Engagement of human host receptors by the antiparasitic drug praziquantel

Abstract

Neglected tropical diseases (NTDs) impact nearly two billion people worldwide. Schistosomiasis is one of the most debilitating NTDs causing a significant healthcare and economic burden in developing countries. The disease is caused by trematodes of the species Schistosoma and is endemic in 78 countries. Treatment is by one broad-spectrum drug, praziquantel (PZQ) which has been the mainstay treatment for the past 40 years. PZQ is clinically administered as a racemic mixture (()-PZQ) of two enantiomers ((R)-PZQ) and (S)-PZQ). The eutomer, (R)-PZQ causes a rapid paralysis of adult schistosome worms and damages the worm tegument. However, the mechanism of action of PZQ is unknown. Thus, identifying relevant molecular targets of PZQ is necessary to aid the development of new antiparasitic therapies and provide treatment alternatives in face of the threat of drug resistance. One obstacle has been the difficulty of heterologously expressing parasite proteins to profile their drug sensitivity. Consequently, I have taken the sidestep of screening PZQ against human targets to attempt to resolve potential targets of PZQ, the identity of which may provide clues to homologs in flatworms. Here, in Chapter 2, I show that PZQ engages human 5-HT2B receptors in vivo, ex vivo on isolated vessels and in vitro by a variety of approaches. PZQ engagement of 5-HT2B R on vascular smooth muscle cells caused vasoconstriction in isolated mouse mesenteric vessels. In Chapter 3, my studies demonstrate that the human transient receptor potential melastatin 8 (hTRPM8) channel is stereoselectively activated by (S)-PZQ. This was evidenced by (S)-PZQ but not (R)-PZQ-evoked increase in Ca2+ signaling in hTRPM8-heterologously expressing cells. Mutational analysis of hTRPM8 binding pocket residues revealed (S)-PZQ activation of hTRPM8 resembles that of the orthosteric ligand, WS-12. These discoveries catalyzed the subsequent discovery of a parasitic flatworm TRPM target in Schistosoma mansoni (Sm.TRPMPZQ) that was stereoselectively activated by (R)-PZQ. Notably, mutation of residues in Sm.TRPMPZQ homologous to those involved in WS-12 binding in hTRPM8 lead to PZQ insensitivity. These findings illustrated that the different PZQ enantiomers bind to human hTRPM8 ((S)-PZQ) and Sm.TRPMPZQ ((R)-PZQ) by engaging a similar binding pocket localized at the base of the first four transmembrane helices (S1-S4) of these TRP channels. Finally, Chapter 4 describes a Ca2+-independent membrane potential (FMP) assay that can be utilized for screening Sm.TRPMPZQ channel as well as Ca2+-impermeable flatworm ion channels with high sensitivity and robust signal. Collectively, this work has provided insight into PZQ action in the definitive human host and had led to the resolution of the target of PZQ in Schistosoma mansoni.