UC San Diego

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, which affects more than 50 million people worldwide resulting in 70,000 deaths annually. Current treatment relies on metronidazole; however, metronidazole-resistant E. histolytica have been induced in the laboratory. Therefore, new drugs based on defined targets are needed to treat amebiasis. Recently, new drugs designed to inhibit E. histolytica cysteine proteinases, which are major virulence factors in amebic invasion and evasion of the host immune system, have been developed. To gain a better understanding of how these inhibitors irreversibly bind their target proteinases, recombinant EhCP1 has been resynthesized and expressed in bacteria to cocrystallize with cysteine proteinase inhibitors. Although soluble rEhCP1 has been expressed, obtaining the necessary large quantity for crystallization is still in progress. Biologically active rEhCP5, the other cysteine proteinase unique to invasive E. histolytica was assayed against vinyl sulfone derivatives of K11777, a cysteine proteinase inhibitor of cruzain that is currently in Phase I clinical trials for treatment of Chagas disease. Through these inhibition assays, both rEhCP5 and rEhCP1 have a substrate preference for positively charged amino acids at the P2 position, more specifically an arginine, despite possessing different amino acid residues at the base of the active pockets. While the readily cell-permeable inhibitors WRR-666 and WRR-668 were effective against rEhCP1 and rEhCP5 in vitro, minimal inhibition of infection by E. histolytica trophozoites was observed in mouse models, possibly due to drug instability. rEhCP1 and rEhCP5 expression and corresponding inhibition assays provide insight into the development of new therapeutics to treat amebiasis