Fighting against malaria is a never-ending battle. Plasmodium parasites continuously develop resistance to the drugs used against them including the artemisinin-based combination therapies as observed recently in Southeast Asia. The main concern now is whether the resistant parasite strains spread to Africa, where most malaria cases are located. To prevent this, we need to think outside the box. To date, there is no allosteric drug for malaria. Hence, allosteric drug targeting sites and modulators might be a new hope for malarial treatment. In Plasmodium falciparum two cysteine proteases, falcipain-2 (FP-2) and falcipain-3 (FP-3), have been identified as the main hemoglobinases, and are considered as attractive drug targets

Bishop, Ozlem Tastan

Musyoka, Thommas

Tastan Bishop, Özlem

South East Academic Libraries System (SEALS)

Thommas M. Musyoka and Özlem Tastan Bishop  Research Unit in Bioinformatics (RUBi), Department of Biochemistry and Microbiology  Rhodes University, Grahamstown, 6140, South Africa E-mail: o.tastanbishop@ru.ac.za Allosteric site modulators:  A case study for falcipains as malarial drug targets Fighting against malaria is a never-ending battle. Plasmodium parasites continuously develop resistance to the drugs used against them including the artemisinin-based combination therapies as observed recently in Southeast Asia.  The main concern now is whether the resistant parasite strains spread to Africa, where most malaria cases are located. To prevent this, we need to think outside the box. To date, there is no allosteric drug for malaria. Hence, allosteric drug targeting sites and modulators might be a new hope for malarial treatment.  In Plasmodium falciparum two cysteine proteases, falcipain-2 (FP-2) and falcipain-3 (FP-3), have been identified as the main hemoglobinases, and are considered as attractive drug targets1. A diverse set of active site inhibitors against these proteases has been identified but are yet to be approved as antimalarial drugs despite their remarkable inhibitory potency of up to nanomolar level2. A key concern is the conserved nature (residue composition and catalytic mechanism) of the active sites between these proteins and human cathepsins leading to non-selectivity. To overcome these challenges, the current work aims to identify and characterize allosteric sites unique to the plasmodial proteases as well as to establish a library of small molecules that can allosterically modulate the catalytic activity of these proteases. In this way, the study provides a platform for the development of a novel class of antimalarial drugs with high specificity and reduced toxicity profiles. Introduction Methodology Results and Discussion Conclusion and Ongoing Work • Several pockets which could potentially be targeted as allosteric sites were identified. • On-going work focuses on the establishment of a chemical library of potential allosteric inhibitors against the plasmodial proteases using high throughput structure based virtual screening approaches to evaluate public natural compound databases as well as synthetic libraries in order to identify hits.  • Ligand based virtual screening approaches are also being implemented using the identified hits to retrieve their analogs with better interaction profiles.  • Ultimately, laboratory based experiments using recombinant enzymes and whole parasites assays to determine the mode of inhibition as well as their antimalarial potency will be undertaken. REFERENCES: (1) Deu, E. Proteases as antimalarial targets: strategies for genetic, chemical, and therapeutic validation. FEBS J. 2017, 284, 2604–2628. (2) Rosenthal, P. J. Falcipains and other cysteine proteases of malaria parasites. Adv. Exp. Med. Biol. 2011, 712, 30–48. (3) Marques, A. F.; Esser, D.; Rosenthal, P. J.; Kassack, M. U.; Lima, L. M. T. R. Falcipain-2 inhibition by suramin and suramin analogues. Bioorg. Med. Chem. 2013, 21, 3667–3673. (4) Novinec M, Korenč M, Caflisch A, Ranganathan R, Lenarčič B, Baici A. A novel allosteric mechanism in the cysteine peptidase cathepsin K discovered by computational methods. Nat Commun. 2014, 5, 3287. Fig 1:  Workflow of the approaches and tools used in the identification of potential allosteric modulators against FP-2 and FP-3. Fig 2:  Binding pocket identification on falcipains and human cathepsins surface. Potential ligand binding pockets identified by SiteMap and FTMAP on A) falcipains and B) human cathepsins. Blind docking results showing the clustering of SANCDB (South African Natural Compounds Database) ligands on the different cavities (C and D) including the active site (red line). Fig 5:  Effect of ligand binding on the communication flow as depicted by ΔBC in A) FP2 and B) FP-3 using the ligand free structures as the reference. Despite the ligands binding on distant sites away from the active pocket, a large population of residues showing significant ΔBC are located in the central α-helix which forms the floor of the active site. This may lead to rearrangement of active site residues which may lead to altered enzyme activity. Fig 3:  Binding pose, residue interaction fingerprint and binding energy of selected compounds on the identified pockets in FP-2 and FP-3. Fig 4:  Global and local residue dynamic stability profiles based on A) RMSD  and B) RMSF for ligand free vs protein complexes and the corresponding ligands during 100 ns MD simulations. Selected ligand-protein complexes exhibited stability throughout the molecular dynamics simulations. • Besides the main active site of plasmodial and human proteases, other distinct pockets were identified.  • Several compounds uniquely binding to the identified pockets in FP-2 and FP-3 were identified by high throughput docking of 628 compounds from SANCDB (https://sancdb.rubi.ru.ac.za/).  • Two previous studies in FP-2 and human Cat-K have identified allosteric inhibitors: Suramin (site 1) and NSC13345 (site 2) respectively3,4.  

