Jefferies R, Yang R, Woh C K, Weldt T, Milech N, Estcourt A, Armstrong T, Hopkins R, Watt P, Reid S, Armson A, Ryan U M
School of Veterinary and Life Sciences, Murdoch University, Western Australia, Australia; Phylogica, Telethon Institute for Child Health Research, Subiaco, Western Australia, Australia.
School of Veterinary and Life Sciences, Murdoch University, Western Australia, Australia.
Exp Parasitol. 2015 Jan;148:40-8. doi: 10.1016/j.exppara.2014.11.003. Epub 2014 Nov 20.
Cryptosporidiosis, a gastroenteric disease characterised mainly by diarrheal illnesses in humans and mammals is caused by infection with the protozoan parasite Cryptosporidium. Treatment options for cryptosporidiosis are limited, with the current therapeutic nitazoxanide, only partly efficacious in immunocompetent individuals. The parasite lacks de novo purine synthesis, and is exclusively dependant on purine salvage from its host. Inhibition of the inosine 5' monophosphate dehydrogenase (IMPDH), a purine salvage enzyme that is essential for DNA synthesis, thereby offers a potential drug target against this parasite. In the present study, a yeast-two-hybrid system was used to identify Phylomer peptides within a library constructed from the genomes of 25 phylogenetically diverse bacteria that targeted the IMPDH of Cryptosporidium parvum (IMPcp) and Cryptosporidium hominis (IMPch). We identified 38 unique interacting Phylomers, of which, 12 were synthesised and screened against C. parvum in vitro. Two Phylomers exhibited significant growth inhibition (81.2-83.8% inhibition; P < 0.05), one of which consistently exhibited positive interactions with IMPcp and IMPch during primary and recapitulation yeast two-hybrid screening and did not interact with either of the human IMPDH proteins. The present study highlightsthe potential of Phylomer peptides as target validation tools for Cryptosporidium and other organisms and diseases because of their ability to bind with high affinity to target proteins and disrupt function.
隐孢子虫病是一种主要以人类和哺乳动物腹泻疾病为特征的胃肠疾病，由原生动物寄生虫隐孢子虫感染引起。隐孢子虫病的治疗选择有限，目前的治疗药物硝唑尼特仅对免疫功能正常的个体部分有效。该寄生虫缺乏从头合成嘌呤的能力，完全依赖于从宿主中挽救嘌呤。抑制肌苷5'-单磷酸脱氢酶（IMPDH），一种对DNA合成至关重要的嘌呤挽救酶，从而为对抗这种寄生虫提供了一个潜在的药物靶点。在本研究中，利用酵母双杂交系统从25种系统发育不同的细菌基因组构建的文库中鉴定靶向微小隐孢子虫（IMPcp）和人隐孢子虫（IMPch）的IMPDH的系统发育组肽。我们鉴定出38种独特的相互作用系统发育组肽，其中12种被合成并在体外针对微小隐孢子虫进行筛选。两种系统发育组肽表现出显著的生长抑制作用（抑制率为81.2-83.8%；P<0.05），其中一种在初次和重复酵母双杂交筛选过程中始终与IMPcp和IMPch表现出阳性相互作用，并且不与任何一种人类IMPDH蛋白相互作用。本研究强调了系统发育组肽作为隐孢子虫及其他生物体和疾病的靶点验证工具的潜力，因为它们能够以高亲和力结合靶蛋白并破坏其功能。
