STUDIES OF THE METALLO BETA LACTAMASE CCrA FROM BACTERIODES FRAGILIS AND A DANSYLATED MONOCYCLIC BETA LACTAM (1-(5-DIMETHYLAMINO-1-NAPTHALENESULFONYL HYDRAZIDO)-3-ACETAMIDO-4-METHOXY-2-AZETIDINONE
Due to their ability to hydrolyze the lactams, the beta lactamases constitute the major mechanism by which bacteria become resistant to beta lactam antibiotics. The Class A beta lactamases (termed serine enzymes) are inhibited by clinically available compounds such as sulbactam, tazobactam and clavulanic acid. However, the metallo or class B enzymes are not affected by inhibitors of the Class A enzymes. Here we present the de novo synthesis and characterization of a dansylated monocyclic beta lactam (1-(5-dimethylamino-1-napthalenesulfonyl hydrazido)-3-acetamido-4-methoxy-2-azetidinone). The dansylated monocyclic beta lactam was characterized via infrared spectroscopy, mass spectrometry, NMR, bioassay, and purified by HPLC. This 2-azetidinone was found to be a covalent, irreversible inhibitor of the metallo beta lactamase CCrA from Bacteriodes fragilis . The CCrA metallo beta lactamase was characterized via HPLC, SDS-PAGE, densitometry, and activity assays. The HPLC elution profile of the beta lactamase showed a consistent retention time of 28.94 minutes with a 1-100% acetonitrile gradient. The enzyme was determined to be inactive (e.g. unable to hydrolyze nitrocefin) in the presence of EDTA. MALDI-TOF analysis of an in-gel tryptic digest of the CCrA beta lactamase gave eight peptides (MH+) that corresponded to the masses of theoretical peptides: 775.398, 1433.651, 1505.730, 1637.778, 1765.912, 2070.961, 2101.977, and 2483.065. The presence of metallo beta lactamases in clinically relevant bacteria has become an obvious threat to the efficacy of existing antibiotics. The ability of bacteria to escape the lethal action of beta lactam antibiotics highlights the importance of the discovery of new compounds that can either escape or inhibit the activity of the beta lactamases, particularly the clinically relevant metallo beta lactamases. There have been no previous reports of beta lactam inhibitors of the metallo beta lactamases.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:metallo beta lactamase enzyme inhibition lactam
Date of Publication:01/01/2001