Allosteric site modulators: a case study for falcipains as malarial drug targets

Rhodes Repository (SEALS)

Thommas M. Musyoka and Özlem Tastan Bishop  
Research Unit in Bioinformatics (RUBi), Department of Biochemistry and Microbiology 
 Rhodes University, Grahamstown, 6140, South Africa 
E-mail: o.tastanbishop@ru.ac.za 
Allosteric site modulators:  
A case study for falcipains as malarial drug targets 
Fighting against malaria is a never-ending battle. Plasmodium parasites continuously develop resistance to 
the drugs used against them including the artemisinin-based combination therapies as observed recently in 
Southeast Asia.  The main concern now is whether the resistant parasite strains spread to Africa, where 
most malaria cases are located. To prevent this, we need to think outside the box. To date, there is no 
allosteric drug for malaria. Hence, allosteric drug targeting sites and modulators might be a new hope for 
malarial treatment.  
In Plasmodium falciparum two cysteine proteases, falcipain-2 (FP-2) and falcipain-3 (FP-3), have been 
identified as the main hemoglobinases, and are considered as attractive drug targets1. A diverse set of 
active site inhibitors against these proteases has been identified but are yet to be approved as antimalarial 
drugs despite their remarkable inhibitory potency of up to nanomolar level2. A key concern is the 
conserved nature (residue composition and catalytic mechanism) of the active sites between these 
proteins and human cathepsins leading to non-selectivity. To overcome these challenges, the current work 
aims to identify and characterize allosteric sites unique to the plasmodial proteases as well as to establish a 
library of small molecules that can allosterically modulate the catalytic activity of these proteases. In this 
way, the study provides a platform for the development of a novel class of antimalarial drugs with high 
specificity and reduced toxicity profiles. 
Introduction 
Methodology 
Results and Discussion 
Conclusion and Ongoing Work 
• Several pockets which could potentially be targeted as allosteric sites were identified. 
• On-going work focuses on the establishment of a chemical library of potential allosteric inhibitors 
against the plasmodial proteases using high throughput structure based virtual screening approaches to 
evaluate public natural compound databases as well as synthetic libraries in order to identify hits.  
• Ligand based virtual screening approaches are also being implemented using the identified hits to 
retrieve their analogs with better interaction profiles.  
• Ultimately, laboratory based experiments using recombinant enzymes and whole parasites assays to 
determine the mode of inhibition as well as their antimalarial potency will be undertaken. 
REFERENCES: 
(1) Deu, E. Proteases as antimalarial targets: strategies for genetic, chemical, and therapeutic validation. FEBS J. 2017, 284, 2604–2628. 
(2) Rosenthal, P. J. Falcipains and other cysteine proteases of malaria parasites. Adv. Exp. Med. Biol. 2011, 712, 30–48. 
(3) Marques, A. F.; Esser, D.; Rosenthal, P. J.; Kassack, M. U.; Lima, L. M. T. R. Falcipain-2 inhibition by suramin and suramin analogues. Bioorg. Med. Chem. 2013, 21, 3667–3673. 
(4) Novinec M, Korenč M, Caflisch A, Ranganathan R, Lenarčič B, Baici A. A novel allosteric mechanism in the cysteine peptidase cathepsin K discovered by computational methods. Nat Commun. 2014, 5, 3287. 
Fig 1:  Workflow of the approaches and tools used in the identification of potential allosteric modulators against FP-2 and FP-3. 
Fig 2:  Binding pocket identification on falcipains and human cathepsins surface. Potential ligand binding pockets identified by 
SiteMap and FTMAP on A) falcipains and B) human cathepsins. Blind docking results showing the clustering of SANCDB (South African 
Natural Compounds Database) ligands on the different cavities (C and D) including the active site (red line). 
Fig 5:  Effect of ligand binding on the communication flow as depicted by ΔBC in A) FP2 and B) FP-3 using the ligand free structures 
as the reference. Despite the ligands binding on distant sites away from the active pocket, a large population of residues showing 
significant ΔBC are located in the central α-helix which forms the floor of the active site. This may lead to rearrangement of active 
site residues which may lead to altered enzyme activity. 
Fig 3:  Binding pose, residue interaction fingerprint and binding energy of selected compounds on the identified pockets in FP-2 
and FP-3. 
Fig 4:  Global and local residue dynamic stability profiles based on A) RMSD  and B) RMSF for ligand free vs protein complexes and 
the corresponding ligands during 100 ns MD simulations. Selected ligand-protein complexes exhibited stability throughout the 
molecular dynamics simulations. 
• Besides the main active site of plasmodial and human proteases, other distinct pockets were identified.  
• Several compounds uniquely binding to the identified pockets in FP-2 and FP-3 were identified by high 
throughput docking of 628 compounds from SANCDB (https://sancdb.rubi.ru.ac.za/).  
• Two previous studies in FP-2 and human Cat-K have identified allosteric inhibitors: Suramin (site 1) and 
NSC13345 (site 2) respectively3,4.  


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Allosteric site modulators: a case study for falcipains as malarial drug targets

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