Serine beta-lactamases are the major resistance mechanism to beta-lactam antibiotics. The long-term goal is to understand how structure encode recognition and function in these enzymes and to use this information to discover novel inhibitors. Such inhibitors may be leads to agents that reverse bacterial resistance to beta-lactams.
The specific aims are: 1. To understand the mechanism the Group I beta-lactamase AmpC. Outstanding questions include: the identity of the catalytic base, the direction of water attack, what separates a beta-lactam substrate from a beta-lactam inhibitor, and overall, how this information is encoded in the structure of the enzyme? Beta- lactam analogs and transition-state analog boronic acids will be synthesized and their structures in complex with Am-C determined by x-ray crystallography. Mutant enzymes will be made to probe the enzyme side of the recognition event. It may be possible to characterize representative structures for each step in the catalytic pathway. The structures and energies that emerge will provide a three-dimensional map for how ligand functionality is recognized by AmpC. 2. To design non-beta-lactam inhibitors of serine beta- lactamases. The motivating idea is that structure can guide the discovery of non-beta-lactam inhibitors of beta-lactamases, and that such novel inhibitors would break the cycle of pre-evolved resistance mechanisms to which beta-lactams are subject. Guided by the functional group """"""""map"""""""" investigated in aim 1, the following questions will be investigated: Can non-beta-lactam inhibitors be found that bind tightly to serine beta-lactamses? Would such non-beta-lactam inhibitors be susceptible to the suite of pre-evolved resistance mechanisms that face new beta-lactam inhibitors? Such non-beta-lactam inhibitors of beta-lactamases would not be hydrolysable by mutant beta-lactamases, and might not up-regulate the expression of beta-lactamases, as do many beta-lactams. Three sorts of inhibitors will be investigated: transition-state analogs, arylboronic acids, and molecules discovered using molecular docking. The Ki values of the new inhibitors will be determined and their structures in complex with AmpC will be solved by x-ray crystallography. The antimicrobial synergy of the new inhibitors will be evaluated, as will their ability to evade pre-evolved resistance mechanisms.
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