Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, 10701 East Blvd., Cleveland, OH 44106, USA.
Antimicrob Agents Chemother. 2010 Feb;54(2):890-7. doi: 10.1128/AAC.00693-09. Epub 2009 Dec 14.
As resistance determinants, KPC beta-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type beta-lactamases are often identified as resistant to standard beta-lactam-beta-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to beta-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (kcat/kinact ratios, where kinact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp2-hybridized C3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: Km of penem 1, 0.06+/-0.01 microM, and Km of penem 2, 0.006+/-0.001 microM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (kcat/kinact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (kinact/Km=6.5+/-0.6 microM(-1) s(-1)). We conclude that KPC-2 is unique among class A beta-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type beta-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current beta-lactamase inhibitors.
作为耐药决定因素，KPCβ-内酰胺酶表现出广泛的底物谱，包括碳青霉烯类、氧亚氨基头孢菌素类和头孢菌素类。此外，在药敏试验中，携带 KPC 型β-内酰胺酶的临床菌株通常被鉴定为对标准β-内酰胺-β-内酰胺酶抑制剂组合具有耐药性。KPC-2 碳青霉烯酶带来了重大的临床挑战，因为 KPC-2 相关表型的机制基础仍然难以捉摸。在这里，我们通过确定克拉维酸、舒巴坦和他唑巴坦分别被 KPC-2 水解的分区比（kcat/kinact 比值，其中 kinact 是酶失活的速率常数）为 2500、1000 和 500，证明了 KPC-2 对β-内酰胺酶抑制剂的耐药性。含有 sp2 杂化 C3 羧酸和双环 R1 侧链的亚甲基青霉（二氢吡唑并[1,5-c][1,3]噻唑[青霉 1]和二氢吡唑并[5,1-c][1,4]噻嗪[青霉 2]）是有效的抑制剂：青霉 1 的 Km 为 0.06+/-0.01 μM，青霉 2 的 Km 为 0.006+/-0.001 μM。我们还证明，青霉 1 和青霉 2 是基于机制的失活剂，它们的分区比（kcat/kinact 比值）分别为 250 和 50。为了了解这些青霉抑制的机制，我们在 KPC-2 的活性部位生成了这两种抑制剂的分子表示。这些模型（i）表明，青霉 1 和青霉 2 与活性部位残基的相互作用方式不同，青霉 2 的羰基位于氧阴离子穴之外，并且水解的位置不如青霉 1 有利，（ii）支持动力学观察结果，即青霉 2 是更好的抑制剂（kinact/Km=6.5+/-0.6 μM(-1) s(-1)）。我们得出结论，KPC-2 是 A 类β-内酰胺酶中唯一能够轻易水解克拉维酸、舒巴坦和他唑巴坦的酶。相比之下，青霉类β-内酰胺酶抑制剂通过表现出独特的活性部位化学性质，可能成为未来开发的重要支架，并为我们目前的β-内酰胺酶抑制剂提供有吸引力的替代方案。
