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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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Growth inhibition.Growth inhibition curves of significant compounds are presented. Parasite cultures were exposed to titration curves of compounds for about 4 days. Parasitemia was evaluated via flow cytometry analysis using a nuclear staining (details in Material and Methods). Ellagic acid, known inhibitor of Plasmodium growth [45], was included as a positive control for the test. NS, Not significant.
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pone.0142509.g013: Growth inhibition.Growth inhibition curves of significant compounds are presented. Parasite cultures were exposed to titration curves of compounds for about 4 days. Parasitemia was evaluated via flow cytometry analysis using a nuclear staining (details in Material and Methods). Ellagic acid, known inhibitor of Plasmodium growth [45], was included as a positive control for the test. NS, Not significant.

Mentions: Despite evidence of cellular internalization, all the synthetized compounds resulted in poor inhibition of parasite growth. The majority of the compounds did not show significant inhibition within the tested range of concentrations, 0.195–200 μM. Some of them gave measurable growth inhibition LD50s ranging from 10 to 150 μM (Fig 13). In these cases the treated cultures appear to die at the trophozoite stage as has been observed with other PmV inhibitors [20]. This may be due to the fact that, once inside the cell, our peptide-based inhibitors are either degraded or mis-localized, and/or that their lipophilic profiles do not guarantee an efficient access to PmV in the endoplasmic reticulum lumen. In fact, despite the detection of the internalization of Compound 36 (Fig 12), the quantity and integrity of the internalized molecules could not be ascertained. The poor in vivo inhibition of the compounds did not allow a proper SAR. Interestingly, however, Compounds 8a and 15a, missing one of the two charges of Compound 1 (due respectively to the lack of the glutamate and the arginine), give measurable LD50s, while both their respective diastereoisomers, 8b and 15b, do not perform as well. Compound 35, carrying an acetyl group at the N-terminus, acts against parasite growth with a LD50 of 16.5 μM, while the best in vitro inhibitor of our series, Compound 37a (containing a more lipophilic N-terminus), up to 200 μM shows no significant activity against the parasites. More importantly, Compound 29 exerted a reproducible lethal effect at ~ 15 μM, one of the lowest effective concentrations. Compound 29, containing a primary amine instead of the guanidinium in P3, showed LogDpH7.4 = −5.99, a better lipophilic profile than Compound 1 (-4.28). Compound 29 was selected to carry out in vivo validation of PmV as target for PExEl-based inhibitors in the following experiments.


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)

Growth inhibition.Growth inhibition curves of significant compounds are presented. Parasite cultures were exposed to titration curves of compounds for about 4 days. Parasitemia was evaluated via flow cytometry analysis using a nuclear staining (details in Material and Methods). Ellagic acid, known inhibitor of Plasmodium growth [45], was included as a positive control for the test. NS, Not significant.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142509.g013: Growth inhibition.Growth inhibition curves of significant compounds are presented. Parasite cultures were exposed to titration curves of compounds for about 4 days. Parasitemia was evaluated via flow cytometry analysis using a nuclear staining (details in Material and Methods). Ellagic acid, known inhibitor of Plasmodium growth [45], was included as a positive control for the test. NS, Not significant.
Mentions: Despite evidence of cellular internalization, all the synthetized compounds resulted in poor inhibition of parasite growth. The majority of the compounds did not show significant inhibition within the tested range of concentrations, 0.195–200 μM. Some of them gave measurable growth inhibition LD50s ranging from 10 to 150 μM (Fig 13). In these cases the treated cultures appear to die at the trophozoite stage as has been observed with other PmV inhibitors [20]. This may be due to the fact that, once inside the cell, our peptide-based inhibitors are either degraded or mis-localized, and/or that their lipophilic profiles do not guarantee an efficient access to PmV in the endoplasmic reticulum lumen. In fact, despite the detection of the internalization of Compound 36 (Fig 12), the quantity and integrity of the internalized molecules could not be ascertained. The poor in vivo inhibition of the compounds did not allow a proper SAR. Interestingly, however, Compounds 8a and 15a, missing one of the two charges of Compound 1 (due respectively to the lack of the glutamate and the arginine), give measurable LD50s, while both their respective diastereoisomers, 8b and 15b, do not perform as well. Compound 35, carrying an acetyl group at the N-terminus, acts against parasite growth with a LD50 of 16.5 μM, while the best in vitro inhibitor of our series, Compound 37a (containing a more lipophilic N-terminus), up to 200 μM shows no significant activity against the parasites. More importantly, Compound 29 exerted a reproducible lethal effect at ~ 15 μM, one of the lowest effective concentrations. Compound 29, containing a primary amine instead of the guanidinium in P3, showed LogDpH7.4 = −5.99, a better lipophilic profile than Compound 1 (-4.28). Compound 29 was selected to carry out in vivo validation of PmV as target for PExEl-based inhibitors in the following experiments.

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