Nucleoside ribohydrolases as targets for Trichomonas vaginalis therapeutic agents. This project will evaluate two essential nucleoside ribohydrolase enzymes as targets for novel antitrichomonal drugs.

项目总结 滴虫病是最常见的非病毒性性传播疾病。虽然临床上 感染的表现通常很轻微，免疫系统可能会受到损害。 滴虫感染的个体对更严重的疾病的易感性更高，例如 宫颈癌、HIV-1和盆腔炎。最近，滴虫感染也出现了 与前列腺癌和良性前列腺增生症有关。致病因素是 寄生原虫阴道毛滴虫。对现有的5-硝基咪唑类抗生素的抗药性 抗滴虫药物近年来显著增加，表明需要新的治疗方法。 具有新的作用机制。阴道毛滴虫所需的核苷挽救途径酶 是开发新型抗滴虫药物的极佳靶点。这条道路的第一步 就是核苷的水解作用，释放出碱基。人体必需的酶 腺苷/鸟苷偏好核苷核糖核糖水解酶和尿苷核苷核糖核糖水解酶 (UNH)已被表征并发现具有不同的底物特异性。两者的抑制物 使用基于核磁共振的活性分析来鉴定酶，以筛选NIH临床收藏。缺欠 两组抑制剂之间的任何显著结构关系进一步表明，这两组抑制剂 核糖水解酶是不同的、可用药的靶标。然而，从基因工程中发现的抑制剂 NIH临床采集的有限化学空间配基效率较低， 因此，开发目标验证所需的化学工具的起点很差。这个项目将 通过使用基于片段的方法来定义最关键的 酶/抑制物相互作用。片段多样性文库包含更广泛的化学成分 比NIH临床收藏中包含的空间更大，并将提供最佳的起点 开发目标验证化学工具。这项提议的具体目标是：1)确定配体- 高效的AGNH和UNH片段抑制剂；2)确定AGNH和UNH的化学工具 用IC50值；1M；和3)通过展示相关性来验证AGNH和UNH作为目标 酶抑制和抗滴虫活性之间的关系。拟议的研究将利用已建立的 核磁共振方法，计算和药物化学，以及阴道毛滴虫生长抑制试验。这个 该项目的总体长期目标是验证AGNH和UNH作为新颖的分子靶标 抗滴虫药物，并鉴定这些酶的抑制剂对5-甲氧基苯丙氨酸和5-甲氧基异丁基异亮氨酸均有抑制作用。 阴道毛滴虫的硝基咪唑敏感株和5-硝基咪唑耐药株。

PROJECT SUMMARY Trichomoniasis is the most prevalent non-viral sexually transmitted disease. Although clinical manifestations of infection are typically mild, the immune system can be concomitantly compromised. Individuals with trichomonal infections have a higher susceptibility to more serious conditions such as cervical cancer, HIV-1, and pelvic inflammatory disease. More recently, trichomonal infection has also been associated with prostate cancer and benign prostatic hyperplasia. The causative agent is the parasitic protozoan Trichomonas vaginalis. Resistance to the existing 5-nitroimidazole class of antitrichomonal drugs has increased markedly in recent years indicating the need for new therapies with novel mechanisms of action. Nucleoside salvage pathway enzymes required by T. vaginalis represent excellent targets for developing novel antitrichomonal agents. The first step in this pathway is the hydrolysis of nucleosides to release the nucleobases. The essential enzymes adenosine/guanosine preferring nucleoside ribohydrolase (AGNH) and uridine nucleoside ribohydrolase (UNH) have been characterized and found to have distinct substrate specificities. Inhibitors of both enzymes were identified using NMR-based activity assays to screen the NIH Clinical Collection. Lack of any significant structural relationship between the two sets of inhibitors further indicates that the two ribohydrolase enzymes are distinct, druggable targets. However, the inhibitors identified from the limited chemistry space sampled by the NIH Clinical Collection have low ligand efficiencies and are thus poor starting points for developing the chemical tools needed for target validation. This project will circumvent this limitation by using a fragment-based approach to define the most critical enzyme/inhibitor interactions. A fragment diversity library encompasses a much broader chemistry space than is contained within the NIH Clinical Collection and will provide the optimal starting points for developing target validation chemical tools. The specific aims of this proposal are: 1) To identify ligand- efficient fragment inhibitors of AGNH and UNH; 2) To identify chemical tools for both AGNH and UNH with IC50 values < 1 M; and 3) To validate AGNH and UNH as targets by demonstrating a correlation between enzyme inhibition and antitrichomonal activity. The proposed studies will utilize established NMR methods, computational and medicinal chemistry, and T. vaginalis growth inhibition assays. The overall long-term goals of this project are to validate AGNH and UNH as molecular targets for novel antitrichomonal agents, and to identify inhibitors of these enzymes that possess efficacy against both 5- nitroimidazole-sensitive and 5-nitroimidazole-resistant strains of T. vaginalis.