Malarial drug targets cysteine proteases as hemoglobinases
- Authors: Mokoena, Fortunate
- Date: 2012
- Subjects: Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4005 , http://hdl.handle.net/10962/d1004065 , Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Description: Malaria has consistently been rated as the worst parasitic disease in the world. This disease affects an estimated 5 billion households annually. Malaria has a high mortality rate leading to distorted socio-economic development of the world at large. The major challenge pertaining to malaria is its continuous and rapid spread together with the emergence of drug resistance in Plasmodium species (vector agent of the disease). For this reason, researchers throughout the world are following new leads for possible drug targets and therefore, investigating ways of curbing the spread of the disease. Cysteine proteases have emerged as potential antimalarial chemotherapeutic targets. These particular proteases are found in all living organisms, Plasmodium cysteine proteases are known to degrade host hemoglobin during the life cycle of the parasite within the human host. The main objective of this study was to use various in silico methods to analyze the hemoglobinase function of cysteine proteases in P. falciparum and P. vivax. Falcipain-2 (FP2) of P. falciparum is the best characterized of these enzymes, it is a validated drug target. Both the three-dimensional structures of FP2 and its close homologue falcipain-3 (FP3) have been solved by the experimental technique X-ray crystallography. However, the homologue falcipain-2 (FP2’)’ and orthologues from P.vivax vivapain-2 (VP2) and vivapain-3 (VP3) have yet to be elucidated by experimental techniques. In an effort to achieve the principal goal of the study, homology models of the protein structures not already elucidated by experimental methods (FP2’, VP2 and VP3) were calculated using the well known spatial restraint program MODELLER. The derived models, FP2 and FP3 were docked to hemoglobin (their natural substrate). The protein-protein docking was done using the unbound docking program ZDOCK. The substrate-enzyme interactions were analyzed and amino acids involved in binding were observed. It is anticipated that the results obtained from the study will help focus inhibitor design for potential drugs against malaria. The residues found in both the P. falciparum and P. vivax cysteine proteases involved in hemoglobin binding have been identified and some of these are proposed to be the main focus for the design of a peptidomimetric inhibitor.
- Full Text:
- Date Issued: 2012
- Authors: Mokoena, Fortunate
- Date: 2012
- Subjects: Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4005 , http://hdl.handle.net/10962/d1004065 , Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Description: Malaria has consistently been rated as the worst parasitic disease in the world. This disease affects an estimated 5 billion households annually. Malaria has a high mortality rate leading to distorted socio-economic development of the world at large. The major challenge pertaining to malaria is its continuous and rapid spread together with the emergence of drug resistance in Plasmodium species (vector agent of the disease). For this reason, researchers throughout the world are following new leads for possible drug targets and therefore, investigating ways of curbing the spread of the disease. Cysteine proteases have emerged as potential antimalarial chemotherapeutic targets. These particular proteases are found in all living organisms, Plasmodium cysteine proteases are known to degrade host hemoglobin during the life cycle of the parasite within the human host. The main objective of this study was to use various in silico methods to analyze the hemoglobinase function of cysteine proteases in P. falciparum and P. vivax. Falcipain-2 (FP2) of P. falciparum is the best characterized of these enzymes, it is a validated drug target. Both the three-dimensional structures of FP2 and its close homologue falcipain-3 (FP3) have been solved by the experimental technique X-ray crystallography. However, the homologue falcipain-2 (FP2’)’ and orthologues from P.vivax vivapain-2 (VP2) and vivapain-3 (VP3) have yet to be elucidated by experimental techniques. In an effort to achieve the principal goal of the study, homology models of the protein structures not already elucidated by experimental methods (FP2’, VP2 and VP3) were calculated using the well known spatial restraint program MODELLER. The derived models, FP2 and FP3 were docked to hemoglobin (their natural substrate). The protein-protein docking was done using the unbound docking program ZDOCK. The substrate-enzyme interactions were analyzed and amino acids involved in binding were observed. It is anticipated that the results obtained from the study will help focus inhibitor design for potential drugs against malaria. The residues found in both the P. falciparum and P. vivax cysteine proteases involved in hemoglobin binding have been identified and some of these are proposed to be the main focus for the design of a peptidomimetric inhibitor.
