Plasmodium falciparum, P. vivax,andP. malariae:A Comparison of the Active Site Properties of Plasmepsins Cloned and Expressed from Three Different Species of the Malaria Parasite

Westling, Jennifer; Yowell, Charles A.; Majer, Pavel; Erickson, John W.; Dame, John B.; Dunn, Ben M.

doi:10.1006/expr.1997.4225

Cited by 54 publications

(42 citation statements)

References 20 publications

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“…Although plasmepsins from each of the four species have been cloned and expressed as recombinant enzymes (12,18,22,44,45), there is still very little known about many of the peptidases recently discovered from the P. falciparum genome-sequencing project (46). The most fully characterized plasmepsin is PfPM2, where numerous structures (18)(19)(20) and kinetic studies (21,31,45,47) have aided in identifying the determinants of substrate specificity.…”

Section: Discussionmentioning

confidence: 99%

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Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

et al. 2005

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Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate [Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009 Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502] and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1′ library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1′-Gln-(Xaa). In both cases, P1 and P1′ were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1′ residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatographymass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1′-Phe pools revealed the favored amino acids at the P3, P2, P2′, and P3′ positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.

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Section: Discussionmentioning

confidence: 99%

“…Genes encoding the zymogens of PfPM2, PmPM4, PoPM4, and PvPM4 were cloned from genomic DNA by PCR amplification (12,21,22). PfPM4 was amplified from genomic DNA and cloned into a pCR2.1 vector (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

et al. 2005

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“…(28,32,41) and Plasmodium sp. (7,40) genomes and are considered potential drug targets (5,6,16). For C. parvum and T. gondii, genomic sequencing revealed the presence of aspartic proteinase-coding regions (5,23).…”

Section: Discussionmentioning

confidence: 99%

Inhibitory Activity of Human Immunodeficiency Virus Aspartyl Protease Inhibitors against Encephalitozoon intestinalis Evaluated by Cell Culture-Quantitative PCR Assay

Menotti

Santillana-Hayat

Cassinat³

et al. 2005

Antimicrob Agents Chemother

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Immune reconstitution might not be the only factor contributing to the low prevalence of microsporidiosis in human immunodeficiency virus (HIV)-infected patients treated with protease inhibitors, as these drugs may exert a direct inhibitory effect against fungi and protozoa. In this study, we developed a cell culture-quantitative PCR assay to quantify Encephalitozoon intestinalis growth in U-373-MG human glioblastoma cells and used this assay to evaluate the activities of six HIV aspartyl protease inhibitors against E. intestinalis. A real-time quantitative PCR assay targeted the E. intestinalis small-subunit rRNA gene. HIV aspartyl protease inhibitors were tested over serial concentrations ranging from 0.2 to 10 mg/liter, with albendazole used as a control. Ritonavir, lopinavir, and saquinavir were able to inhibit E. intestinalis growth, with 50% inhibitory concentrations of 1.5, 2.2, and 4.6 mg/liter, respectively, whereas amprenavir, indinavir, and nelfinavir had no inhibitory effect. Pepstatin A, a reference aspartyl protease inhibitor, could also inhibit E. intestinalis growth, suggesting that HIV protease inhibitors may act through the inhibition of an E. intestinalis-encoded aspartyl protease. These results showed that some HIV protease inhibitors can inhibit E. intestinalis growth at concentrations that are achievable in vivo and that the real-time quantitative PCR assay that we used is a valuable tool for the in vitro assessment of the activities of drugs against E. intestinalis.

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“…Lyophilized porcine pepsin was purchased from Sigma-Aldrich and dissolved in 20 mM sodium formate, pH 3.5. The plasmepsins, PfPM2, PfPM4, PoPM4, PmPM4, and PvPM4, were expressed and purified as previously described (28).…”

Section: Expression and Purificationmentioning

confidence: 99%

Analysis of Binding Interactions of Pepsin Inhibitor-3 To Mammalian and Malarial Aspartic Proteases

et al. 2007

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The nematode Ascaris suum primarily infects pigs, but also causes disease in humans. As part of its survival mechanism in the intestinal tract of the host, the worm produces a number of protease inhibitors, including pepsin inhibitor-3 (PI3), a 17 kDa protein. Recombinant PI3 expressed in E. coli has previously been shown to be a competitive inhibitor of a sub-group of aspartic proteinases: pepsin, gastricsin and cathepsin E. The previously determined crystal structure of the complex of PI3 with porcine pepsin (p. pepsin) showed that there are two regions of contact between PI3 and the enzyme. The first three N-terminal residues (QFL) bind into the prime side of the active site cleft and a polyproline helix (139)(140) in the C-terminal domain of PI3 packs against residues 289-295 that form a loop in p. pepsin. Mutational analysis of both inhibitor regions was conducted to assess their contributions to the binding affinity for p. pepsin, human pepsin (h. pepsin) and several malarial aspartic proteases, the plasmepsins. Overall, the polyproline mutations have a limited influence on the K i values for all the enzymes tested, with the values for p. pepsin remaining in the low nanomolar range. The largest effect was seen with a Q1L mutant, with a 200-fold decrease in K i for plasmepsin 2 from Plasmodium falciparum (PfPM2). Thermodynamic measurements of the binding of PI3 to p. pepsin and PfPM2 showed that inhibition of the enzymes is an entropy-driven reaction. Further analysis of the Q1L mutant showed that the increase in binding affinity to PfPM2 was due to improvements in both entropy and enthalpy.There are over 300 million cases of malaria every year, resulting in at least one million deaths annually (World Health Organization, 2006). Malaria is found in tropical and sub-tropical regions of the world and is caused by one of four species of the plasmodium parasite: P. falciparum, P. vivax, P. ovale, and P. malariae. P. falciparum is the deadliest of the four species and P. vivax is the most common, responsible for 40% to 50% of cases in Latin America and Asia.During the intraerythrocytic stage of the plasmodium parasite's lifecycle in the human host, up to 75% of host cell hemoglobin is degraded (1). In P. falciparum several proteases have been identified in the food vacuole that are involved in hemoglobin degradation, including a family of aspartic proteases known as the plasmepsins (2,3). The four plasmepsins found in the food vacuole of P. falciparum are PfPM1, PfPM2, PfPM4, and the histoaspartic protease (HAP) (3,4). It is now believed that the enzymes from P. vivax (PvPM4), P. ovale (PoPM4), and P. malariae (PmPM4) are orthologs of PfPM4 (5 (3,(6)(7)(8)(9). Therefore, the PfPM4 orthologs would be excellent targets for a single drug therapy directed at all four plasmodium species (4).Most large, proteinaceous inhibitors of aspartic proteases have been isolated from plants (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). For this study, we have analyzed the inhibition of aspartic proteases by a 17 kD inhi...

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Plasmodium falciparum, P. vivax,andP. malariae:A Comparison of the Active Site Properties of Plasmepsins Cloned and Expressed from Three Different Species of the Malaria Parasite

Cited by 54 publications

References 20 publications

Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

Inhibitory Activity of Human Immunodeficiency Virus Aspartyl Protease Inhibitors against Encephalitozoon intestinalis Evaluated by Cell Culture-Quantitative PCR Assay

Analysis of Binding Interactions of Pepsin Inhibitor-3 To Mammalian and Malarial Aspartic Proteases

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