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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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Plasmepsin V activity and inhibition.(A) Compound 1: chemical structure, amino acid composition and correspondence to the PExEl substrate P and P' positions. The PExEl motif is known as RxLx(x) E,Q,D [7] and is processed by PmV, downstream to the third leucine [14, 15, 40]. [Leu-HEA-Ala] indicates the presence of the group ((3S)-3-amino-2-hydroxy-5-methylhexyl)-L-alanine. This fully resembles the leucine and alanine in the third and fourth positions of the PExEl motif, except for the hydroxyethylamino group linking the two simil-amino acids in place of a peptidic bond. (B-E) Plasmepsin V activity and functional validation. (B) Cleavage of DABCYL-LNKRLLHETQ-EDANS, HRPII-derived fluorogenic substrate, by PmV at different concentrations of substrate (from 0.19 to 6 μM), to completion (means of triplicates are shown). Micheal-Menten analysis of initial velocities resulted in KM of 3.48 (±0.7) 10−6 M for DABCYL-LNKRLLHETQ-EDANS. (C) Activity (RFU, relative fluorescent units) was measured in a 96-well plate format for ~50 min after addition of fluorogenic substrate (final concentration ~3 μM). Lines represent linear regressions of data obtained in triplicates. Standard error bars are shown at 3 minute intervals. The black line corresponds to background fluorescence; dark blue, light blue, green, orange and red lines are the activity of PmV’s titration in the order from lowest to highest concentration, approximatively 2, 5, 10, 15, 20 pM; purple and pink lines represent the activity of 20 pM PmV against fluorogenic peptides containing critical mutations of the PExEl motif: L3 → A and R1 → A, respectively; the golden line shows inhibition of 20 pM PmV in the presence of our inhibitor 1 at ~2 μM. (D) Enzymatic parameters (KM and Vmax) were calculated via non-linear curve-fitting analysis taking into account the substrate concentrations and the initial velocities (v0). These were derived from activity curves as shown in (B), acquired in triplicates (as in Materials and Methods). Active enzyme concentration [E]t and, consequently, kcat (calculated) are only approximate values possibly affected by significant errors due to the limits of the applied quantization methodology for active PmV (densitometry of protein gels). Variations of these values in the range of 2–3 fold have been detected in diverse kinetic measurements. (E) A Lineweaver—Burke plot is shown derived from triplicates of PmV-cleavage HRPII-derived fluorogenic substrate, over time at different concentrations of substrate (from 0.19 to 6 μM). (F) PmV activity inhibited by Compound 1. On semi-logarithmic plot a typical titration curve obtained with serial dilutions of 1 is shown. Inhibitor IC50 was calculated by analysis of the sigmoidal fitting of the data. Error bars represent SEM of data.
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pone.0142509.g001: Plasmepsin V activity and inhibition.(A) Compound 1: chemical structure, amino acid composition and correspondence to the PExEl substrate P and P' positions. The PExEl motif is known as RxLx(x) E,Q,D [7] and is processed by PmV, downstream to the third leucine [14, 15, 40]. [Leu-HEA-Ala] indicates the presence of the group ((3S)-3-amino-2-hydroxy-5-methylhexyl)-L-alanine. This fully resembles the leucine and alanine in the third and fourth positions of the PExEl motif, except for the hydroxyethylamino group linking the two simil-amino acids in place of a peptidic bond. (B-E) Plasmepsin V activity and functional validation. (B) Cleavage of DABCYL-LNKRLLHETQ-EDANS, HRPII-derived fluorogenic substrate, by PmV at different concentrations of substrate (from 0.19 to 6 μM), to completion (means of triplicates are shown). Micheal-Menten analysis of initial velocities resulted in KM of 3.48 (±0.7) 10−6 M for DABCYL-LNKRLLHETQ-EDANS. (C) Activity (RFU, relative fluorescent units) was measured in a 96-well plate format for ~50 min after addition of fluorogenic substrate (final concentration ~3 μM). Lines represent linear regressions of data obtained in triplicates. Standard error bars are shown at 3 minute intervals. The black line corresponds to background fluorescence; dark blue, light blue, green, orange and red lines are the activity of PmV’s titration in the order from lowest to highest concentration, approximatively 2, 5, 10, 15, 20 pM; purple and pink lines represent the activity of 20 pM PmV against fluorogenic peptides containing critical mutations of the PExEl motif: L3 → A and R1 → A, respectively; the golden line shows inhibition of 20 pM PmV in the presence of our inhibitor 1 at ~2 μM. (D) Enzymatic parameters (KM and Vmax) were calculated via non-linear curve-fitting analysis taking into account the substrate concentrations and the initial velocities (v0). These were derived from activity curves as shown in (B), acquired in triplicates (as in Materials and Methods). Active enzyme concentration [E]t and, consequently, kcat (calculated) are only approximate values possibly affected by significant errors due to the limits of the applied quantization methodology for active PmV (densitometry of protein gels). Variations of these values in the range of 2–3 fold have been detected in diverse kinetic measurements. (E) A Lineweaver—Burke plot is shown derived from triplicates of PmV-cleavage HRPII-derived fluorogenic substrate, over time at different concentrations of substrate (from 0.19 to 6 μM). (F) PmV activity inhibited by Compound 1. On semi-logarithmic plot a typical titration curve obtained with serial dilutions of 1 is shown. Inhibitor IC50 was calculated by analysis of the sigmoidal fitting of the data. Error bars represent SEM of data.

