Structural Insights into the Inhibition of the Extended-Spectrum β-Lactamase PER-2 by Avibactam

Abstract

The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-β-lactamases. Previous investigations showed that inhibition constants of AVI toward class A PER-2 are reminiscent of values observed for class C and D β-lactamases (i.e., k2/K of ≈103 M−1 s−1) but lower than other class A β-lactamases (i.e., k2/K = 104 to 105 M−1 s−1). Herein, biochemical and structural studies were conducted with PER-2 and AVI to explore these differences. Furthermore, biochemical studies on Arg220 and Thr237 variants with AVI were conducted to gain deeper insight into the mechanism of PER-2 inactivation. The main biochemical and structural observations revealed the following: (i) both amino-acid substitutions in Arg220 and the rich hydrophobic content in the active site hinder the binding of catalytic waters and acylation, impairing AVI inhibition; (ii) movement of Ser130 upon binding of AVI favors the formation of a hydrogen bond with the sulfate group of AVI; and (iii) the Thr237Ala substitution alters the AVI inhibition constants. The acylation constant (k2/K) of PER-2 by AVI is primarily influenced by stabilizing hydrogen bonds involving AVI and important residues such as Thr237 and Arg220. (Variants in Arg220 demonstrate a dramatic reduction in k2/K.) We also observed that displacement of Ser130 side chain impairs AVI acylation, an observation not made in other extended-spectrum β-lactamases (ESBLs). Comparatively, relebactam combined with a β-lactam is more potent against Escherichia coli producing PER-2 variants than β-lactam–AVI combinations. Our findings provide a rationale for evaluating the utility of the currently available DBO inhibitors against unique ESBLs like PER-2 and anticipate the effectiveness of these inhibitors toward variants that may eventually be selected upon AVI usage.

Extended-spectrum β-lactamases: an update on their characteristics, epidemiology and detection

“…These enzymes have been reported to be inhibited by avibactam to a lesser extent than other class A β-lactamases, with significant differences in MIC values for avibactam and relebactam when tested in combination with other β-lactams. 85 , 91 More detailed studies are warranted due to the difference in activity of these two inhibitors of the same class. Recent analysis demonstrated that A. baumannii isolates harbouring PER enzymes can display elevated MIC values against cefiderocol, a siderophore cephalosporin.…”

In vitro synergistic activity of aztreonam (AZT) plus novel and old β-lactamase inhibitor combinations against metallo-β-lactamase-producing AZT-resistant Enterobacterales

“…Only one isolate (K. oxytoca co-harbouring NDM, PER-2 and CIT) showed an AZT-AVI MIC of 8 mg l −1 , which is categorized as intermediate by the CLSI AZT breakpoint. Ruggiero et al [16] demonstrated that PER-2 is not as efficiently inhibited by AVI as other class A β-lactamases. PER-2 represents the second most frequent ESBL in Argentina [17] (5 % of E. coli oxyiminocephalosporin resistant), but its presence in international surveillance studies is minor.…”

Structural Investigations of the Inhibition of Escherichia coli AmpC β-Lactamase by Diazabicyclooctanes

“…The results reveal a distinctive kinetic profile for AmpC EC inhibition by ZID, which differs from those observed for AVI, REL, and NAC. Together with studies on the inhibition of class A, D and other, structurally distinct class C SBLs (16,19,20,(31)(32)(33) the results will help guide further optimization of the DBO scaffold for potent broad spectrum SBL inhibition.…”

“…While the interactions of avibactam with a wide variety of SBLs have been studied (e.g. (24)(25)(26)(27)(28)(29)(30)), structural information on the binding of other DBOs is more limited (16,19,20,(31)(32)(33).…”