Direct Measurement of Nucleoside Ribohydrolase Enzyme Activities in Trichomonas vaginalis Cells Using 19F and 13C-Edited 1H NMR Spectroscopy.
Direct Measurement of Nucleoside Ribohydrolase Enzyme Activities in Trichomonas vaginalis Cells Using 19F and 13C-Edited 1H NMR Spectroscopy.
复制标题
使用 19F 和 13C 编辑的 1H NMR 光谱直接测量阴道毛滴虫细胞中的核苷核糖水解酶活性。
DOI：
10.1021/acs.analchem.2c05330
复制
发表时间：
2023
影响因子：
7.4
通讯作者：
Mahmood,Maham
中科院分区：
文献类型：
--
作者：
Stockman,BrianJ;Ventura,CarlosA;Deykina,ValerieS;KhayanLontscharitsch,Nickolas;Saljanin,Edina;Gil,Ari;Canestrari,Madison;Mahmood,Maham
毛滴虫病是最常见的非病毒性传播感染，全世界约有2.75亿人感染。病原体是寄生原生动物阴道毛滴虫。虽然这种疾病本身通常是温和的，但患有滴虫感染的个体对更严重的疾病有更高的易感性。寄生虫菌株对当前疗法的耐药性的出现需要新的治疗策略。自从。阴道炎是一种需要核苷补救途径的专性寄生虫，必需的核苷核糖水解酶是有希望的新药物靶点。片段筛选和X-射线晶体学已经使这些酶的抑制剂的结构导向设计成为可能。酶和抗原虫活性的联系将是设计新的、基于机制的治疗剂的变革性步骤。虽然与纯化酶的抑制的相关性在机理上是暗示性的，但与细胞内酶活性的抑制的相关性将明确建立这种联系。为了证明这种联系，我们已经翻译了我们的基于NMR的活性测定法，该测定法测量用于阴道毛滴虫细胞的纯化酶的活性。基于19 F NMR的嘧啶特异性酶活性测定直接转化为细胞内测定。然而，基于1H NMR的嘌呤特异性酶活性测定需要从腺苷转换为鸟苷底物，并使用13 C编辑来分辨来自细胞和生长培养基背景信号的底物1H信号。细胞内NMR测定具有耐用性，并已证明可提供供试化合物的抑制数据。这里描述的结果代表了原生动物寄生虫细胞中酶活性的第一次直接测量。
Trichomoniasis is the most common nonviral sexually transmitted infection, affecting an estimated 275 million people worldwide. The causative agent is the parasitic protozoanTrichomonas vaginalis. Although the disease itself is typically mild, individuals with trichomonal infections have a higher susceptibility to more serious conditions. The emergence of parasite strains resistant to current therapies necessitates the need for novel treatment strategies. SinceT. vaginalisis an obligate parasite that requires nucleoside salvage pathways, essential nucleoside ribohydrolase enzymes are promising new drug targets. Fragment screening and X-ray crystallography have enabled structure-guided design of inhibitors for two of these enyzmes. Linkage of enzymatic and antiprotozoal activity would be a transformative step toward designing novel, mechanism-based therapeutic agents. While a correlation with inhibition of purified enzyme would be mechanistically suggestive, a correlation with inhibition of in-cell enzyme activity would definitively establish this linkage. To demonstrate this linkage, we have translated our NMR-based activity assays that measure the activity of purified enzymes for use inT. vaginaliscells. The19F NMR-based activity assay for the pyrimidine-specific enzyme translated directly to in-cell assays. However, the1H NMR-based activity assay for the purine-specific enzyme required a switch from adenosine to guanosine substrate and the use of13C-editing to resolve the substrate1H signals from cell and growth media background signals. The in-cell NMR assays are robust and have been demonstrated to provide inhibition data on test compounds. The results described here represent the first direct measurement of enzyme activity in protozoan parasite cells.