Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase

Abstract

PER ␤-lactamases are an emerging family of extended-spectrum ␤-lactamases (ESBL) found in Gram-negative bacteria. PER ␤-lactamases are unique among class A enzymes as they possess an inverted omega (⍀) loop and extended B3 ␤-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-␤-lactam ␤-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2 /K ϭ 2 ϫ 10 3 Ϯ 0.1 ϫ 10 3 M Ϫ1 s Ϫ1 , where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D ␤-lactamases (i.e., k 2 /K range of Ϸ10 3 M Ϫ1 s Ϫ1 ) and not class A ␤-lactamases (i.e., k 2 /K range, 10 4 to 10 5 M Ϫ1 s Ϫ1 ). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 Ϯ 3 atomic mass units [amu] ¡ 31,604 Ϯ 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the ␤-lactamase and that the sulfate of AVI formed interactions with the ␤-lactam carboxylate binding site of the PER-2 ␤-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 ␤-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k 2 /K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 ␤-lactamase, we tested different ␤-lactam-AVI combinations. By lowering MICs to Յ2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing bla PER-2 . Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.

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

“…An analysis of the ability of AVI to inactivate PER-2 was recently completed (14). Interestingly, the inhibition constants (i.e., k 2 /K ϭ 2 Ϯ 0.1 ϫ 10 3 M Ϫ1 s Ϫ1 ) that were observed when testing AVI were reminiscent of values observed for class C and D ␤-lactamases (i.e., k 2 /K range of Ϸ10 3 M Ϫ1 s Ϫ1 ), but lower than those from class A ␤-lactamases (i.e., k 2 /K range of 10 4 to 10 5 M Ϫ1 s Ϫ1 ) (9,10,14). Once AVI is bound to PER-2 ␤-lactamase, AVI forms a stable complex with PER-2, as observed via mass spectrometry (e.g., from 31,389 Ϯ 3 amu to 31,604 Ϯ 3 amu for 24 h).…”

“…Once AVI is bound to PER-2 ␤-lactamase, AVI forms a stable complex with PER-2, as observed via mass spectrometry (e.g., from 31,389 Ϯ 3 amu to 31,604 Ϯ 3 amu for 24 h). This analysis led to the hypothesis that the hydrophobic nature of the PER-2 active site was responsible in part for the slower acylation rate of AVI (14). In silico molecular modeling suggested key interactions between AVI's sulfate and residues Arg220 and Thr237 in PER-2 (14).…”

“…This analysis led to the hypothesis that the hydrophobic nature of the PER-2 active site was responsible in part for the slower acylation rate of AVI (14). In silico molecular modeling suggested key interactions between AVI's sulfate and residues Arg220 and Thr237 in PER-2 (14). To gain a deeper insight into the mechanism of inactivation of PER-2 by AVI, we conducted structural and biochemical studies with wild-type PER-2 and Arg220 and Thr237 variants of PER-2.…”

Structural Insights into the TLA-3 Extended-Spectrum β-Lactamase and Its Inhibition by Avibactam and OP0595

“…Observed rate constant for inactivation (k obs ) values were calculated on the basis of the fitting of progress curves for avibactam or OP0595 inhibition and were plotted against the inhibitor concentration to obtain the secondorder acylation rate constant (k 2 /K, where k 2 is the acylation rate constant and K is the equilibrium constant) value ( Fig. 2A (17,19,20). These low off rates indicate that diazabicyclooctanes can form stable complexes with TLA-3.…”

“…where v is the velocity of the reaction and [S] is concentration of the substrate. The interaction between TLA-3 and the inhibitors was evaluated as described previously (17,27).…”