Potential targets for new antischistosomal agents

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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.