Substrate and Inhibitor Interactions of Class A b-Lactamases

β-内酰胺类药物是最广泛使用的抗菌药物，对革兰氏阴性菌的感染具有特殊的价值，因为在革兰氏阴性菌中几乎没有替代品。在这些细菌中，产β-内酰胺酶是主要的耐药机制。虽然这些酶种类繁多，种类繁多，但有几种酶的结构和生化特征已经确定，这使得β-内酰胺酶既与人类(和动物)健康有关，也使其成为研究酶催化反应的计算方法的合适模型系统。碳青霉烯类是最新、最有效的β-内酰胺类抗生素，也是治疗严重细菌感染的关键抗生素。虽然大多数β-内酰胺酶都受到碳青霉烯类抗生素的抑制，但能有效降解这些底物的酶(碳青霉烯酶)正在传播。这种活性扩大的分子基础仍然不清楚，碳青霉烯酶对其他β-内酰胺类的活性也是不同的。我们以前曾(Jam Chem Soc 134 18275(2012年)；Chem Comm，50,14736(2014年))使用结构和计算(分子动力学(MD)和量子力学(QM))方法研究β-内酰胺酶与碳青霉烯类抗生素的相互作用，目的既是为了了解这种活性变化的基础，也是为了开发预测未表征系统的活性的方法。我们现在将这项工作扩展到i)通过实验和电子计算机研究碳青霉烯酶和比较酶与不同类别的β-内酰胺酶的相互作用，ii)研究碳青霉烯酶对不同类型的β-内酰胺酶的活性如何受点突变的影响，以及iii)研究β-内酰胺酶与候选抑制剂的相互作用。实验工作包括确定酶的晶体结构：配体(β-内酰胺底物和抑制剂)复合体和突变的酶变体；补充它们与底物和抑制剂的反应的稳态动力学测量。这些数据提供了第一个结构信息，揭示了与人类感染最相关的碳青霉烯酶KPC如何与不同的β-内酰胺类药物相互作用，包括碳青霉烯类，以及与基于机制的抑制剂的相互作用。这些发现还表明，对特定的β-内酰胺底物的活性不同的KPC变体对抑制剂的敏感性也不同，从而确定了通过积累点突变来逃避抑制剂活性的可能性。该项目还应用了最先进的系列飞秒结晶学(SFX)方法来努力表征β-内酰胺酶反应途径上的短期物种。用分子动力学和量子力学/分子力学(QM/MM)方法对β-内酰胺酶复合体进行了计算研究，模拟了反应的脱酰阶段，将碳青霉烯酶的电子性质扩展到了非碳青霉烯类底物(头孢菌素类)的脱酰反应。我们的发现确定了特定的相互作用和配体结合上的结构/动态重组的组合如何控制b-内酰胺酶与配体的相互作用，从而控制底物和抑制剂的活性。

Beta lactams are the most widely prescribed antibacterial drugs, and are of particular value against infections by Gram-negative bacteria, where few alternatives exist. In these organisms b-lactamase production is the main resistance mechanism. While these enzymes are many and varied, several have been structurally and biochemically characterised, making b-lactamases both relevant to human (and animal) health and suitable model systems for developing computational methods to study enzyme-catalysed reactions.Carbapenems are the newest, most potent b-lactams and key antibiotics for severe bacterial infections. While most b-lactamases are inhibited by carbapenems, enzymes that efficiently hydrolyze these substrates (carbapenemases) are disseminating. The molecular basis for this expansion of activity remains obscure, and the activity of carbapenemases against other b-lactam classes is variable. We have previously (J Am Chem Soc 134 18275 (2012); Chem Comm, 50, 14736 (2014)) used structural and computational (molecular dynamics (MD) and quantum mechanical (QM)) approaches to study interaction of b-lactamases with carbapenems with the aim of both understanding the basis for this variation in activity and developing methodologies for predicting the activity of uncharacterised systems. We now extend this work to i) investigate, experimentally and in silico, interactions of carbapenemases and comparator b-lactamases with b-lactams of different classes, ii) study how carbapenemase activity against different b-lactams may be affected by point mutation, and iii) investigate b-lactamase interactions with candidate inhibitors.Experimental work has encompassed crystallographic structure determination for both enzyme:ligand (b-lactam substrate and inhibitor) complexes and mutated enzyme variants; complemented with steady state kinetic measurements of their reactions with substrates and inhibitors. These data provide the first structural information revealing how KPC, the carbapenemase most relevant to human infections, interacts with different b-lactam classes, including carbapenems, and with mechanism-based inhibitors. These findings also indicate that KPC variants differing in their activity towards specific b-lactam substrates also vary in their susceptibility to inhibitors, identifying potential for evasion of inhibitor activity through accumulation of point mutations. The project also applied state-of-the-art serial femtosecond crystallographic (SFX) approaches in efforts to characterise short-lived species on the b-lactamase reaction pathway. b-lactamase complexes were studied computationally using MD and QM/molecular mechanics (QM/MM) approaches to simulate the deacylation stages of the reaction, extending our in silico characterisations of carbapenemases to deacylation of non-carbapenem substrates (cephalosporins). Our findings identify how combinations of specific interactions and structural/dynamic reorganisation on ligand binding control b-lactamase interactions with ligands and hence activity towards substrates and inhibitors.