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Picomolar Inhibition of Plasmepsin V, an Essential Malaria Protease, Achieved Exploiting the Prime Region.

Gambini L, Rizzi L, Pedretti A, Taglialatela-Scafati O, Carucci M, Pancotti A, Galli C, Read M, Giurisato E, Romeo S, Russo I - PLoS ONE (2015)

Bottom Line: It results in an annual death-toll of ~ 600,000.Our work disclosed novel pursuable drug design strategies for highly efficient PmV inhibition highlighting novel molecular elements necessary for picomolar activity against PmV.All the presented data are discussed in respect to human aspartic proteases and previously reported inhibitors, highlighting differences and proposing new strategies for drug development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Università degli Studi di Milano, Milan, Italy.

ABSTRACT
Malaria is an infectious disease caused by Plasmodium parasites. It results in an annual death-toll of ~ 600,000. Resistance to all medications currently in use exists, and novel antimalarial drugs are urgently needed. Plasmepsin V (PmV) is an essential Plasmodium protease and a highly promising antimalarial target, which still lacks molecular characterization and drug-like inhibitors. PmV, cleaving the PExEl motif, is the key enzyme for PExEl-secretion, an indispensable parasitic process for virulence and infection. Here, we describe the accessibility of PmV catalytic pockets to inhibitors and propose a novel strategy for PmV inhibition. We also provide molecular and structural data suitable for future drug development. Using high-throughput platforms, we identified a novel scaffold that interferes with PmV in-vitro at picomolar ranges (~ 1,000-fold more active than available compounds). Via systematic replacement of P and P' regions, we assayed the physico-chemical requirements for PmV inhibition, achieving an unprecedented IC50 of ~20 pM. The hydroxyethylamine moiety, the hydrogen acceptor group in P2', the lipophilic groups upstream to P3, the arginine and other possible substitutions in position P3 proved to be critically important elements in achieving potent inhibition. In-silico analyses provided essential QSAR information and model validation. Our inhibitors act 'on-target', confirmed by cellular interference of PmV function and biochemical interaction with inhibitors. Our inhibitors are poorly performing against parasite growth, possibly due to poor stability of their peptidic component and trans-membrane permeability. The lowest IC50 for parasite growth inhibition was ~ 15 μM. Analysis of inhibitor internalization revealed important pharmacokinetic features for PExEl-based molecules. Our work disclosed novel pursuable drug design strategies for highly efficient PmV inhibition highlighting novel molecular elements necessary for picomolar activity against PmV. All the presented data are discussed in respect to human aspartic proteases and previously reported inhibitors, highlighting differences and proposing new strategies for drug development.

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PmV inhibitory activity of the new compounds.IC50 values, obtained in the presence of 3 μM of HRPII-PExEl substrate, are shown. They are averages of not less than three independent inhibition curves. Standard errors of the calculation of the IC50s by sigmoidal fitting are shown in column ‘±’. HEA indicates the hydroxyethylamino group that links P1, the non-amino acidic leucine analog, (3S)-3-amino-2-hydroxy-5-methylhexyl- group, and P1', the alanine. Sta is statine, an atypical amino acid found in the natural product Pepstatin A. Panel a Activity comparison of the newly generated inhibitor 1 with previously tested compounds [14, 15]. Panel b Activity of two generated diastereoisomers presented in Fig 4. Chemical structures of HIV inhibitors and absolute chirality of Leu-HEA epimers is included. Chemical structures of Lopinavir and Ritonavir are included in panel a; and stereoisomers of Leu-HEA-Ala are shown in panel b.
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pone.0142509.g002: PmV inhibitory activity of the new compounds.IC50 values, obtained in the presence of 3 μM of HRPII-PExEl substrate, are shown. They are averages of not less than three independent inhibition curves. Standard errors of the calculation of the IC50s by sigmoidal fitting are shown in column ‘±’. HEA indicates the hydroxyethylamino group that links P1, the non-amino acidic leucine analog, (3S)-3-amino-2-hydroxy-5-methylhexyl- group, and P1', the alanine. Sta is statine, an atypical amino acid found in the natural product Pepstatin A. Panel a Activity comparison of the newly generated inhibitor 1 with previously tested compounds [14, 15]. Panel b Activity of two generated diastereoisomers presented in Fig 4. Chemical structures of HIV inhibitors and absolute chirality of Leu-HEA epimers is included. Chemical structures of Lopinavir and Ritonavir are included in panel a; and stereoisomers of Leu-HEA-Ala are shown in panel b.

Mentions: Inhibitors of Pepstatin A and HIV-protease have been previously reported as inhibitors of PmV activity [14, 15]. We, therefore, assayed Pepstatin A, Lopinavir and Ritonavir in parallel to Compound 1 obtaining for these molecules IC50s in the range of 9–40 μM (Fig 2 panel a). WEHI916 is another inhibitor of Plasmepsin V, published by Prof A. Cowman’s group [19, 20] while our work was in preparation. Therefore, we also compared side-by-side, Compound 1 and WEHI916, in order to test the IC50-relative differences in the same assay conditions. In our assay format, WEHI916 yielded IC50 of 32.43 (±1.43) nM, close to the published value of 19–20 nM [19, 20], against a confirmed picomolar inhibitory efficiency of Compound 1 (Fig 3). As discussed below, both Compound 1 and WEHI916 are transition-state inhibitors, but with different scaffolds, the first being based on a HEA group, the second on a statine.