Mentions: At the start of this work the available tridimensional structures of aspartic proteases were used to generate 3D-models of P. falciparum Plasmepsin V (gene code PF3D7_1323500, former identification, PF13_0133) via homology modelling platforms [31–33]. Models were constructed using both the full length P. falciparum Plasmepsin V (Pf_PmV) and just its catalytic domain (amino acids 81–500), giving similar results. Two elements were consistently found in all the models: a structural unpredictability of peculiar PmV subdomains that have no or very low homology to other known proteases [17], and a folding of the catalytic domain into two independent subunits. This type of folding is a common trait of aspartic proteases and forms the active site by juxta-positioning the two active aspartates (in Pf_PmV: Asp118, 365), that are contained in each of the subdomains. Despite the high confidence score of the predictions, PmV 3D-models proved to be highly heterogeneous, showing great diversity for the predicted catalytic grooves and no rational criteria for selecting a reference model were suitable (data not shown). In fact, due to the peculiarity of Pf_PmV, none of the analyzed proteases presented a significant similarity to Pf_PmV, yielding values for identity in the range of 18–29%, and homology from 7e-4 to 8e-8 [33]. Plasmodium PmV uniqueness was confirmed by the structural data of P. vivax PmV, a closest homolog of Pf_PmV, that was recently published while this paper was under revision [39]. As the structural uncertainty of the catalytic domain predictions was significant, in order to generate inhibitors we opted for designing inhibitors incorporating minimal modifications of the PmV natural substrate, the PExEl motif (Fig 1a). These molecules were then used to chemically scan PmV catalytic site accessibility and requirements for inhibition.


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)

Plasmepsin V activity and inhibition.(A) Compound 1: chemical structure, amino acid composition and correspondence to the PExEl substrate P and P' positions. The PExEl motif is known as RxLx(x) E,Q,D [7] and is processed by PmV, downstream to the third leucine [14, 15, 40]. [Leu-HEA-Ala] indicates the presence of the group ((3S)-3-amino-2-hydroxy-5-methylhexyl)-L-alanine. This fully resembles the leucine and alanine in the third and fourth positions of the PExEl motif, except for the hydroxyethylamino group linking the two simil-amino acids in place of a peptidic bond. (B-E) Plasmepsin V activity and functional validation. (B) Cleavage of DABCYL-LNKRLLHETQ-EDANS, HRPII-derived fluorogenic substrate, by PmV at different concentrations of substrate (from 0.19 to 6 μM), to completion (means of triplicates are shown). Micheal-Menten analysis of initial velocities resulted in KM of 3.48 (±0.7) 10−6 M for DABCYL-LNKRLLHETQ-EDANS. (C) Activity (RFU, relative fluorescent units) was measured in a 96-well plate format for ~50 min after addition of fluorogenic substrate (final concentration ~3 μM). Lines represent linear regressions of data obtained in triplicates. Standard error bars are shown at 3 minute intervals. The black line corresponds to background fluorescence; dark blue, light blue, green, orange and red lines are the activity of PmV’s titration in the order from lowest to highest concentration, approximatively 2, 5, 10, 15, 20 pM; purple and pink lines represent the activity of 20 pM PmV against fluorogenic peptides containing critical mutations of the PExEl motif: L3 → A and R1 → A, respectively; the golden line shows inhibition of 20 pM PmV in the presence of our inhibitor 1 at ~2 μM. (D) Enzymatic parameters (KM and Vmax) were calculated via non-linear curve-fitting analysis taking into account the substrate concentrations and the initial velocities (v0). These were derived from activity curves as shown in (B), acquired in triplicates (as in Materials and Methods). Active enzyme concentration [E]t and, consequently, kcat (calculated) are only approximate values possibly affected by significant errors due to the limits of the applied quantization methodology for active PmV (densitometry of protein gels). Variations of these values in the range of 2–3 fold have been detected in diverse kinetic measurements. (E) A Lineweaver—Burke plot is shown derived from triplicates of PmV-cleavage HRPII-derived fluorogenic substrate, over time at different concentrations of substrate (from 0.19 to 6 μM). (F) PmV activity inhibited by Compound 1. On semi-logarithmic plot a typical titration curve obtained with serial dilutions of 1 is shown. Inhibitor IC50 was calculated by analysis of the sigmoidal fitting of the data. Error bars represent SEM of data.
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Related In: Results  -  Collection

