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

Inhibition correlates with cellular PmV levels.(A) Western blot analysis of cellular levels of PmV in the parental line, 3D7, and clonal parasites generated by genetic modifications. Immuno-detection of expressed PmV (upper panel) and BiP (lower panel), used as a loading control on the same SDS-PAGE gel, are shown. DC6 clone expresses PmV-GFP chimera under regulation of the native promoter [15]; G6 clone, the PmV-GFP-DD chimera under regulation of the native promoter (this paper) and #3 clone (episomally expressing PmV-GFP chimera under Hsp86-5', a strong and constitutive promoter) [15]. G6 clone is maintained in absence of Shield-1. (B) Growth inhibition curves of parasites expressing different levels of PmV: 3D7 (blue dots and line), DC6 (green dots and line), G6 (PmV knock-down) (red dots and line) and #3 (Overexpressing PmV) (purple dots and line) were analysed in parallel. Growth inhibition was measured in triplicates. Dots represent actual data, lines the sigmoidal fitting obtained by non-linear regression analysis. (C) Shifts of inhibition curves detected by comparison of parasites expressing different levels of PmV. The sensitivities to Compound 29 (bars with solid fill) and Chloroquine (CQ—bars with meshed fill) of 3D7, DC6, G6 (PmV knock-down) and #3 (Overexpressing PmV) were analysed in parallel. CQ sensitivity for #3 is was not determined. Comparisons of IC20, IC50 and IC80 values are shown in Table 1 and derived from curves shown in B. 3D7 and DC6 cultures expressed similar levels of Plasmepsin V; while G6 in the absence of Shield-1 showed decreased, and #3 showed augmented, levels of the cellular enzyme concentration. No significant shift were detected with CQ. Error bars show the standard deviation of the data.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142509.g015: Inhibition correlates with cellular PmV levels.(A) Western blot analysis of cellular levels of PmV in the parental line, 3D7, and clonal parasites generated by genetic modifications. Immuno-detection of expressed PmV (upper panel) and BiP (lower panel), used as a loading control on the same SDS-PAGE gel, are shown. DC6 clone expresses PmV-GFP chimera under regulation of the native promoter [15]; G6 clone, the PmV-GFP-DD chimera under regulation of the native promoter (this paper) and #3 clone (episomally expressing PmV-GFP chimera under Hsp86-5', a strong and constitutive promoter) [15]. G6 clone is maintained in absence of Shield-1. (B) Growth inhibition curves of parasites expressing different levels of PmV: 3D7 (blue dots and line), DC6 (green dots and line), G6 (PmV knock-down) (red dots and line) and #3 (Overexpressing PmV) (purple dots and line) were analysed in parallel. Growth inhibition was measured in triplicates. Dots represent actual data, lines the sigmoidal fitting obtained by non-linear regression analysis. (C) Shifts of inhibition curves detected by comparison of parasites expressing different levels of PmV. The sensitivities to Compound 29 (bars with solid fill) and Chloroquine (CQ—bars with meshed fill) of 3D7, DC6, G6 (PmV knock-down) and #3 (Overexpressing PmV) were analysed in parallel. CQ sensitivity for #3 is was not determined. Comparisons of IC20, IC50 and IC80 values are shown in Table 1 and derived from curves shown in B. 3D7 and DC6 cultures expressed similar levels of Plasmepsin V; while G6 in the absence of Shield-1 showed decreased, and #3 showed augmented, levels of the cellular enzyme concentration. No significant shift were detected with CQ. Error bars show the standard deviation of the data.

