Potential targets for new antischistosomal agents

描述（由申请人提供）：血吸虫病是一种由血吸虫属吸虫扁平虫引起的寄生虫病。它是第二大流行的热带疾病，影响着全世界数亿人，每年造成数十万人死亡。化疗药物用于治疗这种疾病并控制其扩散。吡喹酮（PZQ）是首选药物，副作用最小，对所有血吸虫都有效。然而，耐pzq的血吸虫菌株的报道已经开始出现。此外，PZQ的作用方式尚不清楚，尽管它的作用之一是对寄生虫体内钙（Ca2+）的稳态。我们的证据表明，PZQ作用于血吸虫电压门控Ca2+通道。Ca2+通道是可兴奋细胞的重要组成部分，参与多种Ca2+依赖性过程的调节。它们是由多个亚基组成的膜蛋白复合物，包括孔隙形成、电压传感的α - 1亚基。研究最彻底的辅助亚基是β亚基，它调节Ca2+通道的性质。我们克隆了3个Ca2+通道alpha1亚基和2个β亚基序列（SmCavBetaA, SmCavBetaB）。SmCavBetaA有几个新特性。最引人注目的是，当血吸虫或哺乳动物α 1亚基与非洲爪蟾卵母细胞中的β亚基共表达时，它们在PZQ存在的情况下表现出电流振幅的增加，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Ca2+通道亚基的作用机制。我们将确定SmCavBetaA是否能赋予我们克隆的另外两个血吸虫α 1亚基PZQ敏感性。我们还将测试蛋白激酶C （PKC）对特定β亚基结构域的磷酸化是否在PZQ敏感性中起作用。SmCa43A（和在日本血吸虫中发现的同系物）是唯一已知的在这个关键结构域中缺乏两个共识PKC磷酸化位点的β亚基。在SmCavBetaA中引入这两个位点中的一个或两个，可以抑制PZQ的敏感性。我们将继续研究在这些位点磷酸化的作用，使用几种方法。最后，我们将检测耐PZQ的血吸虫菌株在这些位点的单核苷酸多态性，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新的化疗方法来治疗血吸虫病。

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.