Novel Insights into the Mode of Inhibition of Class A SHV-1 β-Lactamases Revealed by Boronic Acid Transition State Inhibitors

Abstract

Boronic acid transition state inhibitors (BATSIs) are potent class A and C ␤-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 ␤-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 ␤-lactamase with micromolar affinity that is considerably weaker than their inhibition of other ␤-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other ␤-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.Production of ␤-lactamases (EC 3.5.2.6) is one of the major mechanisms by which bacteria develop resistance to ␤-lactam antibiotics. In nature, four classes of ␤-lactamase enzymes exist (classes A to D). Classes A, C, and D are serine-based ␤-lactamases, while class B uses a metal ion (Zn 2ϩ ) to hydrolyze the lactam bond. TEM-1 and SHV-1 ␤-lactamases are among the most commonly observed class A ␤-lactamases found in Escherichia coli and Klebsiella pneumoniae. As such, this family of ␤-lactamases presents a significant clinical threat (3, 21).To counteract ␤-lactamases, mechanism-based inhibitors were developed to be administered in concert with ␤-lactam antibiotics (9). Presently, there are three commercially available ␤-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) which are effective primarily against class A ␤-lactamases (Fig. 1). Unfortunately, bacteria possessing ␤-lactamase enzymes have adapted under continued drug pressure and some variants of class A ␤-lactamases are now "inhibitor resistant" (9). Moreover, class C and D enzymes are poorly inhibited by any of the three commercial inhibitors. Therefore, there is an urgent need to develop novel ␤-lactam antibiotics and new types of ␤-lactamase inhibitors to counteract the current crisis in antimicrobial resistance (9).Based...

New β-Lactamase Inhibitors: a Therapeutic Renaissance in an MDR World

“…Studies have shown analogs of ampicillin, cephalothin, and cefoperazone to be inhibitors of clinically relevant class A and C ␤-lactamases in the nM range (i.e., 9.3 nM, 420 nM, and 11 nM for K. pneumoniae SHV and for AmpCs from P. aeruginosa and Acinetobacter spp., respectively) (Fig. 3b) (53)(54)(55)(56). These compounds are still preclinical.…”

Boronic Acid Transition State Inhibitors Active against KPC and Other Class A β-Lactamases: Structure-Activity Relationships as a Guide to Inhibitor Design

“…This model takes into account the crystallographic intermediates captured in two previous studies (15,17).…”

“…In a parallel quest, BATSIs were studied and optimized for a variety of ␤-lactamase enzymes (7,(13)(14)(15)(16)(17)(18). Mechanistically, the boronic inhibitors act by binding to the active site of the enzyme, where they sterically resemble the quaternary transition state of the ␤-lactam hydrolysis reaction and occupy the active site with high affinity, leading to inhibition in a reversible competitive manner (9).…”

Crystal Structures of KPC-2 and SHV-1 β-Lactamases in Complex with the Boronic Acid Transition State Analog S02030

“…As stated above, an emerging notion in the discovery of novel BLIs is to utilize boronic acid transition state analogs (BATSIs) to inhibit serine ␤-lactamases such as KPC-2 and SHV-1 (12)(13)(14). Such an approach has led to several potent inhibitor compounds (15,16).…”