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

PER beta-lactamases are an emerging family of extended-spectrum beta-lactamases (ESBL) found in Gram-negative bacteria. PER beta-lactamases are unique among class A enzymes as they possess an inverted omega (Omega) loop and extended B3 beta-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-beta-lactam beta-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k(2)/K = 2x 10(3) +/- 0.1 x 10(3) M-1 s(-1), 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 beta-lactamases (i.e., k(2)/K range of approximate to 10(3) M-1 s(-1)) and not class A beta-lactamases (i.e., k(2)/K range, 10(4) to 10(5) M-1 s(-1)). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 +/- 3 atomic mass units [amu] -> 31,604 +/- 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 beta-lactamase and that the sulfate of AVI formed interactions with the beta-lactam carboxylate binding site of the PER-2 beta-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 beta-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 beta-lactamase, we tested different beta-lactam-AVI combinations. By lowering MICs to <= 2 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.