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

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

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Background: 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 Ca§ssup§2+§esup§-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. Methods. 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. Results: 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. Conclusion: 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.

Original languageEnglish (US)
Article number47
JournalMalaria journal
Volume12
Issue number1
DOIs
StatePublished - Feb 6 2013
Externally publishedYes

Fingerprint

Calpain
Plasmodium falciparum
Pharmaceutical Preparations
Parasites
Antimalarials
Malaria
Amino Acids
Plasmodium
Cysteine Proteases
Gelatin
Protease Inhibitors
Catalysis
Fluorescent Dyes
Catalytic Domain
Hemoglobins

Keywords

  • Anti-malarial drug
  • Calpain
  • Cysteine protease
  • Malaria
  • Monomer
  • Plasmodium falciparum
  • Protease inhibitor

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases

Cite this

Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development. / Soh, Byoung Yul; Song, Hyun Ok; Lee, Yoonji; Lee, Junghyun; Kaewintajuk, Kusuma; Lee, Binna; Choi, Yun Young; Cho, Jeong Hoon; Choi, Sun; Park, Hyun.

In: Malaria journal, Vol. 12, No. 1, 47, 06.02.2013.

Research output: Contribution to journalArticle

Soh, Byoung Yul ; Song, Hyun Ok ; Lee, Yoonji ; Lee, Junghyun ; Kaewintajuk, Kusuma ; Lee, Binna ; Choi, Yun Young ; Cho, Jeong Hoon ; Choi, Sun ; Park, Hyun. / Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development. In: Malaria journal. 2013 ; Vol. 12, No. 1.
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abstract = "Background: 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 Ca§ssup§2+§esup§-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. Methods. 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. Results: 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. Conclusion: 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.",
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AU - Song, Hyun Ok

AU - Lee, Yoonji

AU - Lee, Junghyun

AU - Kaewintajuk, Kusuma

AU - Lee, Binna

AU - Choi, Yun Young

AU - Cho, Jeong Hoon

AU - Choi, Sun

AU - Park, Hyun

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N2 - Background: 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 Ca§ssup§2+§esup§-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. Methods. 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. Results: 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. Conclusion: 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.

AB - Background: 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 Ca§ssup§2+§esup§-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. Methods. 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. Results: 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. Conclusion: 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.

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KW - Cysteine protease

KW - Malaria

KW - Monomer

KW - Plasmodium falciparum

KW - Protease inhibitor

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