Probing the molecular origins of the species-selectivity of microtubule-directed fungicides

黑斑病是影响小麦和大麦生产的一种重要病害，在北欧地区尤为严重。目前使用针对真菌微管的抗真菌喷雾剂来控制黄褐斑病菌，而不影响小麦和人类的微管。抑制微管可以阻止真菌细胞分裂并最终杀死它们，而小麦和人类细胞不受影响。这种方法奏效了，但阻力是一个日益严重的问题。我们迫切需要了解耐药性是如何产生的，我们需要开发新的、更好的抗真菌药物，以有效地对抗耐药菌株。先正达正试图做到这一点，理想的情况是能够在SepVictoria分离的微管上测试可能的新药物，将它们的反应与小麦和人类的微管的反应进行比较。理想情况下，先正达希望从耐药细胞中提纯微管，将它们与非耐药细胞中的微管进行比较，并了解为什么耐药细胞的微管不受杀菌剂的影响。这在目前是不可能的--哺乳动物的微管可以相当容易地被提纯，但到目前为止还没有人能够提纯七叶草微管或小麦微管。目前缺乏基于纯化微管的测试限制了先正达和其他地方设计和开发新抗真菌药物的速度。Cross Lab是纯化微管蛋白的专家。例如，我们最近成功地从酵母细胞中纯化了微管蛋白。在这个项目中，我们将与先正达合作，从乌贼细胞、小麦细胞和人类细胞中分离微管蛋白，并开发微型测试，以便可以检查可能的抗真菌化合物，以确保它们针对乌贼微管，同时不影响小麦和人类的微管。先正达将为我们工业批量培养七星细胞，我们将使用我们已经开发的纯化酵母微管蛋白的技术对它们进行处理，以获得微管蛋白。我们还将从小麦幼苗和培养的人类细胞中制造微管蛋白。为了从这些来源中获得大量的微管蛋白，我们将结合我们自己的方法和新的可用的技术来高效纯化微管蛋白。我们将使用现有的微管稳定性测量方法，以及通过直接在光学显微镜下观察微管，来比较不同的抗真菌药物对我们收集的来自乌贼属、抗性乌贼属、小麦和人类的纯化微管的作用。然后我们就可以阐明杀菌剂在分子水平上是如何发挥作用的，以及在分子水平上，抗性和非抗性白僵菌之间的不同之处。我们将通过改造酵母微管蛋白来测试这些想法，使其对我们的抗真菌药物敏感或抗药性。这是我们理解的最终基准--如果我们能够证明我们知道如何从酵母中改造微管蛋白，使其对抗真菌药物具有与SepVictoria蛋白相同的敏感性，那么我们就可以真正说我们了解了抗真菌物质是如何融入微管蛋白并控制其行为的。然后，这些信息可以反馈到开发新的抗真菌药物的工作流程中。几乎所有种植在英国的小麦都会受到黑斑病的影响。据估计，杀菌剂的应用将使产量增加约20%，因此，如果不使用杀菌剂，英国谷物种植者每年将遭受380至4.65亿GB的经济损失(数据来自Rothamsted在线报告)。因此，如果最终成功，这项研究的潜在影响将对英国经济产生重大影响。我们开发的新技术和知识将转移到先正达，从而在项目生命周期内加速发现和开发新的MT导向抗真菌药物。

Septoria leaf blotch is a key disease affecting wheat and barley production, especially in Northern Europe. Septoria is currently controlled using antifungal sprays that target the fungal microtubules whilst leaving wheat and human microtubules unaffected. Inhibiting microtubules prevents the fungal cells from dividing and ultimately kills them, whilst leaving wheat and human cells unaffected. The approach works, but resistance is an increasing problem. We need urgently to understand how resistance arises and we need to develop new and better antifungals that are effective against the resistant strains of the Septoria fungus. Syngenta is trying to do this, and would ideally like to be able to test possible new agents on isolated microtubules from Septoria, comparing their response with that of microtubules from wheat and from humans. Ideally, Syngenta would like to have purified microtubules from resistant Septoria cells, to compare them with those from non-resistant cells and understand why the microtubules from the resistant cells are not affected by the fungicide. Nothing like this is currently possible - mammalian microtubules can be purified fairly readily, but no one has so far been able to purify Septoria microtubules, or wheat microtubules. The lack of tests based on purified microtubules at present limits the rate at which new antifungals can be designed and developed at Syngenta and elsewhere.The Cross lab is expert at purifying microtubule proteins. For example, we have recently succeeded in purifying microtubule protein from yeast cells. In this project we will collaborate with Syngenta to isolate microtubule protein from Septoria cells, wheat cells and human cells, and develop miniaturised tests so that possible antifungal compounds can be checked to make sure they target the Septoria microtubules whilst leaving wheat and human microtubules unaffected. Syngenta will grow Septoria cells for us in industrial quantities, and we will process them to obtain tubulin using the techniques we have already developed for purifying yeast tubulin. We will also make tubulin from wheat seedlings, and from cultured human cells. To obtain large quantities of microtubule protein from all these sources, we will combine our own methods with newly-available techniques for highly efficient purification of microtubule protein. We will compare the actions of different antifungals on our collection of purified microtubules from Septoria, resistant Septoria, wheat and humans, using existing measures of microtubule stability, but also by looking at the microtubules directly by light microscopy. We can then formulate ideas about how the fungicides actually work at the molecular level, and what it is that is different, at the molecular level, between the resistant and non-resistant Septoria. We will test theses ideas by engineering yeast tubulin to make it susceptible or resistant to our antifungal agents. This is the ultimate benchmark of our understanding - if we can prove that we know how to engineer the microtubule protein from yeast so that it has the same susceptibility to antifungals as the Septoria protein, than we can truly say we understand how the antifungal fits into the microtubule protein and controls its behaviour. This information can then be fed back into the workflow for the development of new antifungals. Septoria infection affects virtually all wheat grown in the UK. Application of fungicides is estimated to boost yields by ~20% and therefore without fungicide use UK cereal growers would suffer an economic loss of between £380 and £465 million each year (figures sourced from a Rothamsted on-line report). If ultimately successful, the potential impact of this research would therefore be significant for the UK economy. The new techniques and knowledge that we develop will be transferred to Syngenta, thereby accelerating the discovery and development of new MT-directed antifungals within the project lifetime.