Mentions: A platform of cellular assays was set up in order to test, in vivo, the direct perturbation of both PmV activity and PExEl-secretion, using genetically modified parasites. Four different clones, all deriving from the parental strain 3D7, were used. These clones were: (a) HRPII-GFP, expressing a fluorescent probe for PExEl secretion under control of its native promoter [15]; and three clones producing different levels of PmV: (b) clone #3, expressing PmV-GFP, whose transcription is guided by a strong and constitutive promoter, Hsp86-5' (clone) [15]; (c) clone DC6, expressing PmV-GFP [15] and (d) clone G6 (this work) expressing PmV-GFP-DD, the latter two being under the control of the native PmV promoter. PmV-GFP-DD clone was generated by 3’ integration at the PmV locus of a plasmid carrying DNA encoding a GFP and a destabilization domain (DD) in frame to the PmV, as previously described [15, 23, 24], in order to generate an inducible knock-down (Fig 14a and 14b). The ‘destabilization domain’ (DD) derives from a FK506-binding, protein destabilization domain, which allows the regulation of protein levels [24, 28, 46]. PmV-GFP-DD clone G6 was genotyped by Southern Blot (clone number 3 in Fig 14b). The levels of PmV-knockdown were analysed in comparison to the episomal expression of a cytosolic YFP-DD and the effect on DC6 of the stabilizing drug Shield-1 by western blot (Fig 14d); live microscopy (Fig 14c and 14e); and flow cytometry (Fig 14g). PmV-GFP-DD yielded a reliable ~4–10 fold knockdown of PmV cellular levels (Figs 14f, 14g and 15a). In the absence of Shield-1, PmV-GFP-DD shows a GFP signal mostly localized in the food vacuole (Fig 14c and 14e) and a faint PmV-positive fragment at a molecular weight of about 60 kDa, that is possibly the result of a partial degradation (Fig 14d). The reduction of PmV we obtained does not seem to affect parasite viability to a detectable degree (Fig 14h), as previously observed using an alternative knock-down strategy [19]. We obtained for DC6 and G6, over a long course of growth in absence of Shield-1, growth constants (k) respectively, of 0.028 and 0.0274; doubling times (T) of 24.76 h and 25.3 h and a growth rate of (r) of 2.84 h-1 and 2.78 h-1 (derived from exponential fitting in Fig 14h). In the presence of 0.75 μM Shield-1, for DC6 and G6 the respective k values are 0.0199 and 0.0203; T values are 34.83 h and 34.15 h; and r values are 2.01 h-1 and 2.05 h-1 (Fig 14h).


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)

