Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development

Abstract

BackgroundWith 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.MethodsRecombinant 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.ResultsBased 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.ConclusionThe 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.

Specific calpain activity evaluation in Plasmodium parasites

“…Taken together, the data support Pf-calpain as a potential antimalarial target in P. falciparum. The fact that the full-length Pf-calpain is difficult to express and purify from heterologous systems [17] highlights the importance of the method described in the current work to support the Plasmodium proteolysis studies and screening of novel drugs targeting Pf-calpain.…”

“…In addition, part of Plasmodium protease activities are regulated by cellular events with release calcium [10], which is an important second messenger that regulates various functions in eukaryotic cells such as protein secretion, gene expression, and cellular development [11,12]. Calpain, a calcium-dependent cysteine protease expressed in mammals and other organisms, has an ortholog identified in P. falciparum that has been associated with the development and invasion and egress of the parasite from the host cell [7,[13][14][15][16][17][18]. The Plasmodium genome encodes a unique calpain [15,19] that is expressed during all intraerythrocytic stages and possesses high sequence similarity to Caenorhabditis elegans calpain, which is classified as an atypical calpain [19].…”

A novel flow cytometric application discriminates among the effects of chemical inhibitors on various phases of Babesia divergens intraerythrocytic cycle

“…In this study, we demonstrated that Vybr‐FACS is an effective method not only to analyze the variation of the parasite population in response to normal culturing conditions, but also offers a key tool to characterize B. divergens' response to different drugs and, importantly, to differentiate the stressor effects on the three critical steps of Babesia life cycle: Host invasion, intracellular development and egress, thereby providing information that has not been feasible for any Babesia parasites so far. We chose three drugs that have been widely used in other parasite studies and are well known for their inhibitory effect as a way to illustrate the ability of Vybr‐FACS in identifying and discriminating the specific steps targeted by the compounds: ALLN (as a calpain inhibitor) , EGTA (as chelator of extracellular Ca +2 ) and neuraminidase (to cleave sialic acid residues from RBC). ALLN was previously described in B. bovis as an inhibitor of RBC invasion .…”

Potential Interaction of <i>Plasmodium falciparum</i> Hsp60 and Calpain

“…The rabbit polyclonal P. falciparum ( Pf )-Hsp60 antibody was purchased from LSBio (Seattle, Washington, USA). The rabbit polyclonal anti- Pf -calpain was generated as described in a previous study [ 27 ]. Antisera against GroEL raised in mice were generated in this study.…”