Plasmodium resistance to Artemisinin (ART)-combination therapies poses a major threat to the control of malaria. The need to discover novel antimalarial drug targets, and targets that overcome drug-resistance is paramount.
Recent work has shown that synergistic use of a parasite-selective proteasome inhibitor sensitizes ART-resistant parasites to artemisinin1. Therefore, development of an inhibitor targeting a parasite-specific protein involved in the Plasmodium ubiquitin-proteasome pathway (UPP) could yield a combination treatment to battle artemisinin resistance1. DNA damage-inducible protein 1 (DDI1) is an uncharacterized Plasmodium aspartyl protease. Recent studies have shown that the catalytic domain of human DDI2 upregulates the UPP2. We hypothesize that DDI1 is an active Plasmodium protease and plays a role in the parasite UPP. In this work, we aim to determine if Plasmodium DDI1 is a target to overcome ART resistance.
In P. berghei, Plasmepsin 7 (PM7) has a non-essential but undefined role and is expressed in the mosquito and transmission stages of the parasite lifecycle3. We aim to elucidate the function of PM7 in P. falciparum to validate its potential as a transmission-blocking agent.
We will be utilizing CRISPR-Cas9 technology to tag and knockout DDI1 and PM7 respectively in P. falciparum NF54 parasites. These parasites will be used to assess the localization, essentiality and roles of DD11 and PM7 across the parasite lifecycle. We will also recombinantly express both DDI1 and PM7, characterizing substrate specificity and kinetics for each protein.
Preliminary data suggests that DDI1 is essential to the asexual lifecycle of P. falciparum, whereas PM7 is confirmed to be redundant in this stage4. Localization and phenotypic studies are currently underway. DDI1-RVP protein was produced using an E. coli expression system and shows proteolytic activity on a distinct set of peptide substrates. Protein construct expression and purification is ongoing to enable structural characterization of DDI1.