Zhai Zongzhen, Jiang Peng, Wang Dongqiang, Chen Tao, Yin Jigang, Zhu Guan
State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun, 130062, China.
Int J Parasitol Drugs Drug Resist. 2025 Jun 4;28:100601. doi: 10.1016/j.ijpddr.2025.100601.
Cryptosporidium parvum is an intestinal protozoan parasite, the causative agent of the diarrheal cryptosporidiosis in humans and animals for which fully effective treatments are yet unavailable. The C. parvum genome encodes highly streamlined metabolic pathways, lacking enzymes to synthesize any amino acids de novo. However, it possesses a standalone type A asparagine synthetase (CpAsnA) that catalyzes the ammonia/ATP-dependent synthesis of asparagine from aspartate. Here, we expressed recombinant CpAsnA and characterized its enzyme functional parameters towards aspartate. We screened 5000 bioactive compounds using a thermal shift assay (TSA) and identified 31 hits showing high binding affinity to CpAsnA. Four of the 31 TSA hits exhibited lower micromolar activity against CpAsnA enzyme activity, including XD14, SB225002, histone acetyltransferase inhibitor II (HATi-II) and tolcapone. Among the four CpAsnA inhibitors, three displayed lower micromolar in vitro efficacy against the growth of C. parvum in vitro with satisfactory selectivity indices as primary antiparasitic hits. Our data suggest that CpAsnA merits further investigation as a potential drug target in the parasite.
微小隐孢子虫是一种肠道原生动物寄生虫，是人类和动物腹泻性隐孢子虫病的病原体，目前尚无完全有效的治疗方法。微小隐孢子虫基因组编码高度简化的代谢途径，缺乏从头合成任何氨基酸的酶。然而，它拥有一种独立的A型天冬酰胺合成酶（CpAsnA），可催化由天冬氨酸依赖氨/ATP合成天冬酰胺。在此，我们表达了重组CpAsnA，并对其针对天冬氨酸的酶功能参数进行了表征。我们使用热位移分析（TSA）筛选了5000种生物活性化合物，鉴定出31种与CpAsnA具有高结合亲和力的命中化合物。31种TSA命中化合物中的4种对CpAsnA酶活性表现出低微摩尔活性，包括XD14、SB225002、组蛋白乙酰转移酶抑制剂II（HATi-II）和托卡朋。在这四种CpAsnA抑制剂中，三种在体外对微小隐孢子虫的生长表现出低微摩尔效力，具有令人满意的选择性指数，作为主要的抗寄生虫命中化合物。我们的数据表明，CpAsnA作为该寄生虫的潜在药物靶点值得进一步研究。
