, continue to pose a serious threat to public health due to the emergence and spread of increasingly antibiotic resistant bacteria. Penicillin- binding proteins (PBPs) are cell wall synthesizing enzymes that are the targets of the beta-lactam antibiotics. Each bacterial species has a number of PBPs, E. coli having at having at least eight. These enzymes can be divided into two groups, high molecular weight (HMW) PBPs, and low molecular weight (LMW) PBPs. HMW PBPs are lethal targets for the beta-lactam antibiotics, whereas the LMW PBPs are not lethal targets. The LMW PBPs give detectible activity against cell wall related substrates, whereas the HMW PBPs do not. Enzymatic assays against cell wall related substrates therefore do not exist for the most medically important PBPs. Even for LMW PBPs, currently available assay methods are inadequate. As observed by Waxman and Strominger (1983) """"""""The absence of a simple, rapid and sensitive assay has made detailed kinetic and enzymological studies of purified CPases (PBPs) slow and difficult"""""""". In preliminary studies precise and sensitive UV-vis based coupled enzyme assays have been established for PBP activity, for both D-Ala and D-Lac based substrates, and used to partially characterize the activity of E. coli PBP5 amd the previously uncharacterized N. gonorrhoeae PBP3. These assays are substantially more precise than previously described PBP assays. A synthetic approach to D-Ala based substrates has been developed and significant progress toward the synthesis of D-Lac substrates has been made. The proposed research is directed towards: 1) the development of high sensitivity PBP assays, 2) the determination of PBP substrate specificity with synthetic cell wall related substrates, 3) the characterization of PBP steady-state kinetics using precise assays, and 4) preparation and testing of several possible new classes of PBP inhibitors. These studies will established improved methods for the study of the PBPs, determine essential features of PBP substrate specificity, and use this information to design new PBP inhibitors.
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