Unravelling a Novel Mode of Multiple Antibiotic Resistance: Mechanism and Inhibition of Radical-SAM RNA Methyltransferases

许多化学反应在实验室中很难完成。然而，在许多情况下，生物系统已经进化出专门的机制，使它们能够执行这些本质上困难的过程。因此，在自然界中能够产生的化学结构的范围比目前在实验室中能够产生的要大得多。由于许多这样的结构具有有用的性质，如药物活性，了解这些结构在自然界中是如何形成的将扩大我们制造具有广泛潜在应用的新分子的能力。现在很清楚，自然界通过特定的生物催化剂-酶的作用实现了许多这些本质上不利的反应。特别是，被称为自由基SAM酶的一大群负责催化各种各样的这些“困难”化学反应。在这里，我们建议研究一种这样的酶，称为Cfr，它对细菌核糖体进行了特定的修饰。核糖体是细菌细胞的组成部分，负责合成蛋白质，因此对细菌的生存力至关重要。许多抗生素通过毒害细菌核糖体来发挥作用，但Cfr酶会对核糖体进行特异性修饰，使含Cfr的细菌抵抗它们的活性。特别是，Cfr使细菌对利奈唑胺产生耐药性，利奈唑胺是一种特别重要的抗生素，因为它代表了对许多细菌的最后一道防线，例如耐甲氧西林金黄色葡萄球菌（MRSA），这些细菌不再容易用其他类型的药物治疗。Cfr开始在细菌群体中传播，可能威胁到我们使用利奈唑胺治疗严重感染的能力。然而，Cfr修饰核糖体的方式的许多方面仍有待研究。提高我们对Cfr等酶功能的理解，既可以使我们开发阻断其活性的药物，也可以使我们利用它们进行“困难”化学反应的能力来合成新的和潜在有用的分子。我们将使用这些来获得有关Cfr如何识别核糖体的特定部分以及该位点如何被选择性修饰的基本信息。我们还将使用我们开发的方法来筛选有限的合成分子，目的是鉴定一些能够阻断Cfr活性的分子。我们将建立，在近原子水平的细节，我们确定的分子是如何结合的Cfr，以及Cfr如何识别核糖体。我们获得的信息将确定对抗Cfr活性的策略，这可能会延长利奈唑胺的治疗有效期，该申请还将加强英国在自由基SAM酶家族方面的知识和技能基础。自由基SAM酶正吸引越来越多的关注，由于它们可以催化的反应范围非常广泛，但在这一领域活跃的英国研究小组相对较少。我们的提案将在临床和生物技术相关性日益增强的领域进行能力建设。

Many chemical reactions are difficult to accomplish in the laboratory. However, in many cases biological systems have evolved specialised mechanisms that enable them to carry out these intrinsically difficult processes. Consequently the range of chemical structures able to be produced in nature is considerably greater than can currently be produced in the laboratory. As many such structures have useful properties, such as pharmaceutical activity, understanding how these are made in nature will expand our ability to make new molecules with a wide range of potential applications.It is now clear that nature achieves many of these intrinsically unfavourable reactions through the action of specific biological catalysts- enzymes. In particular, a large group known as the radical SAM enzymes are responsible for catalysing a diverse range of these "difficult" chemistries. Here we propose to study one such enzyme, known as Cfr, that introduces a specific modification to the bacterial ribosome. The ribosome is the component of the bacterial cell that is responsible for synthesising proteins and as such is essential to the viability of the bacterium. Many antibiotics act by poisoning the bacterial ribosome, but the Cfr enzyme causes a specific modification to the ribosome that makes Cfr-containing bacteria resist their activity. In particular, Cfr makes bacteria resistant to linezolid, an antibiotic that is particularly important as it represents a last line of defence against many bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) that are no longer easily treatable with other types of drugs. Cfr is beginning to spread through the bacterial population, potentially threatening our ability to use linezolid to treat serious infections. However, many aspects of the way that Cfr modifies the ribosome remain to be investigated. Improving our understanding of how enzymes like Cfr function may both permit us to develop drugs that block their activity, and enable us to exploit their ability to perform "difficult" chemical reactions to synthesise new and potentially useful moleculesIn this proposal, we will develop tools to investigate the activity of Cfr. We will use these to obtain fundamental information about how Cfr recognises a specific portion of the ribosome and how this site is selectively modified. We will also use the methods that we develop to screen a limited selection of synthetic molecules with the intention of identifying some that are able to block Cfr activity. We will establish, at a near atomic level of detail, how the molecules we identify are bound by Cfr, and how Cfr recognises the ribosome. The information we obtain will identify strategies for countering the activity of Cfr, that may prolong the therapeutically useful lifetime of linezolid, The application will also strengthen the UK knowledge and skills base with respect to the radical SAM enzyme family. Radical SAM enzymes are attracting increasing attention, due to the extraordinary range of reactions that they can catalyse, but there remain relatively few UK research groups active in this area. Our proposal will build capacity in an area of growing clinical and biotechnological relevance.