Thio-dependent Antioxidants: A Novel Drug Target for Toxoplasma Gondii

描述(由申请人提供)：中枢神经系统的弓形虫病可能会有潜在的破坏性的长期影响。它是由弓形虫引起的，在美国食源性病原体中是导致住院(8%)和死亡(24%)的第二大原因。不幸的是，目前可用的药物有很大的毒性，对 缓虫体/潜伏期，以及对这些药物出现耐药性的迫在眉睫的威胁，使发现新的治疗靶点成为优先事项。在这项建议中，我们证明了金诺芬在体外对弓形虫具有活性，并诱导感染宿主细胞中的活性氧积累，而在体内，它可以防止100%的鸡胚死亡(急性弓形虫病模型)，并调节宿主免疫反应，防止压倒性的炎症反应。Auranofin可能的目标是依赖硫醇的抗氧化系统，正如我们对溶组织内阿米巴(硫氧还蛋白还原酶)以及其他寄生虫(如婴儿利什曼原虫(锥虫硫代还原酶)和曼氏血吸虫(谷胱甘肽-硫氧还蛋白还原酶))的观察所证明的那样。这项建议的长期目标是阐明硫醇依赖的抗氧化系统在弓形虫生物学中的作用，这些作用在很大程度上是未知的。中心假设是弓形虫依赖硫醇的抗氧化系统是弓形虫生物学所必需的，并且是一个可行的药物靶点。具体目标1将决定T。 贡地依赖硫醇的抗氧化酶(一个硫氧还蛋白还原酶和两个硫氧还蛋白依赖的过氧化物酶)是金诺芬的靶标。将产生这些酶的弓形虫敲除菌株来证实这些观察结果。重组酶将被表达，并将评估它们与金诺芬在体外相互作用的药代动力学。具体目的2将评估Auranofin在急性和慢性弓形虫病标准小鼠模型中的体内活性，并将通过质谱仪测量CNS中Auranofin的浓度，以评估血脑屏障的穿透。特指目标3将评估金诺芬对 在鸡胚急性弓形虫病模型中，给予宿主免疫应答，具有显著的清除寄生虫和减轻宿主组织炎症的作用。利用GFP-LC3小鼠追踪自噬将是可能的，这对研究这一细胞过程起到了重要作用。将进行体外和体内试验，以确定在金诺芬存在下和在弓形虫感染期间自噬的激活和进展。该项目有可能确定一种新的抗寄生虫药物靶点--弓形虫依赖硫醇的抗氧化系统，该系统可用于治疗弓形虫感染的药物开发。重新调整FDA批准的具有公认安全性的药物的用途可以极大地加快临床试验的时间。

DESCRIPTION (provided by applicant): Toxoplasmosis of the central nervous system can have potentially devastating long term effects. It is caused by Toxoplasma gondii and it is the second leading cause of hospitalizations (8%) and deaths (24%) among food borne pathogens in the US. Unfortunately, current available drugs have significant toxicity and have no effect over the bradyzoite/latent form while the impending threat of emergence of resistance to these drugs makes the discovery of new therapeutic targets a priority. In this proposal, we show that auranofin has in vitro activity against T. gondii and induces accumulation of reactive oxygen species in infected host cells while in vivo, it prevents death in 100% of chicken embryos (acute toxoplasmosis model) and modulates the host immune response preventing an overwhelming inflammatory reaction. Auranofin's likely target is the thiol-dependent anti-oxidant system as demonstrated by our own observations on Entamoeba histolytica (thioredoxin reductase), and on other parasites such as Leishmania infantum (trypanothione reductase), and Schistosoma mansoni (glutathione-thioredoxin reductase). The long term goal of this proposal is to elucidate the roles of the thiol-dependent antioxidant system in T. gondii biology which are largely unknown. The central hypothesis is that T. gondii thiol-dependent antioxidant system is essential for T. gondii biology, and is a viable drug target. Specific Aim 1 will determine that T. gondii thiol-dependent anti-oxidant enzymes (one thioredoxin reductase and two thioredoxin-dependent peroxidases) are auranofin targets. T. gondii knockout strains of these enzymes will be generated to corroborate these observations. Recombinant enzymes will be expressed and their pharmacokinetics of interaction with auranofin in vitro will be evaluated. Specific Aim 2 wil evaluate auranofin's in vivo activity in standard mouse models of acute and chronic toxoplasmosis and will measure auranofin concentrations in CNS by mass spectrometry to assess blood brain barrier penetration. Specific Aim 3 will evaluate the effect of auranofin on the host immune response given that it has an impressive effect on parasite clearance and decreasing host tissue inflammation in vivo chicken embryo model of acute toxoplasmosis. Tracking autophagy will be possible using GFP-LC3 mice which have been instrumental to study this cellular process. In vitro and in vivo assays will be conducted to determine autophagy activation and progression in the presence of auranofin and during T. gondii infection. This project has the potential of identifying a new anti-parasitic drug target, the T. gondii thiol-dependent anti-oxidant system, that can be exploited in drug development for the treatment of toxoplasma infection. Re-purposing of a FDA-approved drug with a proven safety profile can dramatically accelerate the time to clinical trials.