Substrate and Inhibitor Interactions of Class A b-Lactamases

β-内酰胺是最广泛使用的抗菌药物，对革兰氏阴性菌感染具有特别价值，而革兰氏阴性菌的替代品很少。在这些微生物中，产生β-内酰胺酶是主要的耐药机制。虽然这些酶种类繁多，但其中一些酶的结构和生物化学特征已得到证实，这使得β-内酰胺酶既与人类（和动物）健康相关，又适合用于开发计算方法以研究酶催化反应的模型系统。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b-内酰胺酶被碳青霉烯类抑制，但有效水解这些底物的酶（碳青霉烯酶）正在传播。这种活性扩展的分子基础仍然不清楚，碳青霉烯酶对其他b-内酰胺类的活性是可变的。我们先前已经（J Am Chem Soc 134 18275（2012）;化学通讯，50岁，14736（2014））使用结构和计算（分子动力学（MD）和量子力学（QM））方法来研究B-目的是了解这种活性变化的基础，并开发预测碳青霉烯类抗生素活性的方法。无特征的系统我们现在将这项工作扩展到i）通过实验和计算机模拟研究碳青霉烯酶和比较b-内酰胺酶与不同类别b-内酰胺的相互作用，ii）研究碳青霉烯酶对不同b-内酰胺的活性如何受到点突变的影响，iii）研究b-内酰胺酶与候选抑制剂的相互作用。实验工作包括两种酶的晶体结构测定：配体（β-内酰胺底物和抑制剂）复合物和突变的酶变体;辅以它们与底物和抑制剂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KPC（与人类感染最相关的碳青霉烯酶）如何与不同的b-内酰胺类（包括碳青霉烯类）和基于机制的抑制剂相互作用。这些发现还表明，KPC变体对特定b-内酰胺底物的活性不同，对抑制剂的敏感性也不同，确定了通过点突变累积逃避抑制剂活性的可能性。该项目还应用了最先进的连续飞秒晶体学（SFX）方法，以研究b-内酰胺酶反应途径上的短寿命物种。使用MD和QM/分子力学（QM/MM）方法对b-内酰胺酶复合物进行了计算研究，以模拟反应的脱酰阶段，将碳青霉烯酶的计算机表征扩展到非碳青霉烯底物（头孢菌素）的脱酰。我们的研究结果确定了如何结合特定的相互作用和结构/动态重组的配体结合控制b-内酰胺酶与配体的相互作用，因此对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.