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

M. RUGGIERO; K. M. PAPP-WALLACE; M. TARACILA; M. MOJICA; C. BETHEL; S, RUDIN; E. ZEISER; G. GUTKIND; R. A. BONOMO; P. POWER

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY

AMER SOC MICROBIOLOGY

Lugar: Washington; Año: 2017

0066-4804

PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted Ω loop and extended B3 β-strand. These singular structural differences 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-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k2/K = 2 ± 0.1 × 103 M-1s-1) that were obtained when testing AVI were reminiscent of values observed testing class C and D β-lactamases (i.e., k2/K range ≈103 M-1s-1) and not class A β-lactamases (i.e., k2/K range, 104-105 M-1s-1). Once AVI was bound, AVI formed a stable complex with PER-2 as observed via mass spectrometry (e.g., 31,389 ± 3 amu 31,604 ± 3 amu for 24 hr). Molecular modeling of PER-2 with AVI showed that thecarbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase. However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) may affect the binding of necessary catalytic water molecules thus slowing acylation (k2/K) of AVI onto PER-2. As a result, the magnitude of k2/K resembles class D enzymes. Similarities in electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested β-lactam-AVI combinations. As a result of lowering MICs to ≤ 2 mg/L, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing blaPER-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.