Potential targets for new antischistosomal agents

描述(申请人提供)：血吸虫病是一种由血吸虫属扁虫引起的寄生虫病。作为第二种最流行的热带疾病，它影响着全世界数亿人，每年导致数十万人死亡。化疗药物被用来治疗这种疾病并控制其传播。吡喹酮(PZQ)是首选药物，副作用最小，对所有血吸虫物种都有效。然而，对PZQ耐药的血吸虫菌株的报告已经开始出现。此外，PZQ的作用模式尚不清楚，尽管它的作用之一是影响寄生虫的钙(Ca~(2+))稳态。我们的证据表明，PZQ作用于血吸虫电压门控钙通道。钙通道是可兴奋细胞的重要组成部分，参与多种钙依赖过程的调节。它们是由多个亚基组成的膜蛋白复合体，包括形成孔洞的、电压敏感的α1亚基。研究最深入的辅助亚基是调节钙通道特性的β亚基。我们从曼氏链霉菌中克隆了3个钙通道α1亚基和2个β亚基序列(SmCavBetaA、SmCavBetaB)。SmCavBetaA具有几个新的性质。最引人注目的是，当血吸虫或哺乳动物的α1亚基与这个Beta亚基在非洲爪哇卵母细胞中共表达时，它们在PZQ存在的情况下表现出电流幅度的增加，这一反应与该药物的临床效果一致。在本项目中，我们将更详细地探讨PZQ对血吸虫钙通道亚单位的作用机制。我们将确定SmCavBetaA是否能赋予我们克隆的另外两个血吸虫α1亚基PZQ敏感性。我们还将测试蛋白激酶C(PKC)对特定Beta亚单位结构域的磷酸化是否在PZQ敏感性中发挥作用。SmCa43A(和在日本血吸虫中发现的同源物)是唯一已知的在这个关键区域缺乏两个共识的PKC磷酸化位点的Beta亚基。将这两个位点中的一个或两个引入SmCavBetaA会导致PZQ敏感性的抑制。我们将继续研究这些位点的磷酸化作用，使用几种方法。最后，我们将检查耐PZQ的血吸虫菌株在这些位点上的单核苷酸多态性，并测试抑制或激活PKC的药物是否可以改变这些蠕虫对PZQ的敏感性。这些实验可能最终导致治疗血吸虫病的新的化疗方法。

DESCRIPTION (provided by the applicant): Schistosomiasis is a parasitic disease caused by trematode flatworms of the genus Schistosoma. The second most prevalent tropical disease, it affects hundreds of millions of people worldwide, killing hundreds of thousands each year. Chemotherapeutic agents are used to treat the disease and control its spread. Praziquantel (PZQ), the drug of choice, has minimal side effects and is effective against all schistosome species. However, reports of PZQ-resistant strains of schistosome have begun to emerge. Furthermore, the mode of PZQ action is unknown, although one of its effects is on calcium (Ca2+) homeostasis in the parasite. Our evidence indicates that PZQ acts on schistosome voltage-gated Ca2+ channels. Ca2+ channels are crucial components of excitable cells and participate in the regulation of several Ca2+-dependent processes. They are membrane protein complexes that consist of multiple subunits, including the pore-forming, voltage-sensing alpha1 subunit. The most thoroughly studied auxiliary subunit is the Beta subunit, which modulates Ca2+ channel properties. We have cloned 3 Ca2+ channel alpha1 subunits and 2 Beta subunit sequences (SmCavBetaA, SmCavBetaB) from S. mansoni. SmCavBetaA has several novel properties. Most strikingly, when schistosome or mammalian alpha1 subunits are co-expressed with this Beta subunit in Xenopus oocytes, they show increases in current amplitude in the presence of PZQ, a response that is consistent with the clinical effects of the drug. In this project we will pursue the mechanism of PZQ action on schistosome Ca2+ channel subunits in greater detail. We will determine whether SmCavBetaA can confer PZQ sensitivity to the two other schistosome alpha1 subunits we have cloned. We will also test whether phosphorylation of a particular Beta subunit domain by protein kinase C (PKC) plays a role in PZQ sensitivity. SmCa43A (and a homolog found in S. japonicum) are the only known Beta subunits lacking two consensus PKC phosphorylation sites in this critical domain. Introduction of either or both of these sites into SmCavBetaA results in a suppression of PZQ sensitivity. We will continue to examine the role of phosphorylation at these sites, using several approaches. Finally, we will examine PZQ-resistant strains of schistosome for single nucleotide polymorphisms at these sites and test whether agents that inhibit or activate PKC can alter sensitivity of these worms to PZQ. These experiments may eventually lead to new chemotherapeutic approaches to treating schistosomiasis.