Limits...
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.

Show MeSH

Related in: MedlinePlus

PmV inhibitory activity of the generated compounds (2nd set).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. The 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. Relevant modifications follow: Panel a, Shorter molecules; b, modifications of the N-terminus. The dash at a given position indicates absence of the amino acidic residue in that position. Amino acids are three-letter and colour coded but ‘[Leu-HEA-…]’ corresponding to the (3S)-3-amino-2-hydroxy-5-methylhexyl- group, as follows: hydrophobic aa are in shades of yellow; aromatic aa, orange; positively charged aa, purple; polar aa, blue; and acidic aa, grey. Two GABA units were used as linker for the Biotinylated Compound 36. NS = Not significant; ND = not determined; NA = not applicable.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4643876&req=5

pone.0142509.g006: PmV inhibitory activity of the generated compounds (2nd set).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. The 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. Relevant modifications follow: Panel a, Shorter molecules; b, modifications of the N-terminus. The dash at a given position indicates absence of the amino acidic residue in that position. Amino acids are three-letter and colour coded but ‘[Leu-HEA-…]’ corresponding to the (3S)-3-amino-2-hydroxy-5-methylhexyl- group, as follows: hydrophobic aa are in shades of yellow; aromatic aa, orange; positively charged aa, purple; polar aa, blue; and acidic aa, grey. Two GABA units were used as linker for the Biotinylated Compound 36. NS = Not significant; ND = not determined; NA = not applicable.

Mentions: Bioinformatics analysis of the PExEl motifs in P. falciparum shows high variability for the P' region [7]. A region expected to be less determinant for enzyme affinity. Therefore, we decided to progressively trim this region in order to minimize the dimensions of our molecules and to test the importance of this region for inhibition. We first studied the importance of the alanine in P3' within compounds containing either alanine or tyrosine in P1'. In both cases, when P3' alanine was removed approximately the same activity was detected, as shown by Compound 22 compared to 1, 2a to 7a and 26a to 9a (Fig 6 panela). More pronounced increases in IC50 (2.5 and 8 fold) were observed in Compounds 23 and 29 when compared to their closest cognate molecules 8a and 13a. However, we consider these differences to be of lesser significance, as the comparison is between pure ‘a’ diastereoisomers and racemic mixtures. The amidation of the new C-terminus does not substantially change the activity (24 compared to 23).


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 generated compounds (2nd set).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. The 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. Relevant modifications follow: Panel a, Shorter molecules; b, modifications of the N-terminus. The dash at a given position indicates absence of the amino acidic residue in that position. Amino acids are three-letter and colour coded but ‘[Leu-HEA-…]’ corresponding to the (3S)-3-amino-2-hydroxy-5-methylhexyl- group, as follows: hydrophobic aa are in shades of yellow; aromatic aa, orange; positively charged aa, purple; polar aa, blue; and acidic aa, grey. Two GABA units were used as linker for the Biotinylated Compound 36. NS = Not significant; ND = not determined; NA = not applicable.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142509.g006: PmV inhibitory activity of the generated compounds (2nd set).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. The 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. Relevant modifications follow: Panel a, Shorter molecules; b, modifications of the N-terminus. The dash at a given position indicates absence of the amino acidic residue in that position. Amino acids are three-letter and colour coded but ‘[Leu-HEA-…]’ corresponding to the (3S)-3-amino-2-hydroxy-5-methylhexyl- group, as follows: hydrophobic aa are in shades of yellow; aromatic aa, orange; positively charged aa, purple; polar aa, blue; and acidic aa, grey. Two GABA units were used as linker for the Biotinylated Compound 36. NS = Not significant; ND = not determined; NA = not applicable.
Mentions: Bioinformatics analysis of the PExEl motifs in P. falciparum shows high variability for the P' region [7]. A region expected to be less determinant for enzyme affinity. Therefore, we decided to progressively trim this region in order to minimize the dimensions of our molecules and to test the importance of this region for inhibition. We first studied the importance of the alanine in P3' within compounds containing either alanine or tyrosine in P1'. In both cases, when P3' alanine was removed approximately the same activity was detected, as shown by Compound 22 compared to 1, 2a to 7a and 26a to 9a (Fig 6 panela). More pronounced increases in IC50 (2.5 and 8 fold) were observed in Compounds 23 and 29 when compared to their closest cognate molecules 8a and 13a. However, we consider these differences to be of lesser significance, as the comparison is between pure ‘a’ diastereoisomers and racemic mixtures. The amidation of the new C-terminus does not substantially change the activity (24 compared to 23).

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