Ruggiero Melina, Papp-Wallace Krisztina M, Taracila Magdalena A, Mojica Maria F, Bethel Christopher R, Rudin Susan D, Zeiser Elise T, Gutkind Gabriel, Bonomo Robert A, Power Pablo
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología y Biotecnología, Cátedra de Microbiología, Buenos Aires, Argentina.
Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA.
Antimicrob Agents Chemother. 2017 May 24;61(6). doi: 10.1128/AAC.02476-16. Print 2017 Jun.
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 omega (Ω) loop and extended B3 β-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-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., / = 2 × 10 ± 0.1 × 10 M s, where is the rate constant for acylation (carbamylation) and 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 β-lactamases (i.e., / range of ≈10 M s) and not class A β-lactamases (i.e., / range, 10 to 10 M s). 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 β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (/) 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 β-lactamase, we tested different β-lactam-AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against expressing 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.
PER β-内酰胺酶是在革兰氏阴性菌中发现的一类新出现的超广谱β-内酰胺酶（ESBL）。PER β-内酰胺酶在A类酶中独具特色，因为它们具有一个反向的ω（Ω）环和延长的B3 β链。据推测，这些独特的结构特征有助于其对氧亚氨基头孢菌素（如头孢噻肟和头孢他啶）的水解特性。在此，我们测试了新型非β-内酰胺β-内酰胺酶抑制剂阿维巴坦（AVI）使PER-2失活的能力。有趣的是，测试AVI时获得的PER-2抑制常数（即/ = 2 × 10 ± 0.1 × 10 M s，其中是酰化（氨甲酰化）速率常数，是平衡常数）让人想起测试AVI对C类和D类β-内酰胺酶抑制作用时观察到的值（即/范围约为10 M s），而不是A类β-内酰胺酶（即/范围为10至10 M s）。一旦AVI结合，通过质谱观察到与PER-2形成稳定复合物（例如，24小时内从31,389 ± 3原子质量单位[amu]变为31,604 ± 3 amu）。PER-2与AVI的分子模拟显示，AVI的羰基位于β-内酰胺酶的氧阴离子洞中，且AVI的硫酸盐与PER-2 β-内酰胺酶的β-内酰胺羧酸酯结合位点（R220和T237）形成相互作用。然而，观察到PER-2活性位点附近（由Ser70和B3 - B4 β链构成）存在疏水区域，这可能会影响必需催化水分子的结合，从而减缓AVI在PER-2上的酰化（/）。在OXA-48和PER-2之间也观察到活性位点类似的静电和疏水性，而CTX-M-15更具亲水性。为证明AVI克服PER-2 β-内酰胺酶增强的头孢菌素酶活性的能力，我们测试了不同的β-内酰胺 - AVI组合。通过将最低抑菌浓度降低至≤2 mg/升，头孢洛林 - AVI组合可能是针对表达的的一种有利治疗选择。我们的研究确定了AVI对PER-2 ESBL的失活作用，并表明活性位点的生物物理特性有助于确定失活效率。