Picomolar Inhibition of Plasmepsin V, an Essential Malaria Protease, Achieved Exploiting the Prime Region.

Gambini L, Rizzi L, Pedretti A, Taglialatela-Scafati O, Carucci M, Pancotti A, Galli C, Read M, Giurisato E, Romeo S, Russo I - PLoS ONE (2015)

PmV inhibitory activity of the new compounds.IC50 values, obtained in the presence of 3 μM of HRPII-PExEl substrate, are shown. They are averages of not less than three independent inhibition curves. Standard errors of the calculation of the IC50s by sigmoidal fitting are shown in column ‘±’. HEA indicates the hydroxyethylamino group that links P1, the non-amino acidic leucine analog, (3S)-3-amino-2-hydroxy-5-methylhexyl- group, and P1', the alanine. Sta is statine, an atypical amino acid found in the natural product Pepstatin A. Panel a Activity comparison of the newly generated inhibitor 1 with previously tested compounds [14, 15]. Panel b Activity of two generated diastereoisomers presented in Fig 4. Chemical structures of HIV inhibitors and absolute chirality of Leu-HEA epimers is included. Chemical structures of Lopinavir and Ritonavir are included in panel a; and stereoisomers of Leu-HEA-Ala are shown in panel b.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4643876&req=5

pone.0142509.g002: PmV inhibitory activity of the new compounds.IC50 values, obtained in the presence of 3 μM of HRPII-PExEl substrate, are shown. They are averages of not less than three independent inhibition curves. Standard errors of the calculation of the IC50s by sigmoidal fitting are shown in column ‘±’. HEA indicates the hydroxyethylamino group that links P1, the non-amino acidic leucine analog, (3S)-3-amino-2-hydroxy-5-methylhexyl- group, and P1', the alanine. Sta is statine, an atypical amino acid found in the natural product Pepstatin A. Panel a Activity comparison of the newly generated inhibitor 1 with previously tested compounds [14, 15]. Panel b Activity of two generated diastereoisomers presented in Fig 4. Chemical structures of HIV inhibitors and absolute chirality of Leu-HEA epimers is included. Chemical structures of Lopinavir and Ritonavir are included in panel a; and stereoisomers of Leu-HEA-Ala are shown in panel b.
Mentions: Inhibitors of Pepstatin A and HIV-protease have been previously reported as inhibitors of PmV activity [14, 15]. We, therefore, assayed Pepstatin A, Lopinavir and Ritonavir in parallel to Compound 1 obtaining for these molecules IC50s in the range of 9–40 μM (Fig 2 panel a). WEHI916 is another inhibitor of Plasmepsin V, published by Prof A. Cowman’s group [19, 20] while our work was in preparation. Therefore, we also compared side-by-side, Compound 1 and WEHI916, in order to test the IC50-relative differences in the same assay conditions. In our assay format, WEHI916 yielded IC50 of 32.43 (±1.43) nM, close to the published value of 19–20 nM [19, 20], against a confirmed picomolar inhibitory efficiency of Compound 1 (Fig 3). As discussed below, both Compound 1 and WEHI916 are transition-state inhibitors, but with different scaffolds, the first being based on a HEA group, the second on a statine.

Bottom Line: It results in an annual death-toll of ~ 600,000.Our work disclosed novel pursuable drug design strategies for highly efficient PmV inhibition highlighting novel molecular elements necessary for picomolar activity against PmV.All the presented data are discussed in respect to human aspartic proteases and previously reported inhibitors, highlighting differences and proposing new strategies for drug development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Università degli Studi di Milano, Milan, Italy.

ABSTRACT
Malaria is an infectious disease caused by Plasmodium parasites. It results in an annual death-toll of ~ 600,000. Resistance to all medications currently in use exists, and novel antimalarial drugs are urgently needed. Plasmepsin V (PmV) is an essential Plasmodium protease and a highly promising antimalarial target, which still lacks molecular characterization and drug-like inhibitors. PmV, cleaving the PExEl motif, is the key enzyme for PExEl-secretion, an indispensable parasitic process for virulence and infection. Here, we describe the accessibility of PmV catalytic pockets to inhibitors and propose a novel strategy for PmV inhibition. We also provide molecular and structural data suitable for future drug development. Using high-throughput platforms, we identified a novel scaffold that interferes with PmV in-vitro at picomolar ranges (~ 1,000-fold more active than available compounds). Via systematic replacement of P and P' regions, we assayed the physico-chemical requirements for PmV inhibition, achieving an unprecedented IC50 of ~20 pM. The hydroxyethylamine moiety, the hydrogen acceptor group in P2', the lipophilic groups upstream to P3, the arginine and other possible substitutions in position P3 proved to be critically important elements in achieving potent inhibition. In-silico analyses provided essential QSAR information and model validation. Our inhibitors act 'on-target', confirmed by cellular interference of PmV function and biochemical interaction with inhibitors. Our inhibitors are poorly performing against parasite growth, possibly due to poor stability of their peptidic component and trans-membrane permeability. The lowest IC50 for parasite growth inhibition was ~ 15 μM. Analysis of inhibitor internalization revealed important pharmacokinetic features for PExEl-based molecules. Our work disclosed novel pursuable drug design strategies for highly efficient PmV inhibition highlighting novel molecular elements necessary for picomolar activity against PmV. All the presented data are discussed in respect to human aspartic proteases and previously reported inhibitors, highlighting differences and proposing new strategies for drug development.

Show MeSH
Related in: MedlinePlus