Targeting Trypanosome Cation Pumps and Channels

人类非洲锥虫病(HAT，昏睡病)是一种被忽视但致命的媒介传播疾病 由布氏锥虫引起。(T.brucei)。6000万人面临感染HAT的风险，以及 在撒哈拉以南的非洲，每年报告的新病例和相同数量的死亡人数。唯一的 市场上可用的抗锥虫药物毒性极大，并会导致治疗后 接受治疗的患者的脑病。理想的抗锥虫药物将针对重要的生理 过程和/或非变异寄生虫衍生分子，而不会对人类宿主产生不利影响。我们 另有研究表明，布氏毛滴虫血液期细胞内钙离子浓度[Ca~(2+)]_i为4~ 比寄主细胞外环境低10个数量级，寄生虫需要有效的 维持宿主内[Ca~(2+)]动态平衡、存活和增殖的机制。在我们之前的拨款中， 我们鉴定并鉴定了两个关键的质膜型阳离子泵(ATPase；TBCA1和TBCA2)。 布氏毛滴虫用于生存并产生抗体，使其在血液期和昆虫中免疫定位 阶段寄生虫。我们使用瞬时RNAi确定了它们在昆虫和血期寄生虫中的功能作用 缓蚀技术和合成缓蚀剂检测。我们随后构建了重组反泵 基于一种新的细菌幽灵疫苗递送技术的疫苗，由莫尔豪斯学院开发 药物，对小鼠的寄生虫攻击有部分保护作用。我们的结果显示，TBCA1 TBCA2类似于真菌的K+/Na+-ATPase，而TBCA2是一种质膜型的Ca2+-ATPase。RNAi 抑制这些靶标会导致寄生虫死亡率增加。此外，我们通过抑制来确定 布氏毛滴虫钙稳态不仅受钙三磷酸腺苷酶的调节，还受L类型的调控 钙离子通道。由于以钙泵为靶点的RNAi抑制增加了寄生虫的死亡率和 接种TBCA2疫苗可显著降低感染小鼠的寄生虫血症和存活率，我们建议 靶向关键阳离子泵(Tbca1和Tbca2)以及L类型的钙离子通道 合成抑制剂药物或疫苗，将足以抑制布氏毛滴虫的增殖，并提供完全 预防布氏毛滴虫感染。在这项竞争性更新建议中，我们更进一步， 假设锥虫利用阳离子泵和通道在体内建立 哺乳动物宿主血液和靶向特定药物同时抑制和阻断 接种疫苗将阻止布氏毛滴虫的增殖和发育。提出了两个具体目标： 具体目标1.对布氏毛滴虫和毛滴虫的L钙通道进行功能鉴定和定位。 确定其在钙动态平衡中的作用。在具体目标2.我们将构建和测试各种钙离子 泵/通道基因作为抗原在新型细菌幽灵疫苗系统中的应用 并确定它们对布氏毛滴虫感染的保护水平。我们的长期计划 目标是在我们既定的原则证明的基础上，开发和交付一种新型的小型 分子药物和/或免疫疗法能够抑制T。 布鲁斯在发育过程中。我们还计划通过此应用程序生成足够的数据，以申请 RQ-1型赠款，用于资助未来抗锥虫药物的研究。

Human African Trypanosomiasis (HAT, sleeping sickness) is a neglected but fatal vector borne disease caused by Trypanosoma brucei ssp. (T. brucei). Sixty million people are at risk of infection with HAT, and 50,000 new case's and an equal number of deaths are reported annually in subsaharan Africa. The only available anti-trypanosomal drugs on the market are extremely toxic, and result in post-treatment encephalopathy in treated patients. The ideal anti-trypanosomal agents will target vital physiological processes and/or non-variant parasite-derived molecules without adversely affecting the human host. We and others have shown that that cytosolic calcium ion concentration [Ca2+ ]i in blood stages of T. brucei is 4- 10 orders of magnitude below that encountered in host extracellular milieu and that parasites require effective mechanisms to maintain [Ca2+ ]\ homeostasis, survival, and proliferation in their host. In our previous grant, we identified and characterized two key plasma-membrane-like cation pumps (ATPases; TBCA1 and TBCA2) utilized by T. brucei for survival and generated antibodies to immunolocalize them in bloodstage and insect stage parasites. We determined their functional role in insect and bloodstage parasites using transient RNAi inhibition technology and synthetic inhibitor assays. We subsequently constructed recombinant anti-pump vaccines based on a novel bacterial ghost vaccine delivery technology, developed at Morehouse School of Medicine, which partially protected against parasite challenge in mice. Our results revealed that TBCA1 resembled a fungal K+/Na+-ATPase while TBCA2 was a plasma-membrane-like Ca2+ ATPase. RNAi inhibition of these targets resulted in increased parasite mortality. Furthermore, we determined by inhibition studies that Ca2+ homeostasis in T. brucei is not only regulated by the Ca2+ ATPases but also by L-type calcium ion channels. Since targeting the Ca2+ pumps by RNAi inhibition increased parasite mortality and vaccination with TBCA2 significantly reduced parasitemia and survival of infected mice, we propose that targeting the key cation pumps (TBCA1 and TBCA2) as well as L-type Ca2+ channels together with either synthetic inhibitor drugs or vaccines, will be sufficient to inhibit proliferation of T. brucei and provide complete protection against T. brucei infection. In this competitive renewal proposal, we have gone a step further to hypothesize that trypanosomes utilize cation pumps and channels to mediate establishment in mammalian host blood and that simultaneous inhibition by target specific drugs and blocking by vaccination will prevent T. brucei proliferation and development. Two specific aims are proposed: In Specific aim 1. we will functionally characterize and localize the L-type Ca2+ channel in T. brucei and determine its role in Ca2+ homeostasis. In Specific aim 2. we will construct and test various Ca2+ pump/channel gene constructs as antigens in a novel bacterial ghost based vaccine system against infections by T. brucei and determine their levels of protection against T. brucei infection. Our longterm goal is to build on our established proof of principle to develop and deliver a novel class of small molecule drugs and/or immunotherapeutics capable of inhibiting the essential Ca2+ pumps and channels of T. brucei during development. We also plan to generate enough data, through this application, to apply for an RQ-1 type grant to fund future studies on anti-trypanosome drugs.