Design, synthesis and testing of novel phytopathogenic fungicides.

酶是一种促进反应的蛋白质，使生物体能够获得生长、繁殖和维持所需的能量。要了解其中一类酶，即替代氧化酶（AOX）的结构-功能关系，一个关键的挑战在于鉴定其底物和抑制剂结合位点及其作用机制。对这种结合位点性质的详细了解是很重要的，因为它将揭示是否存在一种可以应用于底物和抑制剂结合位点的通用结构，从而提供对结合机制的深入了解。更重要的是，这些知识将有助于适当合理地设计专门针对替代氧化酶的植物致病和抗寄生虫药物。替代氧化酶不仅存在于真菌和植物中，也广泛存在于人类寄生虫中，如布氏锥虫（非洲昏睡病的病原体）、肠道寄生虫，如细小隐孢子虫（导致空气传播的肠道感染隐孢子虫病）和机会性人类病原体，如白色念珠菌（引起念珠菌病或鹅口疮）。关于AOX在真菌中的作用，植物真菌病原体对农用化学品产生抗性是一个影响所有主要作物的国际问题。事实上，抗杀菌剂是英国谷物种植成功的一个重要因素。据估计，英国谷物杀菌剂市场约为2.5亿英镑（全球30亿美元），冬小麦是主要作物。杀菌剂用于防治多种病害，其中冬小麦的主要病害是由黑穗病引起的。用于处理英国谷物的杀菌剂的主要化学类别包括甾醇生物合成抑制剂。这些杀菌剂中最重要和最成功的一类是strobilurin杀菌剂，它专门针对线粒体呼吸链（Qo位点），从而抑制真菌的呼吸作用。不幸的是，对这种杀菌剂的耐药性经常发展导致无法通过继续使用来控制真菌病原体。尽管对Qo杀菌剂产生耐药性的机制仍存在争议，但越来越多的证据表明，在真菌病原体中添加抑制剂（如偶氮嘧菌酯）会导致选择性氧化酶（AOX）的强烈诱导。AOX是一种线粒体末端氧化酶，绕过Qo位点，在所有植物、真菌病原体和原生生物中都能被诱导。我们之前已经证明，真菌植物病原体，如小麦Septoria tritici（一种引起小麦主要叶斑病的真菌）和小麦“全食”真菌Gaeumannomyces graminis var. tritici，在使用呼吸抑制剂处理时具有表达AOX的能力，从而允许抗strobilurin的呼吸途径的发展，这可能解释了strobilurin杀菌剂的不同效果。本研究的主要目的是鉴定含有抑制真菌替代氧化酶和/或主要呼吸链的新化合物的配方，从而为有效控制谷物真菌疾病提供一种技术。我们已经设计了许多这样的化合物（PCT/GB2015/050148和PCT/GB2013/051030），这些化合物已被证明是白灰枯死真菌的有效抑制剂，并且已经证明这些化合物对稻瘟病也有效。我们将在酵母中表达真菌rAOX酶，这将使我们不仅能够快速筛选化合物的抑制效果，而且还可以通过真菌酶的遗传操作确定酶对我们合成的化合物产生抗性的程度。

Enzymes are proteins that facilitate the reactions that enable living organisms to acquire energy for growth, reproduction and maintenance. A key challenge in understanding the structure-function relationship of one such group of enzymes, the alternative oxidases (AOX), rests upon the identification of its substrate and inhibitor-binding site and its mechanism of action. A detailed knowledge of the nature of this binding site is important since it will reveal whether or not there is a common architecture that can be applied to substrate and inhibitor-binding sites in general and hence provide an insight into the mechanism of binding. More importantly, this knowledge will assist in the suitable rational design of phytopathogenic and anti-parasitic drugs that are specifically targeted to the alternative oxidase. The alternative oxidase is not only present in fungi and plants but also widespread amongst human parasites such as Trypanosoma brucei (the causative agent of African sleeping sickness), intestinal parasites such as Cryptosporidium parvum (responsible for an airborne intestinal infection cryptosporidiosis) and opportunistic human pathogens such as Candida albicans (causes candidiasis or 'thrush'). With respect to the role of AOX in fungi, the development of resistance to agrochemicals by plant fungal pathogens is an international problem that affects all major crops. Indeed fungicide resistance is an important factor in the successful cultivation of cereals in the UK. It is estimated that the UK market for fungicides in cereals is approximately £250M (worldwide $3bn) with winter wheat being the main crop. Fungicides are used against a number of diseases, the major one of winter wheat being caused by Septoria tritici. The main chemical classes of fungicides used to treat UK cereals include the sterol biosynthesis inhibitors. The most important and successful group of these fungicides that have proved effective in the control of plant pathogens are the strobilurin fungicides which are specifically targeted to the mitochondrial respiratory chain (Qo site) thereby inhibiting fungal respiration. Unfortunately resistance to this fungicide often develops resulting in an inability to control fungal pathogens through continued application. Although the mechanism for conferring resistance to Qo fungicides is still controversial there is growing evidence to suggest that the addition of inhibitors, such as azoxystrobin, to fungal pathogens results in a strong induction of the alternative oxidase (AOX). AOX is a mitochondrial terminal oxidase which by-passes the Qo site and is induced in all plants, fungal pathogens and protists following stress induction. We have previously demonstrated that fungal plant pathogens such as Septoria tritici, a fungus that causes major leaf spot diseases in wheat & the wheat "Take-all" fungus, Gaeumannomyces graminis var. tritici have the capacity to express AOX when treated with respiratory inhibitors thereby allowing a strobilurin-resistant respiratory pathway to develop which may account for the varying efficacy of strobilurin fungicides. The major objective of this study is to identify formulations containing novel compounds which inhibit the fungal alternative oxidase and/or the main respiratory chain thereby providing a technique for the effective control of fungal diseases in cereal crops. We have already designed a number of these compounds (PCT/GB2015/050148 & PCT/GB2013/051030) which have proved to be potent inhibitors of the Ash dieback fungus and have demonstrated that these compounds are also effective against Septoria tritici. We will express the fungal rAOX enzyme in a yeast which will enable us to not only to quickly screen the compounds inhibitory effectiveness but also through genetic manipulation of the fungal enzyme ascertain the extent to which the enzyme may become resistant to the compounds we have synthesised.