The complexed structure and antimicrobial activity of a non-β-lactam inhibitor of AmpC β-lactamase

Abstract

β-Lactamases are the major resistance mechanism to β-lactam antibiotics and pose a growing threat to public health. Recently, bacteria have become resistant to β-lactamase inhibitors, making this problem pressing. In an effort to overcome this resistance, non-β-lactam inhibitors of β-lactamases were investigated for complementarity to the structure of AmpC β-lactamase from Escherichia coli. This led to the discovery of an inhibitor, benzo(b)thiophene-2-boronic acid (BZBTH2B), which inhibited AmpC with a K_i of 27 nM. This inhibitor is chemically dissimilar to β-lactams, raising the question of what specific interactions are responsible for its activity. To answer this question, the X-ray crystallographic structure of BZBTH2B in complex with AmpC was determined to 2.25 Å resolution. The structure reveals several unexpected interactions. The inhibitor appears to complement the conserved, R1-amide binding region of AmpC, despite lacking an amide group. Interactions between one of the boronic acid oxygen atoms, Tyr150, and an ordered water molecule suggest a mechanism for acid/base catalysis and a direction for hydrolytic attack in the enzyme catalyzed reaction. To investigate how a non-β-lactam inhibitor would perform against resistant bacteria, BZBTH2B was tested in antimicrobial assays. BZBTH2B significantly potentiated the activity of a third-generation cephalosporin against AmpC-producing resistant bacteria. This inhibitor was unaffected by two common resistance mechanisms that often arise against β-lactams in conjunction with β-lactamases. Porin channel mutations did not decrease the efficacy of BZBTH2B against cells expressing AmpC. Also, this inhibitor did not induce expression of AmpC, a problem with many β-lactams. The structure of the BZBTH2B/AmpC complex provides a starting point for the structure-based elaboration of this class of non-β-lactam inhibitors.