Assessing the Potency of β-Lactamase Inhibitors with Diverse Inactivation Mechanisms against the PenA1 Carbapenemase from Burkholderia multivorans.
Assessing the Potency of β-Lactamase Inhibitors with Diverse Inactivation Mechanisms against the PenA1 Carbapenemase from Burkholderia multivorans.
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DOI：
10.1021/acsinfecdis.0c00682
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发表时间：
2021-04-09
影响因子：
5.3
通讯作者：
Papp-Wallace KM
中科院分区：
文献类型：
--
作者：
Nukaga M;Yoon MJ;Taracilia MA;Hoshino T;Becka SA;Zeiser ET;Johnson JR;Papp-Wallace KM
洋葱伯克霍尔德菌复合体（Bcc）对囊性纤维化或免疫系统受损的人构成严重的健康威胁。感染通常由Bcc菌株引起，该菌株对许多种类的抗生素，包括β-内酰胺具有高度耐药性。Bcc的β-内酰胺抗性主要通过pena样β-内酰胺酶获得。阿维巴坦先前被证明是一种有效的PenA1灭活剂。在这里，我们使用不同作用机制的β-内酰胺酶抑制剂（β-内酰胺型、重氮杂环辛烷型和硼酸盐型）研究了多佛氏伯克霍尔德菌a类丝氨酸碳青霉烯酶PenA1的失活机制。在全细胞实验中，阿维巴坦、乐巴坦、恩美唑巴坦和瓦波巴坦恢复了哌拉西林对大肠杆菌中表达的PenA1的敏感性。根据kinact/KI或k2/K值（范围为3.4 × 102 ~ 2 × 106 M−1 s−1），对PenA1的体外失活效序为阿维巴坦>恩美他唑巴坦>他唑巴坦>雷巴坦>克拉维酸>瓦博巴坦。利用定点诱变技术评估了PenA1中选定的氨基酸（S70、K73、S130、E166、N170、R220、K234、T237和D276）对失活的贡献。PenA1的S130A、R220A和K234A变体对阿维巴坦失活的敏感性较低。R220A变体被纯化并通过稳态抑制动力学进行评估，发现与PenA1相比，对所有测试抑制剂具有增加的KI -app值和降低的kinact/KI或k2/K值。阿维巴坦受丙氨酸替代的影响最大，其乙酰化速率降低了近400倍。对R220A的x射线晶体结构进行了解析，发现残基237和276之间的氢键网络丢失，在活性位点留下了一个空隙，取而代之的是水分子。生成Michaelis - Menten复合物以阐明vaborbactam对PenA1 （k2/K, 3.4 × 102 M−1 s−1）较差的体外抑制谱的分子贡献，并将其与KPC-2（一种被vaborbactam强烈抑制的a类碳青霉烯酶）进行比较。PenA1的活性位点比KPC-2的活性位点大，这影响了vaborbactam形成有利相互作用的能力，导致vaborbactam的羧酸盐被PenA1中的K234/T235吸引，取代了硼酸靠近亲核S70。此外，在PenA1中，与KPC-2中的色氨酸相比，位于105位的酪氨酸更灵活，旋转超过90°，因此PenA1的Y105与vaborbactam的环硼酸盐和噻吩部分竞争结合，进一步排除了vaborbactam对PenA1的抑制作用。考虑到与PenA1相比，R220A变体的k2/K降低了400倍，通过分子建模生成了酰基-酶复合物，并与PenA1-avibactam晶体结构进行了比较。与PenA1相比，占据R220A变体活性位点的水分子无法稳定活性位点的T237和D276区域，从而改变了阿维巴坦形成有利相互作用的能力。前者可能会影响所有抑制剂有效地酰基化这种变异酶的能力。综上所述，这些较新的β-内酰胺酶抑制剂（如乐巴坦、恩美唑巴坦、阿维巴坦和瓦博巴坦）与β-内酰胺联合用于对抗产生PenA1和R220A变体的多芽胞杆菌是有希望的。
Burkholderia cepacia complex (Bcc) poses a serious health threat to people with cystic fibrosis or compromised immune systems. Infections often arise from Bcc strains, which are highly resistant to many classes of antibiotics, including β-lactams. β-Lactam resistance in Bcc is conferred largely via PenA-like β-lactamases. Avibactam was previously shown to be a potent inactivator of PenA1. Here, we examined the inactivation mechanism of PenA1, a class A serine carbapenemase from Burkholderia multivorans using β-lactamase inhibitors (β-lactam-, diazabicyclooctane-, and boronate-based) with diverse mechanisms of action. In whole cell based assays, avibactam, relebactam, enmetazobactam, and vaborbactam restored susceptibility to piperacillin against PenA1 expressed in Escherichia coli. The rank order of potency of inactivation in vitro based on kinact/KI or k2/K values (range: 3.4 × 102 to 2 × 106 M−1 s−1) against PenA1 was avibactam > enmetazobactam > tazobactam > relebactam > clavulanic acid > vaborbactam. The contribution of selected amino acids (S70, K73, S130, E166, N170, R220, K234, T237, and D276) in PenA1 toward inactivation was evaluated using site-directed mutagenesis. The S130A, R220A, and K234A variants of PenA1 were less susceptible to inactivation by avibactam. The R220A variant was purified and assessed via steady-state inhibition kinetics and found to possess increased Ki-app values and decreased kinact/KI or k2/K values against all tested inhibitors compared to PenA1. Avibactam was the most affected by the alanine replacement at 220 with a nearly 400-fold decreased acylation rate. The X-ray crystal structure of the R220A variant was solved and revealed loss of the hydrogen bonding network between residues 237 and 276 leaving a void in the active site that was occupied instead by water molecules. Michaelis−Menten complexes were generated to elucidate the molecular contributions of the poorer in vitro inhibition profile of vaborbactam against PenA1 (k2/K, 3.4 × 102 M−1 s−1) and was compared to KPC-2, a class A carbapenemase that is robustly inhibited by vaborbactam. The active site of PenA1 is larger than that of KPC-2, which impacted the ability of vaborbactam to form favorable interactions, and as a result the carboxylate of vaborbactam was drawn toward K234/T235 in PenA1 displacing the boronic acid from approaching the nucleophilic S70. Moreover, in PenA1, the tyrosine at position 105 compared to tryptophan in KPC-2, was more flexible rotating more than 90°, and as a result PenA1’s Y105 competed for binding with the cyclic boronate vs the thiophene moiety of vaborbactam, further precluding inhibition of PenA1 by vaborbactam. Given the 400-fold decreased k2/K for the R220A variant compared to PenA1, acyl−enzyme complexes were generated via molecular modeling and compared to the PenA1-avibactam crystal structure. The water molecules occupying the active site of the R220A variant are unable to stabilize the T237 and D276 region of the active site altering the ability of avibactam to form favorable interactions compared to PenA1. The former likely impacts the ability of all inhibitors to effectively acylate this variant enzyme. Based on the summation of all evidence herein, the utility of these newer β-lactamase inhibitors (i.e., relebactam, enmetazobactam, avibactam, and vaborbactam) in combination with a β-lactam against B. multivorans producing PenA1 and the R220A variant is promising.
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作者：
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通讯作者：
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影响因子：
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DOI：
10.1107/s2059798319011471
发表时间：
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影响因子：
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