Inhibition of the class C β-lactamase from Acinetobacter spp. Insights into effective inhibitor design

The need to develop β-lactamase inhibitors against class C cephalosporinases of Gram-negative pathogens represents an urgent clinical priority. To respond to this challenge, five boronic acid derivatives, including a new cefoperazone analogue, were synthesized and tested against the class C cephalosporinase of Acinetobacter baumannii [Acinetobacter-derived cephalosporinase (ADC)]. The commercially available carbapenem antibiotics were also assayed. In the boronic acid series, a chiral cephalothin analogue with a meta-carboxyphenyl moiety corresponding to the C ₃/C ₄ carboxylate of β-lactams showed the lowest Ki (11 ± 1 nM). In antimicrobial susceptibility tests, this cephalothin analogue lowered the ceftazidime and cefotaxime minimum inhibitory concentrations (MICs) of Escherichia coli DH10B cells carrying bla _ADC from 16 to 4 μg/mL and from 8 to 1 μg/mL, respectively. On the other hand, each carbapenem exhibited a K _i of <20 μM, and timed electrospray ionization mass spectrometry (ESI-MS) demonstrated the formation of adducts corresponding to acyl-enzyme intermediates with both intact carbapenem and carbapenem lacking the C ₆ hydroxyethyl group. To improve our understanding of the interactions between the β-lactamase and the inhibitors, we constructed models of ADC as an acyl-enzyme intermediate with (i) the meta-carboxyphenyl cephalothin analogue and (ii) the carbapenems, imipenem and meropenem. Our first model suggests that this chiral cephalothin analogue adopts a novel conformation in the β-lactamase active site. Further, the addition of the substituent mimicking the cephalosporin dihydrothiazine ring may significantly improve affinity for the ADCβ-lactamase. In contrast, theADC-carbapenem models offer a novel role for theR ₂ side group and also suggest that elimination of the C ₆ hydroxyethyl group by retroaldolic reaction leads to a significant conformational change in the acyl-enzyme intermediate. Lessons from the diverse mechanisms and structures of the boronic acid derivatives and carbapenems provide insights for the development of new β-lactamase inhibitors against these critical drug resistance targets.