Exploring Novel Targeting Strategies for AIDS Protozoal Pathogens

 描述（由申请人提供）：世界卫生组织估计，截至 2012 年底，超过 3500 万人感染了 HIV-1（人类免疫缺陷病毒）。由于高效抗逆转录病毒（HAART）疗法的进步，与艾滋病相关的死亡人数已经减少，人们的寿命也更长了。尽管HAART取得了成功，但机会性感染仍然是艾滋病患者死亡的主要原因之一。其中最常见的是原生动物寄生虫病原体，隐孢子虫。隐孢子虫病是一种以严重慢性腹泻为特征的艾滋病定义疾病，目前尚无有效的治疗方法，也是发展中国家引起腹泻疾病的最常见病原体之一。 上一个资助期间的工作重点是一种独特的双功能酶胸苷酸合酶二氢叶酸还原酶（TS-DHFR），该酶仅在原生动物病原体中发现，作为抑制剂设计的可能分子靶标。这些人型隐孢子虫 (Ch)TS-DHFR 的概念验证研究已经确定了这种双功能酶中独特的物种特异性和变构/非活性目标区域，突变分析和机制研究已验证这些位点对于催化功能至关重要。 PI 实验室及其合作者开发了一种独特且成功的计算和机械引导方法，用于发现新的 ChTS-DHFR 抑制剂。这种计算方法和详细实验方法的结合是一种独特的策略，可以在设计中构建最佳的理化和药理学参数。然后对这些分子进行实验测试，并通过机械、结构和细胞评估迭代完善设计。分子对接和虚拟筛选与结构分析相结合，发现了 CH TS-DHFR 的新型抑制剂，其中一些在细胞培养物中具有纳摩尔效力和抗隐孢子虫活性。当前的资助期将重点关注先导化合物优化和寄生虫药物输送的新型纳米技术策略，以开发治疗隐孢子虫感染的新疗法。

 DESCRIPTION (provided by applicant): The World Health Organization estimated that at the end of 2012 over 35 million individuals were infected with HIV-1 (Human Immunodeficiency Virus). Due to the advancements in highly active antiretroviral (HAART) therapy the number of AIDS-related deaths has been reduced and individuals are living longer. Despite successes with HAART, opportunistic infections remain one of major causes of death in AIDS patients. Among the most prevalent is the protozoal parasite pathogen, Cryptosporidium. Cryptosporidiosis is one of the AIDS- defining illnesses characterized by a severe chronic diarrhea for which there are currently no effective therapies and among the most frequent pathogens causing diarrheal diseases in developing countries. Efforts during the previous grant period have focused on a unique bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) found only in protozoal pathogens as a possible molecular target for inhibitor design. These proof of concept studies in Cryptosporidium hominis (Ch)TS-DHFR have identified unique species specific and allosteric/non-active target regions in this bifunctional enzymes and mutational analysis and mechanistic studies have validated these sites as essential for catalytic function. The PIs lab and their collaborators have developed a distinctive and successful computationally and mechanistically guided approach for the discovery of new inhibitors of ChTS-DHFR. This partnership of computational and detailed experimental methodologies is a unique strategy that builds optimal physiochemical and pharmacological parameters into the design. These molecules are then experimentally tested and the design iteratively refined through mechanistic, structural and cellular evaluation. Molecular docking and virtual screening coupled with structural analyses have discovered novel inhibitors of CH TS-DHFR including some with nanomolar potency and anti-Cryptosporidial activity in cell culture. The current grant period will focus on lead optimization and a novel nanotechnology strategy for parasite drug delivery to develop new therapies to treat Cryptosporidial infections.