- Full Text:
- Date Issued: 2012
Structural analysis of prodomain inhibition of cysteine proteases in plasmodium species
- Authors: Njuguna, Joyce Njoki
- Date: 2012
- Subjects: Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4021 , http://hdl.handle.net/10962/d1004081 , Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Description: Plasmodium is a genus of parasites causing malaria, a virulent protozoan infection in humans resulting in over a million deaths annually. Treatment of malaria is increasingly limited by parasite resistance to available drugs. Hence, there is a need to identify new drug targets and authenticate antimalarial compounds that act on these targets. A relatively new therapeutic approach targets proteolytic enzymes responsible for parasite‟s invasion, rupture and hemoglobin degradation at the erythrocytic stage of infection. Cysteine proteases (CPs) are essential for these crucial roles in the intraerythrocytic parasite. CPs are a diverse group of enzymes subdivided into clans and further subdivided into families. Our interest is in Clan CA, papain family C1 proteases, whose members play numerous roles in human and parasitic metabolism. These proteases are produced as zymogens having an N-terminal extension known as the prodomain which regulates the protease activity by selectively inhibiting its active site, preventing substrate access. A Clan CA protease Falcipain-2 (FP-2) of Plasmodium falciparum is a validated drug target but little is known of its orthologs in other malarial Plasmodium species. This study uses various structural bioinformatics approaches to characterise the prodomain‟s regulatory effect in FP-2 and its orthologs in Plasmodium species (P. vivax, P. berghei, P. knowlesi, P. ovale, P. chabaudi and P. yoelii). This was in an effort to discover short peptides with essential residues to mimic the prodomain‟s inhibition of these proteases, as potential peptidomimetic therapeutic agents. Residues in the prodomain region that spans over the active site are most likely to interact with the subsite residues inhibiting the protease. Sequence analysis revealed conservation of residues in this region of Plasmodium proteases that differed significantly in human proteases. Further prediction of the 3D structure of these proteases by homology modelling allowed visualisation of these interactions revealing differences between parasite and human proteases which will lead to significant contribution in structure based malarial inhibitor design.
- Full Text:
- Date Issued: 2012
- Authors: Njuguna, Joyce Njoki
- Date: 2012
- Subjects: Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4021 , http://hdl.handle.net/10962/d1004081 , Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Description: Plasmodium is a genus of parasites causing malaria, a virulent protozoan infection in humans resulting in over a million deaths annually. Treatment of malaria is increasingly limited by parasite resistance to available drugs. Hence, there is a need to identify new drug targets and authenticate antimalarial compounds that act on these targets. A relatively new therapeutic approach targets proteolytic enzymes responsible for parasite‟s invasion, rupture and hemoglobin degradation at the erythrocytic stage of infection. Cysteine proteases (CPs) are essential for these crucial roles in the intraerythrocytic parasite. CPs are a diverse group of enzymes subdivided into clans and further subdivided into families. Our interest is in Clan CA, papain family C1 proteases, whose members play numerous roles in human and parasitic metabolism. These proteases are produced as zymogens having an N-terminal extension known as the prodomain which regulates the protease activity by selectively inhibiting its active site, preventing substrate access. A Clan CA protease Falcipain-2 (FP-2) of Plasmodium falciparum is a validated drug target but little is known of its orthologs in other malarial Plasmodium species. This study uses various structural bioinformatics approaches to characterise the prodomain‟s regulatory effect in FP-2 and its orthologs in Plasmodium species (P. vivax, P. berghei, P. knowlesi, P. ovale, P. chabaudi and P. yoelii). This was in an effort to discover short peptides with essential residues to mimic the prodomain‟s inhibition of these proteases, as potential peptidomimetic therapeutic agents. Residues in the prodomain region that spans over the active site are most likely to interact with the subsite residues inhibiting the protease. Sequence analysis revealed conservation of residues in this region of Plasmodium proteases that differed significantly in human proteases. Further prediction of the 3D structure of these proteases by homology modelling allowed visualisation of these interactions revealing differences between parasite and human proteases which will lead to significant contribution in structure based malarial inhibitor design.
