Targeting Apicomplexan Atg8 for Rational Drug Design

Abstract

Apicomplexan pathogens present significant health and economic burdens globally. Species of Plasmodium, the causative agent of malaria, result in more than half a million deaths annually, with the primary victims being children under the age of five. Other organisms in this phylum present a threat to immunocompromised individuals (e.g., Toxoplasma gondii, Cryptosporidium spp.) or to the productivity of the livestock industry (e.g., Eimeria spp., Neospora caninum). Autophagy is the process by which cells recycle intracellular material by encapsulating them in a double membrane bound vesicle, the autophagosome, which then fuses with the lysosome. The autophagy pathway has been shown to be essential to parasite survival in both Plasmodium and Toxoplasma. Autophagy-related protein 8 (Atg8), which is required for the formation of the autophagosomal membrane, is a ubiquitin-like protein whose conjugation pathway is conserved in Apicomplexa. Within Apicomplexans, Atg8 has a conserved loop region not present in human homologues. In Plasmodium falciparum, this loop was shown to be essential to the interaction between Atg8 and its E2 conjugating enzyme, Atg3, and may represent a viable pan-Apicomplexan drug target. We are pursuing this possibility using x-ray crystallography, SPR, and virtual library screening to identify small compound drug leads which can then be chemically diversified and optimized. Having solved the structure of PfAtg8, we performed two screens against this protein: a screen of the MMV Malaria box of compounds and a virtual library screen. We pursued hits from both screens and tested them via SPR against five pairs of Atg3 and Atg8 homologues: Plasmodium falciparum, Homo sapiens, Cryptosporidium parvum, Eimeria tenella, and Neospora caninum. We have now also obtained protein crystals for the Atg8 homologues in E. tenella, C. parvum, and N. caninum, which will facilitate future virtual library screens against these specific proteins.