Rational Design, Synthesis and Biological Evaluation of Benzimidazoles; Towards a Novel Therapy Selectively Targeting C. neoformans beta-tubulin

需要：新生隐球菌(C.neformans)是一种酵母样真菌，在人体内表现为脑膜炎，较少发生肺炎。在撒哈拉沙漠以南的非洲地区，每年新生念珠菌导致的死亡人数超过结核病。新生芽孢杆菌是艾滋病患者真菌感染的最常见原因，据估计，全世界每年有100万例隐球菌性脑膜炎。目前用于治疗这种疾病的药物是两性霉素B和5-氟胞嘧啶2周疗程，然后是长期氟康唑疗程。两性霉素B和5-氟胞嘧啶与毒性有关，氟康唑可以抑制真菌的生长，但不能完全将其从体内清除。因此，非常需要新药来治疗新生葡萄球菌感染。这些药物必须具有较低的毒性，并能更好地对抗这种疾病。这项建议的目的是完成早期药物发现活动，最终开发可用于撒哈拉以南非洲的隐球菌性脑膜脑炎新药。概念：之前有科学论文报道，一类名为苯并咪唑的化合物(广泛用于治疗人类蠕虫感染和农业中的杀菌剂)在实验室对新生隐球菌有良好的活性，这一点随后得到利物浦大学(UOL)的工作证实。苯并咪唑已被证明通过与β-微管蛋白结合来干扰新生葡萄球菌的细胞分裂。β-微管蛋白是一种对细胞分裂和生长至关重要的蛋白质，当苯并咪唑与β-微管蛋白结合时，真菌细胞就不能再生长了。最近，在UOL进行的未发表的研究表明，氟苯达唑在小鼠模型中对新生葡萄球菌有活性。人类也有β-微管蛋白，所以针对β-微管蛋白靶标开发的任何新药都对新生葡萄球菌β-微管蛋白具有选择性，这是至关重要的。药物化学(药物化学结构的修饰)可以用来提高对β-微管蛋白的选择性和对新生葡萄球菌的活性。解决方案：针对已知的靶标，利用已建立的全细胞新生葡萄球菌和动物模型活性来鉴定一类活性化合物，为药物化学修饰提供了一个很好的起点。到目前为止，苯并咪唑类化合物从未进行过药物化学优化，以建立β-微管蛋白的选择性或提高新生葡萄球菌的活性。两种最常用的苯并咪唑药物(氟苯达唑和阿苯达唑)在体内广泛代谢，从胃进入血液的能力有限。新的化合物将使用计算机模型中的信息来设计，以预测它们在体内的可能行为。为了改善新设计化合物的安全性，将在实验室测量它们对新生杆菌β-微管蛋白相对于人β-微管蛋白的选择性(以及对新生杆菌的活性)，并建立两种微管蛋白结合部位的计算机模型，以研究新化合物如何可能与新生杆菌β-微管蛋白结合得更好。一旦确定了新生葡萄球菌的活性、对β-微管蛋白的选择性和提高了循环药物的水平，这些化合物就会进入新生葡萄球菌感染的小鼠模型。影响：长期目标是开发一种单一的治疗方法，不需要随后使用氟康唑进行长期治疗，并且可以口服和注射。考虑到受新生芽孢杆菌影响的患者的数量，这个项目有可能对个人和社会产生巨大的有益影响。发展：在小鼠模型中显示活性的化合物将成为进一步资助申请的基础，以在药物开发管道中进展新药。

The Need:Cryptococcus neoformans (C. neoformans) is a yeast like fungus which manifests as meningitis and less frequently pneumonia within the human body. In sub-Saharan Africa C. neoformans causes more deaths annually than tuberculosis. C. neoformans is the most common cause of fungal infection in patients with AIDS and there are an estimated 1 million cases of cryptococcal meningitis worldwide each year. The current drugs used to treat this disease are a 2 week course of amphotericin B and 5-flucytosine followed by a long term course of fluconazole. Amphotericin B and 5-flucytosine are associated with toxicity and fluconazole suppresses the growth of the fungus but does not completely remove it from the body. There is a therefore a great need for new drugs to treat C. neoformans infections. These drugs must have less toxicity and work better against the disease. The aim of this proposal is to complete the early stage drug discovery activities required to ultimately develop new drugs for cryptococcal meningoencephalitis that can be used in sub-Saharan Africa.The Concept:It has previously been reported within scientific papers that a class of compound called the benzimidazoles (widely used to treat worm infections in humans and as fungicides in agriculture) have good activity against C. neoformans in a laboratory and this has subsequently been confirmed by work here at the University of Liverpool (UoL). Benzimidazoles have been shown to interfere with cell division in C. neoformans through binding to beta-tubulin. Beta-tubulin is a protein that is essential for cell division and growth, when benzimidazoles bind to beta-tubulin the fungal cell can no longer grow. More recently, unpublished studies carried out at UoL have shown flubendazole to be active against C. neoformans in the mouse model. Humans also have beta-tubulin so it is essential that any new drug developed against the beta-tubulin target is selective for C. neoformans beta-tubulin. Medicinal chemistry (modification of the chemical structure of a drug) can be used to improve both selectivity for beta-tubulin and activity against C. neoformans.The Solution:Identification of an active class of compound, against a known target, with established whole cell C. neoformans and animal model activity provides an excellent starting point for medicinal chemistry modification. To date benzimidazole compounds have never undergone medicinal chemistry optimisation to establish beta-tubulin selectivity or to improve C. neoformans activity. The two most commonly use benzimidazole drugs (Flubendazole and albendazole) are extensively metabolised within the body and have a limited ability to move from the stomach into the blood stream. New compounds will be designed using information from computer models to predict how they are likely to behave within the body. In order to improve the safety profile of the newly designed compounds their selectivity for C. neoformans beta-tubulin over human beta-tubulin will be measured in the laboratory (alongside activity against C. neoformans) and computer models of the binding sites of the two tubulins used to look at how new compounds are likely to bind better with C. neoformans beta-tubulin. Once C. neoformans activity, selectivity for beta-tubulin and improved levels of circulating drug have been established the compounds will move into a mouse model of C. neoformans infection. Impact:The long term aim is to develop a single therapy that does not require subsequent long term treatment with fluconazole and that can be given both orally and by injection. Given the number of patients affected by C. neoformans this project has the potential to have a dramatic beneficial impact on individuals and societies.Development:Compounds demonstrating activity in the mouse model will form the basis of further funding applications to progress the new drugs through the drug development pipeline.