Inhibition of <i>Acinetobacter</i>-Derived Cephalosporinase: Exploring the Carboxylate Recognition Site Using Novel β‑Lactamase Inhibitors

Boronic acids are attracting a lot of attention as β-lactamase inhibitors, and in particular, compound <b>S02030</b> (<i>K</i><sub>i</sub> = 44 nM) proved to be a good lead compound against ADC-7 (<i>Acinetobacter</i>-derived cephalosporinase), one of the most significant resistance determinants in <i>A. baumannii</i>. The atomic structure of the ADC-7/<b>S02030</b> complex highlighted the importance of critical structural determinants for recognition of the boronic acids. Herein, to elucidate the role in recognition of the R2-carboxylate, which mimics the C<sub>3</sub>/C<sub>4</sub> found in β-lactams, we designed, synthesized, and characterized six derivatives of <b>S02030</b> (<b>3a</b>). Out of the six compounds, the best inhibitors proved to be those with an explicit negative charge (compounds <b>3a</b>–<b>c</b>, <b>3h</b>, and <b>3j</b>, <i>K</i><sub>i</sub> = 44–115 nM), which is in contrast to the derivatives where the negative charge is omitted, such as the amide derivative <b>3d</b> (<i>K</i><sub>i</sub> = 224 nM) and the hydroxyamide derivative <b>3e</b> (<i>K</i><sub>i</sub> = 155 nM). To develop a structural characterization of inhibitor binding in the active site, the X-ray crystal structures of ADC-7 in a complex with compounds <b>3c</b>, <b>SM23</b>, and <b>EC04</b> were determined. All three compounds share the same structural features as in <b>S02030</b> but only differ in the carboxy-R2 side chain, thereby providing the opportunity of exploring the distinct binding mode of the negatively charged R2 side chain. This cephalosporinase demonstrates a high degree of versatility in recognition, employing different residues to directly interact with the carboxylate, thus suggesting the existence of a “carboxylate binding region” rather than a binding site in ADC enzymes. Furthermore, this class of compounds was tested against resistant clinical strains of <i>A. baumannii</i> and are effective at inhibiting bacterial growth in conjunction with a β-lactam antibiotic.