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

pone.0142509.g001: Plasmepsin V activity and inhibition.(A) Compound 1: chemical structure, amino acid composition and correspondence to the PExEl substrate P and P' positions. The PExEl motif is known as RxLx(x) E,Q,D [7] and is processed by PmV, downstream to the third leucine [14, 15, 40]. [Leu-HEA-Ala] indicates the presence of the group ((3S)-3-amino-2-hydroxy-5-methylhexyl)-L-alanine. This fully resembles the leucine and alanine in the third and fourth positions of the PExEl motif, except for the hydroxyethylamino group linking the two simil-amino acids in place of a peptidic bond. (B-E) Plasmepsin V activity and functional validation. (B) Cleavage of DABCYL-LNKRLLHETQ-EDANS, HRPII-derived fluorogenic substrate, by PmV at different concentrations of substrate (from 0.19 to 6 μM), to completion (means of triplicates are shown). Micheal-Menten analysis of initial velocities resulted in KM of 3.48 (±0.7) 10−6 M for DABCYL-LNKRLLHETQ-EDANS. (C) Activity (RFU, relative fluorescent units) was measured in a 96-well plate format for ~50 min after addition of fluorogenic substrate (final concentration ~3 μM). Lines represent linear regressions of data obtained in triplicates. Standard error bars are shown at 3 minute intervals. The black line corresponds to background fluorescence; dark blue, light blue, green, orange and red lines are the activity of PmV’s titration in the order from lowest to highest concentration, approximatively 2, 5, 10, 15, 20 pM; purple and pink lines represent the activity of 20 pM PmV against fluorogenic peptides containing critical mutations of the PExEl motif: L3 → A and R1 → A, respectively; the golden line shows inhibition of 20 pM PmV in the presence of our inhibitor 1 at ~2 μM. (D) Enzymatic parameters (KM and Vmax) were calculated via non-linear curve-fitting analysis taking into account the substrate concentrations and the initial velocities (v0). These were derived from activity curves as shown in (B), acquired in triplicates (as in Materials and Methods). Active enzyme concentration [E]t and, consequently, kcat (calculated) are only approximate values possibly affected by significant errors due to the limits of the applied quantization methodology for active PmV (densitometry of protein gels). Variations of these values in the range of 2–3 fold have been detected in diverse kinetic measurements. (E) A Lineweaver—Burke plot is shown derived from triplicates of PmV-cleavage HRPII-derived fluorogenic substrate, over time at different concentrations of substrate (from 0.19 to 6 μM). (F) PmV activity inhibited by Compound 1. On semi-logarithmic plot a typical titration curve obtained with serial dilutions of 1 is shown. Inhibitor IC50 was calculated by analysis of the sigmoidal fitting of the data. Error bars represent SEM of data.
Mentions: At the start of this work the available tridimensional structures of aspartic proteases were used to generate 3D-models of P. falciparum Plasmepsin V (gene code PF3D7_1323500, former identification, PF13_0133) via homology modelling platforms [31–33]. Models were constructed using both the full length P. falciparum Plasmepsin V (Pf_PmV) and just its catalytic domain (amino acids 81–500), giving similar results. Two elements were consistently found in all the models: a structural unpredictability of peculiar PmV subdomains that have no or very low homology to other known proteases [17], and a folding of the catalytic domain into two independent subunits. This type of folding is a common trait of aspartic proteases and forms the active site by juxta-positioning the two active aspartates (in Pf_PmV: Asp118, 365), that are contained in each of the subdomains. Despite the high confidence score of the predictions, PmV 3D-models proved to be highly heterogeneous, showing great diversity for the predicted catalytic grooves and no rational criteria for selecting a reference model were suitable (data not shown). In fact, due to the peculiarity of Pf_PmV, none of the analyzed proteases presented a significant similarity to Pf_PmV, yielding values for identity in the range of 18–29%, and homology from 7e-4 to 8e-8 [33]. Plasmodium PmV uniqueness was confirmed by the structural data of P. vivax PmV, a closest homolog of Pf_PmV, that was recently published while this paper was under revision [39]. As the structural uncertainty of the catalytic domain predictions was significant, in order to generate inhibitors we opted for designing inhibitors incorporating minimal modifications of the PmV natural substrate, the PExEl motif (Fig 1a). These molecules were then used to chemically scan PmV catalytic site accessibility and requirements for inhibition.

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