Purification, characterization, and kinetic analysis of various metallo-β-lactamases and discovery of novel inhibitors of these enzymes.

Abstract

Pathogenic bacteria are rapidly becoming antibiotic resistant, at a rate much faster than the production and FDA approval of new antibiotics that can combat these extreme resistant infections. One way to overcome antibiotic resistance is to design inhibitors that shut down current resistance pathways in order for traditionally useful antibiotics to be reintroduced in combination with the new inhibitor to cure these types of infections. One major contributor to antibiotic resistance are enzymes known as β-lactamases, which render the current most widely prescribed class of antibiotics inactive. This work describes the purification of the enzyme Bla2, a metallo-β-lactamase which confers antibiotic resistance to Bacillus anthracis. Aspects of the enzyme’s activity and stability are investigated to find an optimal setting in which potential inhibitors can be analyzed. Three classes of inhibitors are investigated against this enzyme: compounds containing hydroxamate residues, a compound containing a zinc binding group, and a set of DNA/PNA aptamers previously developed by SELEX technology. The hydroxamate compound N-hydroxy-3-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide exhibited promising inhibitory activity against Bla2 as well as inhibition of growth of Escherichia coli cells expressing the enzyme. The compound that contains the zinc binding group, known as thiomaltol, inhibits Bla2 more strongly than when employed against other metal containing enzymes and is a reversible, slow-binding inhibitor of Bla2, which may provide a clinical advantage for this compound when co-administered with a β-lactam antibiotic. Also, DNA/PNA aptamers were examined against Bla2 and have some inhibitory activity against the enzyme as well as Bacillus subtilis cells expressing Bla2. In addition to Bla2, a truncated version of the metallo-β-lactamase NDM-1 from Klebsiella pneumoniae was purified and kinetically characterized in order to aid in future inhibitor design of this potent enzyme. In conclusion, various metallo-β-lactamases were successfully purified, characterized, and various classes of inhibitors were investigated against Bla2 which have potential as lead compounds to direct future efforts in novel antibiotic design.