Influence of the α-Methoxy Group on the Reaction of Temocillin with Pseudomonas aeruginosa PBP3 and CTX-M-14 β-Lactamase.
Influence of the α-Methoxy Group on the Reaction of Temocillin with Pseudomonas aeruginosa PBP3 and CTX-M-14 β-Lactamase.
复制标题
α-甲氧基基团对替莫西林与铜绿假单胞菌 PBP3 和 CTX-M-14 β-内酰胺酶反应的影响。
DOI：
10.1128/aac.01473-19
复制
发表时间：
2019
影响因子：
4.9
通讯作者：
Chen,Yu
中科院分区：
文献类型：
--
作者：
Sacco,MichaelD;Kroeck,KyleG;Kemp,MTrent;Zhang,Xiujun;Andrews,LoganD;Chen,Yu
多药耐药铜绿假单胞菌的流行导致了对较老的“被遗忘”的药物（如替莫西林）的重新检查，以检查其对抗耐药微生物的能力。替莫西林是替卡西林的6-α-甲氧基类似物，替卡西林是一种具有良好抗假单胞菌特性的羧基青霉素。α-甲氧基修饰赋予对丝氨酸β-内酰胺酶的抗性，然而替莫西林对铜绿假单胞菌生长无效。替莫西林对铜绿假单胞菌的较差抗菌特性的起源仍然相对未被探索。在这里，我们分析的反应动力学，蛋白质的稳定性，并结合构象的替莫西林和替卡西林与青霉素结合蛋白3（PBP 3），一个必不可少的PBP在铜绿假单胞菌。我们表明，6-α-甲氧基扰乱PBP 3酰基酶的稳定性，这表现在生物化学测定中比较替莫西林与替卡西林的解离速率常数（koff）升高。与PBP 3的复杂晶体结构揭示了两种药物的相似结合模式，但具有重要差异。最值得注意的是，6-α-甲氧基破坏了一个高质量的氢键，其中一个保守的残基对配体结合很重要，同时也被插入到一个拥挤的活性位点，可能使活性位点不稳定，并使水分子从大量溶剂中进入并切割酰基-酶键。这一假设得到以下观察结果的支持：与替卡西林相比，替莫西林的酰基-酶复合物具有降低的热稳定性。此外，我们还利用CTX-M-14 A类丝氨酸β-内酰胺酶的高分辨率复合物结构探讨了替莫西林抑制β-内酰胺酶的机制。结果表明，α-甲氧基通过将化合物锁定为意想不到的构象来防止水解，该构象阻止催化水进入酰基-酶加合物。
The prevalence of multidrug-resistant Pseudomonas aeruginosa has led to the reexamination of older “forgotten” drugs, such as temocillin, for their ability to combat resistant microbes. Temocillin is the 6-α-methoxy analogue of ticarcillin, a carboxypenicillin with well-characterized antipseudomonal properties. The α-methoxy modification confers resistance to serine β-lactamases, yet temocillin is ineffective against P. aeruginosa growth. The origins of temocillin’s inferior antibacterial properties against P. aeruginosa have remained relatively unexplored. Here, we analyze the reaction kinetics, protein stability, and binding conformations of temocillin and ticarcillin with penicillin-binding protein 3 (PBP3), an essential PBP in P. aeruginosa. We show that the 6-α-methoxy group perturbs the stability of the PBP3 acyl-enzyme, which manifests in an elevated off-rate constant (koff) in biochemical assays comparing temocillin with ticarcillin. Complex crystal structures with PBP3 reveal similar binding modes of the two drugs but with important differences. Most notably, the 6-α-methoxy group disrupts a high-quality hydrogen bond with a conserved residue important for ligand binding while also being inserted into a crowded active site, possibly destabilizing the active site and enabling water molecule from bulk solvent to access and cleave the acyl-enzyme bond. This hypothesis is supported by the observation that the acyl-enzyme complex of temocillin has reduced thermal stability compared with ticarcillin. Furthermore, we explore temocillin’s mechanism of β-lactamase inhibition with a high-resolution complex structure of CTX-M-14 class A serine β-lactamase. The results suggest that the α-methoxy group prevents hydrolysis by locking the compound into an unexpected conformation that impedes access of the catalytic water to the acyl-enzyme adduct.