Structural insights into the inhibition of the extended-spectrum beta-lactamase PER-2 by Avibactam

MELINA RUGGIERO; KRISZTINA M. PAPP-WALLACE; FLORENCIA BRUNETTI; MELISSA D. BARNES; ROBERT A. BONOMO; GABRIEL GUTKIND; SEBASTIÁN KLINKE *CO-CORRESPONDING AUTHOR; PABLO POWER *CO-CORRESPONDING AUTHOR

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY

AMER SOC MICROBIOLOGY

Año: 2019 vol. 63

0066-4804

The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-beta-lactamases. Previous investigations showed that inhibition constants of AVI towards class A PER-2 are reminiscent of values observed for class C and D beta-lactamases (i.e., k2/K ≈10^3 M-1s-1), but lower than other class A beta-lactamases (i.e., k2/K 10^4-10^5 M-1s-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; (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 beta-lactamases (ESBLs). Comparatively, relebactam combined with a beta-lactam is more potent against Escherichia coli producing PER-2 variants than beta-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.