Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, 77843-4467, USA.
College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China.
Int J Parasitol Drugs Drug Resist. 2018 Apr;8(1):43-49. doi: 10.1016/j.ijpddr.2018.01.003. Epub 2018 Jan 30.
Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI) and determined its Michaelis constant towards fructose-6-phosphate (K = 0.309 mM, V = 31.72 nmol/μg/min). We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC = 8.33 μM; K = 36.33 μM), while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC = 165 μM) at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC on HCT-8 cells = 700 μM). Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target.
微小隐孢子虫是一种经水和食物传播的顶复门寄生虫病原体。它是发展中国家五岁以下儿童四大腹泻病因之一，也是免疫功能低下个体中的机会性寄生虫病原体。与其他顶复门寄生虫不同，微小隐孢子虫缺乏克雷布斯循环和细胞色素呼吸，因此主要依赖糖酵解产生 ATP。在这项研究中，我们描述了微小隐孢子虫葡萄糖-6-磷酸异构酶（CpGPI）的主要生化特征，并确定了其对果糖-6-磷酸的米氏常数（K = 0.309 mM，V = 31.72 nmol/μg/min）。我们还发现，通过对 1200 种已知药物进行高通量筛选，有机硒药物依布硒啉是 CpGPI 的选择性抑制剂。依布硒啉作为变构非竞争性抑制剂作用于 CpGPI（IC = 8.33 μM；K = 36.33 μM），但未能完全抑制 CpGPI 活性。依布硒啉还可以抑制体外微小隐孢子虫的生长（EC = 165 μM），而对宿主细胞无毒，尽管体外安全性窗口相对较小，为 4.2（HCT-8 细胞的细胞毒性 TC 值= 700 μM）。此外，依布硒啉可能还靶向寄生虫中的其他酶，导致寄生虫生长减少。因此，尽管依布硒啉在研究 CpGPI 酶活性抑制方面很有用，但仍需要进一步的化学和/或遗传证据来验证 CpGPI 作为药物靶点的合理性。