Inhibition correlates with cellular PmV levels.(A) Western blot analysis of cellular levels of PmV in the parental line, 3D7, and clonal parasites generated by genetic modifications. Immuno-detection of expressed PmV (upper panel) and BiP (lower panel), used as a loading control on the same SDS-PAGE gel, are shown. DC6 clone expresses PmV-GFP chimera under regulation of the native promoter [15]; G6 clone, the PmV-GFP-DD chimera under regulation of the native promoter (this paper) and #3 clone (episomally expressing PmV-GFP chimera under Hsp86-5', a strong and constitutive promoter) [15]. G6 clone is maintained in absence of Shield-1. (B) Growth inhibition curves of parasites expressing different levels of PmV: 3D7 (blue dots and line), DC6 (green dots and line), G6 (PmV knock-down) (red dots and line) and #3 (Overexpressing PmV) (purple dots and line) were analysed in parallel. Growth inhibition was measured in triplicates. Dots represent actual data, lines the sigmoidal fitting obtained by non-linear regression analysis. (C) Shifts of inhibition curves detected by comparison of parasites expressing different levels of PmV. The sensitivities to Compound 29 (bars with solid fill) and Chloroquine (CQ—bars with meshed fill) of 3D7, DC6, G6 (PmV knock-down) and #3 (Overexpressing PmV) were analysed in parallel. CQ sensitivity for #3 is was not determined. Comparisons of IC20, IC50 and IC80 values are shown in Table 1 and derived from curves shown in B. 3D7 and DC6 cultures expressed similar levels of Plasmepsin V; while G6 in the absence of Shield-1 showed decreased, and #3 showed augmented, levels of the cellular enzyme concentration. No significant shift were detected with CQ. Error bars show the standard deviation of the data.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142509.g015: Inhibition correlates with cellular PmV levels.(A) Western blot analysis of cellular levels of PmV in the parental line, 3D7, and clonal parasites generated by genetic modifications. Immuno-detection of expressed PmV (upper panel) and BiP (lower panel), used as a loading control on the same SDS-PAGE gel, are shown. DC6 clone expresses PmV-GFP chimera under regulation of the native promoter [15]; G6 clone, the PmV-GFP-DD chimera under regulation of the native promoter (this paper) and #3 clone (episomally expressing PmV-GFP chimera under Hsp86-5', a strong and constitutive promoter) [15]. G6 clone is maintained in absence of Shield-1. (B) Growth inhibition curves of parasites expressing different levels of PmV: 3D7 (blue dots and line), DC6 (green dots and line), G6 (PmV knock-down) (red dots and line) and #3 (Overexpressing PmV) (purple dots and line) were analysed in parallel. Growth inhibition was measured in triplicates. Dots represent actual data, lines the sigmoidal fitting obtained by non-linear regression analysis. (C) Shifts of inhibition curves detected by comparison of parasites expressing different levels of PmV. The sensitivities to Compound 29 (bars with solid fill) and Chloroquine (CQ—bars with meshed fill) of 3D7, DC6, G6 (PmV knock-down) and #3 (Overexpressing PmV) were analysed in parallel. CQ sensitivity for #3 is was not determined. Comparisons of IC20, IC50 and IC80 values are shown in Table 1 and derived from curves shown in B. 3D7 and DC6 cultures expressed similar levels of Plasmepsin V; while G6 in the absence of Shield-1 showed decreased, and #3 showed augmented, levels of the cellular enzyme concentration. No significant shift were detected with CQ. Error bars show the standard deviation of the data.
Mentions: A platform of cellular assays was set up in order to test, in vivo, the direct perturbation of both PmV activity and PExEl-secretion, using genetically modified parasites. Four different clones, all deriving from the parental strain 3D7, were used. These clones were: (a) HRPII-GFP, expressing a fluorescent probe for PExEl secretion under control of its native promoter [15]; and three clones producing different levels of PmV: (b) clone #3, expressing PmV-GFP, whose transcription is guided by a strong and constitutive promoter, Hsp86-5' (clone) [15]; (c) clone DC6, expressing PmV-GFP [15] and (d) clone G6 (this work) expressing PmV-GFP-DD, the latter two being under the control of the native PmV promoter. PmV-GFP-DD clone was generated by 3’ integration at the PmV locus of a plasmid carrying DNA encoding a GFP and a destabilization domain (DD) in frame to the PmV, as previously described [15, 23, 24], in order to generate an inducible knock-down (Fig 14a and 14b). The ‘destabilization domain’ (DD) derives from a FK506-binding, protein destabilization domain, which allows the regulation of protein levels [24, 28, 46]. PmV-GFP-DD clone G6 was genotyped by Southern Blot (clone number 3 in Fig 14b). The levels of PmV-knockdown were analysed in comparison to the episomal expression of a cytosolic YFP-DD and the effect on DC6 of the stabilizing drug Shield-1 by western blot (Fig 14d); live microscopy (Fig 14c and 14e); and flow cytometry (Fig 14g). PmV-GFP-DD yielded a reliable ~4–10 fold knockdown of PmV cellular levels (Figs 14f, 14g and 15a). In the absence of Shield-1, PmV-GFP-DD shows a GFP signal mostly localized in the food vacuole (Fig 14c and 14e) and a faint PmV-positive fragment at a molecular weight of about 60 kDa, that is possibly the result of a partial degradation (Fig 14d). The reduction of PmV we obtained does not seem to affect parasite viability to a detectable degree (Fig 14h), as previously observed using an alternative knock-down strategy [19]. We obtained for DC6 and G6, over a long course of growth in absence of Shield-1, growth constants (k) respectively, of 0.028 and 0.0274; doubling times (T) of 24.76 h and 25.3 h and a growth rate of (r) of 2.84 h-1 and 2.78 h-1 (derived from exponential fitting in Fig 14h). In the presence of 0.75 μM Shield-1, for DC6 and G6 the respective k values are 0.0199 and 0.0203; T values are 34.83 h and 34.15 h; and r values are 2.01 h-1 and 2.05 h-1 (Fig 14h).

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