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

Byoung Yul Soh,Junghyun Lee,Yoonji Lee,Jeong Hoon Cho,Binna Lee,Kusuma Kaewintajuk,Sun Choi,Hyun Park,Yun-Young Choi,Hyun-Ok Song

doi:10.1186/1475-2875-12-47

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.

Highlights

With the increasing resistance of malaria parasites to available drugs, there is an urgent demand to develop new anti-malarial drugs
Since Plasmodium plasmepsin and falcipain are involved in haemoglobin degradation, which is necessary for parasite proliferation in the host, they have been targeted for development of anti-malarial drugs for decades [5,16,17,18,19]
The catalytic domain IIa is sufficient for enzymatic activity of Pf-calpain To characterize the Plasmodium calpain protein, the full length genomic Pf-calpain gene (6.2 kb) was firstly cloned from P. falciparum FCR-3 strain (Figure 1A; Accession No HQ386136)

Summary

Introduction

With the increasing resistance of malaria parasites to available drugs, there is an urgent demand to develop new anti-malarial drugs. ALLN, is proposed to inhibit parasite proliferation by suppressing haemoglobin degradation This provides Plasmodium calpain as a potential target for drug development. In P. falciparum, three different classes of proteases are mainly responsible for the haemoglobin degradation; they include aspartic proteases (plasmepsin I, II, IV and HAP), cysteine proteases (falcipain-1, -2 and −3) and the metalloprotease (falcilysin) [6,7,8,9]. Several exopeptidases such as dipeptidyl aminopeptidase 1 (DPAP1) and three metallo-aminopeptidases (A-M1, APP and LAP) have essential roles in haemoglobin degradation [10,11,12]. Its antimalarial activity is likely due primarily to the inhibition of falcipain, it still opens the possibility that calpain could be the one of the mediators for haemoglobin degradation and, thereby, a potential anti-malarial drug target

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Results

Discussion

Conclusion