Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development
- Equal contributors
1 Laboratory of Cell & Molecular Biology, Department of Life Science, Seonam University, 590-711, Namwon, Jeonbuk, Republic of Korea
2 Zoonosis Research Center, Department of Infection Biology, Wonkwang University School of medicine, 570-749, Iksan, Jeonbuk, Republic of Korea
3 National Leading Research Laboratory (NLRL) of Molecular Modeling & Drug Design, College of Pharmacy, Division of Life and Pharmaceutical Sciences, and National Core Research Center for Cell Signaling and Drug Discovery Research, Ewha Womans University, 120-750, Seoul, Republic of Korea
4 Division of Biology Education, College of Education, Chosun University, 501-759, Gwangju, Republic of Korea
Malaria Journal 2013, 12:47 doi:10.1186/1475-2875-12-47Published: 4 February 2013
With the increasing resistance of malaria parasites to available drugs, there is an urgent demand to develop new anti-malarial drugs. Calpain inhibitor, ALLN, is proposed to inhibit parasite proliferation by suppressing haemoglobin degradation. This provides Plasmodium calpain as a potential target for drug development. Pf-calpain, a cysteine protease of Plasmodium falciparum, belongs to calpain-7 family, which is an atypical calpain not harboring Ca2+-binding regulatory motifs. In this present study, in order to establish the screening system for Pf-calpain specific inhibitors, the active form of Pf-calpain was first identified.
Recombinant Pf-calpain including catalytic subdomain IIa (rPfcal-IIa) was heterologously expressed and purified. Enzymatic activity was determined by both fluorogenic substrate assay and gelatin zymography. Molecular homology modeling was carried out to address the activation mode of Pf-calpain in the aspect of structural moiety.
Based on the measurement of enzymatic activity and protease inhibitor assay, it was found that the active form of Pf-calpain only contains the catalytic subdomain IIa, suggesting that Pf-calpain may function as a monomeric form. The sequence prediction indicates that the catalytic subdomain IIa contains all amino acid residues necessary for catalytic triad (Cys-His-Asn) formation. Molecular modeling suggests that the Pf-calpain subdomain IIa makes an active site, holding the catalytic triad residues in their appropriate orientation for catalysis. The mutation analysis further supports that those amino acid residues are functional and have enzymatic activity.
The identified active form of Pf-calpain could be utilized to establish high-throughput screening system for Pf-calpain inhibitors. Due to its unique monomeric structural property, Pf-calpain could be served as a novel anti-malarial drug target, which has a high specificity for malaria parasite. In addition, the monomeric form of enzyme may contribute to relatively simple synthesis of selective inhibitors.