Structure Elucidation and Characterization of the Venus Kinase Receptors of a Philippine Isolate of Schistosoma japonicum

在世界范围内，数百万人患有一种名为血吸虫病的寄生虫病。这种疾病是由血吸虫属的双基因吸虫引起的。这种疾病在菲律宾是地方病。血吸虫病是一种由血吸虫引起的寄生虫病。这种疾病是通过感染了人类感染阶段的称为血吸虫尾蚴的寄生虫的淡水蜗牛传播的。这种疾病的特点是肝脏和脾脏发炎等病态症状。血吸虫病在菲律宾流行。由于它的病态，每年的经济损失高达数百万菲律宾比索。250万人有活动性感染，使1200万人面临感染风险。在菲律宾，日本血吸虫是血吸虫病的唯一病原体。该病流行于12个地区，对应28个省。菲律宾的血吸虫病控制计划包括大规模药物管理。疾病流行率高的地方只有一种药物--吡喹酮。一年一次或两年一次。然而，对这种药物的抗药性引发了合理的恐惧。在实验室条件下，用亚疗效药物浓度处理的曼氏血吸虫对吡喹酮产生抗药性。此外，在塞内加尔(1988年)，大规模服用吡喹酮的寄生虫学治愈率未能达到预期的70%-90%。这种疾病的病理是由血吸虫卵沉积在人类宿主的不同器官中引起的。血吸虫卵是血吸虫病的病原体。因此，干预血吸虫卵的产生可能导致减少疾病负担和传播控制。近年来，曼氏血吸虫Venus Kinase受体(VKRs)的发现为血吸虫病的研究开辟了新的方向。已发现VKRs直接参与鸡蛋生产。因此，可以利用VKR蛋白的抑制作用来开发抗血吸虫病的药物。为了开发有效的抗血吸虫新药，需要对VKR蛋白进行分子理解。这项提议旨在通过在蛋白质组学、药物设计和膜蛋白结晶学领域应用尖端科学和技术来承担这一复杂的任务。我们将在大肠杆菌、酿酒酵母或杆状病毒表达系统中表达和纯化重组形式的VKR蛋白。我们将采用实验和计算两种方法来表征VKR蛋白的二聚化潜力、配体结合和抑制剂相互作用。VKR蛋白的高分辨结构对VKR抑制剂的开发具有重要意义。我们将尝试在含有和不含有其假定的脂类和抑制剂的情况下使VKR蛋白结晶。VKR结构将用于筛选小分子VKR抑制剂，并将测试与VKR蛋白和磷酸酶活性的结合。抗血吸虫病药物开发的影响需要数年时间才能交付给最终用户。但是，这一提议是朝着实现这一目标迈出的一大步。研究结果的直接受益者将是学术团体，使他们能够获得VKR蛋白质3D结构的免费数据。有了VKR坐标，将促进全球抗血吸虫病药物开发的同步工作。这项研究中确定的先导化合物可以被制药公司利用，以加快对血吸虫病患者的影响。

Worldwide, millions of people suffer from the parasitic disease called schistosomiasis. This disease is caused by the digenetic trematode of the genus Schistosoma. The disease is endemic in the Philippines. Schistosomiasis is a parasitic disease caused by blood dwelling worms. This disease is spread by freshwater snails infected with the human infective stage of the parasite called schistosome cercariae. The disease is characterized by morbidities like inflammation of the liver and the spleen. Schistosomiasis is endemic in the Philippines. Due to its morbidities, economic loss amount to millions of Philippine Peso per year. Two and a half million people have active infections, posing 12 million individuals at risk of infection. In the Philippines, S. japonicum is the only causative agent of schistosomiasis. The disease is widespread in 12 Regions, corresponding to 28 Provinces. Schistosomiasis control program in the Philippines include mass drug administration. Endemic places with high disease prevalence rates are treated with a single drug, praziquantel. A single dose is given once annually or biannually. However, resistance to the drug poses legitimate fear. Under laboratory conditions, S. mansoni worms treated with subcurative drug concentration developed resistance against praziquantel. Moreover, in Senegal (1988), mass drug administration of praziquantel failed to meet the expected parasitological cure rate of 70-90%. The disease pathologies are caused by schistosome eggs deposited in different organs in the human host. Schistosome eggs are the causative agents of schistosomiasis. As such, interventions of schistosome egg production may lead to reduction of disease burden and transmission control. In recent years, the discovery of Venus Kinase Receptors (VKRs) in S. mansoni opened new directions in schistosomiasis research. It has been found that VKRs are directly involved in egg production. Thus, inhibition of VKR proteins can be exploited to develop anti-schistosomiasis therapeutics.In order to develop new drugs active against schistosome worms, molecular understanding of VKR proteins will be needed. This proposal aims to undertake this complex task by employing cutting edge science and technology in the field of proteomics, drug design and membrane protein crystallography. We will express and purify the recombinant form of VKR proteins in either E. coli, S. cerivisiae or Bacullovirus expression systems. We will employ both experimental and computational approches to charcaterize VKR proteins in terms of their dimerization potential, ligand binding, and inhibitor interactions. Important to VKR inhibitor development is high resolution structure of VKR proteins. We will attempt to crystallize VKR proteins with and without their putative liagnds and inhibitors. The VKR structure will be used to screen small molecule VKR inhibitors, and will be tested for binding to VKR proteins and phosphorylase activity.The impact of anti-schistosomiasis drug development takes years to deliver to end users. But, this proposal is a huge leap towards its realization. The immediate beneficiaries of study outcomes will be the academic groups, allowing them to access free data of the 3D structure of VKR proteins. With VKR coordinates available, simultaneous work on the development of anti-schistosomiasis drugs worldwide will be facilitated. Lead compounds identified in the study can be exploited by pharmaceutical companies to speed up delivery of impact to suffering individiduals due to schistosomiasis.