Elucidating mechanisms of beta-lactam antibiotic resistance through serial crystallography and molecular simulations

细菌抗生素耐药性是一个全球性的突发公共卫生事件，已经造成全世界每年120万人死亡，预计到2050年将达到1000万人。内酰胺类（青霉素类及相关药物）是使用最广泛的抗生素。在革兰氏阴性细菌如大肠杆菌（英国血液感染的主要原因）中，β -内酰胺耐药性通常是由于β -内酰胺酶切割β -内酰胺环上的酰胺结合并取消抗生素活性。该建议应用x射线晶体学和化学反应计算模拟的最新方法来研究β -内酰胺酶降解β -内酰胺的机制，这些信息将指导开发阻断β -内酰胺活性的小分子抑制剂，并恢复β -内酰胺对产生β -内酰胺酶的细菌的有效性。x射线晶体学提供了蛋白质的近原子分辨率结构信息，以及它们与小分子的相互作用，但传统上是一种静态技术，无法描述瞬时存在的物种或捕获动态信息，例如化学反应中状态的相互转换。最近开发的一系列方法，其中微米级晶体中的反应是通过快速混合或依赖于光的化学反应引发的，然后在规定的时间间隔后收集每个晶体的单个图像，但是现在可以在毫秒时间尺度上产生结构信息。这提供了创造“分子电影”的潜力，这些电影由沿着反应途径在不同时间点的一系列结构快照组成，首次使酶催化反应中短暂的、机械重要状态的结构描述成为可能。在这个项目中，我们将应用一系列技术，与钻石光源的科学家合作，研究β -内酰胺酶与其抗生素底物和候选抑制剂的反应。我们获得的结构将用于分子模拟，以了解酶结合物种的动态特性，并研究它们相互转化的能量学。基于经典（牛顿）力学的模拟将研究沿着反应途径的单个状态的构象灵活性，而量子力学方法将使我们能够计算控制它们相互转换的能量垒，从而评估单个复合物的反应性。结合起来，这两种方法将揭示不同的β -内酰胺酶如何采用不同的机制来降解共同的底物，以及不同的酶对不同抗生素的反应性和对不同抑制剂的敏感性如何以及为什么不同。这些信息对于确定β -内酰胺如何被修饰以逃避β -内酰胺酶的活性，以及β -内酰胺酶抑制剂如何被优化以针对最广泛的β -内酰胺酶靶标的活性具有价值。

Bacterial antibiotic resistance is a global public health emergency, already responsible for >1.2 m deaths per annumworldwide with up to 10 m predicted by 2050. Beta-lactams (penicillins and related drugs) are the most widely usedantibiotics. In Gram-negative bacteria such as Escherichia coli (the leading cause of bloodstream infections in the U.K.)beta-lactam resistance is usually due to beta-lactamase enzymes that cleave the amide bond in the beta-lactam ring andabolish antibiotic activity.This proposal applies state-of-the-art approaches in X-ray crystallography and computational simulations of chemicalreactions to study the mechanism(s) by which beta-lactamases degrade beta-lactams, information that will guidedevelopment of small molecule inhibitors that block their activity and restore beta-lactam effectiveness against beta-lactamase producing bacteria. X-ray crystallography provides near-atomic resolution structural information on proteins,and their interactions with small molecules, but is traditionally a static technique unable to describe transientlypresent species or capture dynamic information on e.g. interconversion of states in chemical reactions. Recentlydeveloped serial methods, where reactions in micron-scale crystals are initiated by rapid mixing or light-dependent chemistry and a single image per crystal subsequently collected after a defined time interval, can however nowyield structural information on the millisecond time scale. This offers potential to create "molecular movies" composed ofa series of structural snapshots at different time points along a reaction pathway, for the first time enabling structuraldescriptions of transient, mechanistically important states in enzyme-catalysed reactions. In this project we will applyserial techniques, working together with scientists at Diamond Light Source, to investigate reactions of beta-lactamases with their antibiotic substrates and with inhibitor candidates. Structures that we obtain will be used inmolecular simulations to understand the dynamic properties of enzyme bound species and to investigate theenergetics of their interconversion. Simulations based on classical (Newtonian) mechanics will investigate theconformational flexibility of individual states along a reaction pathway, while quantum mechanical methods will enableus to calculate the energy barriers controlling their interconversion and so assess the reactivity of individual complexes.In combination, these two approaches will reveal how different beta-lactamases employ different mechanisms to degrade common substrates, and how and why different enzymes vary in their reactivity varies towards different antibioticsand susceptibility towards different inhibitors. This information will be valuable in identifying how beta-lactams maybe modified to evade beta-lactamase activity, and how beta-lactamase inhibitors may be optimised for activity againstthe widest range of beta-lactamase targets.