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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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Interaction between PmV and Compound 36.(A) PmV detection after pulldown with 36. Compound 36 bound to streptavidin-resin was incubated with cellular lysates of DC6, that expresses PMV-GFP. Immuno-detection of GFP is shown. Controls: total cellular extract, immuno-precipitated PmV and resin not incubated with DC6 extracts. In the presence of 36-streptavidin resin, PmV is pulled down, and this binding is inhibited by the presence of Compound 1. (B) Detection of Compound 36 in immuno-precipitated PmV. After incubation of cell lysate with 50 μM Compound 36 in the absence or presence of Compound 1 (6th and 7th dots, respectively), PmV-GFP was immuno-precipitated with anti-GFP antibody 3E6. Biotin positivity is detected only in samples containing PmV (as shown in panel B, lane 2), indicating interaction between PmV and 36. Similarly to panel B, 36-binding is inhibited in the presence of 1. Controls: 36 solution in presence and absence of DC6 cellular lysate (first two dots on the left), immuno-precipitation in absence of DC6 lysate (3rd and 4th dots) and immuno-precipitation in the absence of 36 (5th dot), are included.
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pone.0142509.g011: Interaction between PmV and Compound 36.(A) PmV detection after pulldown with 36. Compound 36 bound to streptavidin-resin was incubated with cellular lysates of DC6, that expresses PMV-GFP. Immuno-detection of GFP is shown. Controls: total cellular extract, immuno-precipitated PmV and resin not incubated with DC6 extracts. In the presence of 36-streptavidin resin, PmV is pulled down, and this binding is inhibited by the presence of Compound 1. (B) Detection of Compound 36 in immuno-precipitated PmV. After incubation of cell lysate with 50 μM Compound 36 in the absence or presence of Compound 1 (6th and 7th dots, respectively), PmV-GFP was immuno-precipitated with anti-GFP antibody 3E6. Biotin positivity is detected only in samples containing PmV (as shown in panel B, lane 2), indicating interaction between PmV and 36. Similarly to panel B, 36-binding is inhibited in the presence of 1. Controls: 36 solution in presence and absence of DC6 cellular lysate (first two dots on the left), immuno-precipitation in absence of DC6 lysate (3rd and 4th dots) and immuno-precipitation in the absence of 36 (5th dot), are included.

Mentions: In order to assess the cellular permeability of our molecules and their interaction with PmV, we created Compound 36, a biotinylated version of Compound 1, that showed an in vitro inhibitory activity of ~ 42 nM (Fig 6 panel b). Complementary pull-down experiments using crude parasite lysates of clone DC6 [15] (that constitutively expresses PmV-GFP) in the presence or absence of Compound 36, confirmed the interaction between our inhibitor and PmV (Fig 11a and 11b). Compound 36 was detected in association with immuno-precipitated PmV (Fig 11a). Conversely, PmV was detected after pull-down using Compound 36 bound to streptavidin-resin (Fig 11b). We also show that Compound 1, acting as competitor, negatively affects the interaction between Compound 36 and PmV (Fig 11a and 11b).


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)

Interaction between PmV and Compound 36.(A) PmV detection after pulldown with 36. Compound 36 bound to streptavidin-resin was incubated with cellular lysates of DC6, that expresses PMV-GFP. Immuno-detection of GFP is shown. Controls: total cellular extract, immuno-precipitated PmV and resin not incubated with DC6 extracts. In the presence of 36-streptavidin resin, PmV is pulled down, and this binding is inhibited by the presence of Compound 1. (B) Detection of Compound 36 in immuno-precipitated PmV. After incubation of cell lysate with 50 μM Compound 36 in the absence or presence of Compound 1 (6th and 7th dots, respectively), PmV-GFP was immuno-precipitated with anti-GFP antibody 3E6. Biotin positivity is detected only in samples containing PmV (as shown in panel B, lane 2), indicating interaction between PmV and 36. Similarly to panel B, 36-binding is inhibited in the presence of 1. Controls: 36 solution in presence and absence of DC6 cellular lysate (first two dots on the left), immuno-precipitation in absence of DC6 lysate (3rd and 4th dots) and immuno-precipitation in the absence of 36 (5th dot), are included.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142509.g011: Interaction between PmV and Compound 36.(A) PmV detection after pulldown with 36. Compound 36 bound to streptavidin-resin was incubated with cellular lysates of DC6, that expresses PMV-GFP. Immuno-detection of GFP is shown. Controls: total cellular extract, immuno-precipitated PmV and resin not incubated with DC6 extracts. In the presence of 36-streptavidin resin, PmV is pulled down, and this binding is inhibited by the presence of Compound 1. (B) Detection of Compound 36 in immuno-precipitated PmV. After incubation of cell lysate with 50 μM Compound 36 in the absence or presence of Compound 1 (6th and 7th dots, respectively), PmV-GFP was immuno-precipitated with anti-GFP antibody 3E6. Biotin positivity is detected only in samples containing PmV (as shown in panel B, lane 2), indicating interaction between PmV and 36. Similarly to panel B, 36-binding is inhibited in the presence of 1. Controls: 36 solution in presence and absence of DC6 cellular lysate (first two dots on the left), immuno-precipitation in absence of DC6 lysate (3rd and 4th dots) and immuno-precipitation in the absence of 36 (5th dot), are included.
Mentions: In order to assess the cellular permeability of our molecules and their interaction with PmV, we created Compound 36, a biotinylated version of Compound 1, that showed an in vitro inhibitory activity of ~ 42 nM (Fig 6 panel b). Complementary pull-down experiments using crude parasite lysates of clone DC6 [15] (that constitutively expresses PmV-GFP) in the presence or absence of Compound 36, confirmed the interaction between our inhibitor and PmV (Fig 11a and 11b). Compound 36 was detected in association with immuno-precipitated PmV (Fig 11a). Conversely, PmV was detected after pull-down using Compound 36 bound to streptavidin-resin (Fig 11b). We also show that Compound 1, acting as competitor, negatively affects the interaction between Compound 36 and PmV (Fig 11a and 11b).

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