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 Ki
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.