Early Stage Discovery of Natural Products Targeting Anaerobic Protozoal Pathogen

 描述(申请人提供)：阴道毛滴虫是美国最流行的性传播传染病，约有700万人受到影响。感染阴道毛滴虫的人面临着患宫颈癌、不孕不育、盆腔炎性疾病、前列腺炎和艾滋病毒易感性的风险增加。硝基咪唑类药物是唯一可用于治疗滴虫病的药物，但它们有几个严重的副作用。此外，对硝基咪唑类药物的耐药性的出现和蔓延正成为一个令人担忧的问题。因此，迫切需要开发新的治疗药物来对抗阴道毛滴虫，而针对这种RFA而设计的拟议研究为发现和开发满足这一需求的生物活性分子提供了一种机制。药物发现计划成功的关键是获得一个杰出的化学多样性物质库，这些物质非常适合成为早期的线索。天然产品满足了这一基本要求，来自真菌的代谢物以前没有被筛选出来对抗阴道毛滴虫。因此，这项工作是创新的，因为我们将把一个新的天然产物来源与一个新的高含量筛选系统结合起来，从一个大型真菌次生代谢物库中筛选出有前景的生物活性物质。在这项研究的R21阶段，我们将重点关注以下目标，以确保治疗阴道毛滴虫感染的新化合物：(I)实施高含量筛选系统，以检测抑制阴道毛滴虫感染的天然产品。 (2)测试独一无二的纯天然产品、预分提材料和富含次生代谢物的真菌提取物的阴道毛滴虫抑制物文库；(3)利用哺乳动物细胞的反筛选技术从筛选中去除非选择性毒素；以及(4)使用真菌分类信息和次生代谢物去复制工具来选择提取物和级分以供进一步研究。从R21研究中脱颖而出的最有希望的药物将通过下列目标进行详细的R33研究：(1)从真菌部分和提取物混合物中纯化生物活性天然产物；(2)建立生物活性代谢物的结构和生物评价的安全毫克量；(3)建立免费的高含量筛选试验，以评估每个被击中的分子相对于其他厌氧原生动物靶标(肠贾第鞭毛虫和溶组织内阿米巴)的作用；以及(4)进行最有希望的被击中分子的体外代谢研究，并测试其代谢物的毒性和生物活性。这些目标的成功实现有望产生一系列集中的天然产品，抑制阴道毛滴虫的活性，对人类细胞的毒性最小。预计在随后的R01/SBIR研究中，将获得3-5个有希望的抑制剂，作为生物活性先导立即进行研究。在R21/R33研究期间，重点是确保高质量的生物活性化合物，预计将有助于产生一流的分子，应用于治疗阴道毛滴虫和其他厌氧原生动物。

 DESCRIPTION (provided by applicant): Trichomonas vaginalis is the most prevalent sexually-transmitted infectious organism in the United States with roughly 7 million people affected. Individuals infected with T. vaginalis face an increased risk of cervical cancer, infertility, pelvc inflammatory disease, prostatitis, and susceptibility to HIV. The nitroimidazoles are the only class of drugs available to treat trichomoniasis, but they suffer from several serious side effects Additionally, the emergence and spread of resistance to nitroimidazoles is becoming a concern. Consequently, there is a critical need for the development of new therapeutic agents to combat T. vaginalis and the proposed studies, designed in response to this RFA, provide a mechanism for the discovery and development of bioactive molecules that meet this need. Vital to the success of a drug discovery program is access to an outstanding library of chemically diverse substances that are well suited to become early stage leads. Natural products meet this essential requirement with metabolites from fungi having not been previously screened against T. vaginalis. Thus, this work is innovative since we will couple a novel natural product source with a new high-content screening system to identify promising bioactive substances from a large fungal secondary metabolite library. During the R21 phase of this investigation, we will focus on the following objectives to secure new compounds for treating T. vaginalis infections: (i) implement a high-content screening system for the detection of natural products that inhibit T. vaginalis viability; (ii) test a one-of-a-kind library of pure natural products, pre-fractionated materials, and secondary-metabolite-enriched fungal extracts for inhibitors of T. vaginalis; (iii) employ a mammalian-cell-based counter-screen to remove non-selective toxins from screening; and (iv) use fungal taxonomic information and secondary metabolite dereplication tools to select extracts and fractions for further studies. The most promising agents emerging from the R21 studies will be subjected to detailed R33 studies via the following aims: (i) purify bioactive natural products from fungal fractions and extract mixtures, (ii) establish the structures of bioactive metabolites and secure milligram quantities for biological evaluation, (iii) develop a complimentary high-content screening assay to evaluate each hit molecule against additional anaerobic protozoan targets (Giardia intestinalis and Entamoeba histolytica), and (iv) conduct in vitro metabolism studies of the most promising hit molecules and test their metabolites for toxicity and bioactivity. The successful accomplishment of these aims is expected to yield a focused set of natural products that inhibit T. vaginalis viability with minimal toxicity to human cells. It is anticipated that 3-5 promising inhibitors will be obtained that will serve as bioactiv leads for immediate investigation during subsequent R01/SBIR studies. The focus on securing high-quality bioactive compounds during the R21/R33 study is anticipated to help generate first-in-class molecules with applications for treating T. vaginalis and other anaerobic protozoans.