- Full Text:
- Date Issued: 2012
Marine anti-malarial isonitriles : a synthetic and computational study
- Authors: Adendorff, Matthew Ralph
- Date: 2011 , 2010-05-17
- Subjects: Isocyanides , Isocyanates , Marine pharmacology , Antimalarials , Antimalarials -- Development , Drug development
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4398 , http://hdl.handle.net/10962/d1006674 , Isocyanides , Isocyanates , Marine pharmacology , Antimalarials , Antimalarials -- Development , Drug development
- Description: The development of Plasmodium falciparum malarial resistance to the current armoury of anti-malarial drugs requires the development of new treatments to help combat this disease. The marine environment is a well established source of potential pharmaceuticals. Of interest to us are isonitrile, isocyanate and isothiocyanate compounds isolated from marine sponges and molluscs which have exhibited nano-molar anti-plasmodial activities. Through quantitative structure-activity relation studies (QSAR), a literature precedent exists for a pseudoreceptor model from which a pharmacophore for the design of novel anti-malarial agents was proposed. The current theory suggests that these marine compounds exert their inhibitory action through interfering with the heme detoxification pathway in P. falciparum. We propose that the computational methods used to draw detailed conclusions about the mode of action of these marine compounds were inadequate. This thesis addresses this problem using contemporary computational methodologies and seeks to propose a more robust method for the rational design of new anti-malarial drug compounds that inhibit heme polymerization to hemozoin. In order to investigate the interactions of the marine compounds with their heme targets, a series of modern computational procedures were formulated, validated and then applied to theoretical systems. The validations of these algorithms, before their application to the marine compound-heme systems, were achieved through two case studies. The first was used to investigate the applicability of the statistical docking algorithm AutoDock to be used for the exploration of conformational space around the heme target. A theoretical P. falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) enzyme model, constructed by the Biochemistry Department at Rhodes University, provided the ideal model to validate the AutoDock program. The protein model was accordingly subjected to rigorous docking simulations with over 30 different ligand molecules using the AutoDock algorithm which allowed for the docking algorithm’s limitations to be ascertained and improved upon. This investigation facilitated the successful validation of the protein model, which can now be used for the rational design of new PfDXR-inhibiting anti-plasmodial compounds, as well as enabling us to propose an improvement of the docking algorithm for application to the heme systems. The second case study was used to investigate the applicability of an ab initio molecular dynamics algorithm for simulation of bond breaking/forming events between the marine compounds and their heme target. This validation involved the exploration of intermolecular interactions in a naturally occurring nonoligomeric zipper using the Car-Parrinello Molecular Dynamics (CPMD) method. This study allowed us to propose a model for the intermolecular forces responsible for zipper self-assembly and showcased the CPMD method’s abilities to simulate and predict bond forming/breaking events. Data from the computational analyses suggested that the interactions between marine isonitriles, isocyanates and isothiocyanates occur through bond-less electrostatic attractions rather than through formal intermolecular bonds as had been previously suggested. Accordingly, a simple bicyclic tertiary isonitrile (5.14) was synthesized using Kitano et al’s relatively underutilized isonitrile synthetic method for the conversion of tertiary alcohols to their corresponding isonitriles. This compound’s potential for heme detoxification inhibition was then explored in vitro via the pyridine-hemochrome assay. The assay data suggested that the synthesized isonitrile was capable of inhibiting heme polymerization in a similar fashion to the known inhibitor chloroquine. Attempts to synthesize tricyclic analogues of 5.14 were unsuccessful and highlighted the limitation of Kitano et al’s isonitrile synthetic methodology.
- Full Text:
- Date Issued: 2011
- Authors: Adendorff, Matthew Ralph
- Date: 2011 , 2010-05-17
- Subjects: Isocyanides , Isocyanates , Marine pharmacology , Antimalarials , Antimalarials -- Development , Drug development
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4398 , http://hdl.handle.net/10962/d1006674 , Isocyanides , Isocyanates , Marine pharmacology , Antimalarials , Antimalarials -- Development , Drug development
- Description: The development of Plasmodium falciparum malarial resistance to the current armoury of anti-malarial drugs requires the development of new treatments to help combat this disease. The marine environment is a well established source of potential pharmaceuticals. Of interest to us are isonitrile, isocyanate and isothiocyanate compounds isolated from marine sponges and molluscs which have exhibited nano-molar anti-plasmodial activities. Through quantitative structure-activity relation studies (QSAR), a literature precedent exists for a pseudoreceptor model from which a pharmacophore for the design of novel anti-malarial agents was proposed. The current theory suggests that these marine compounds exert their inhibitory action through interfering with the heme detoxification pathway in P. falciparum. We propose that the computational methods used to draw detailed conclusions about the mode of action of these marine compounds were inadequate. This thesis addresses this problem using contemporary computational methodologies and seeks to propose a more robust method for the rational design of new anti-malarial drug compounds that inhibit heme polymerization to hemozoin. In order to investigate the interactions of the marine compounds with their heme targets, a series of modern computational procedures were formulated, validated and then applied to theoretical systems. The validations of these algorithms, before their application to the marine compound-heme systems, were achieved through two case studies. The first was used to investigate the applicability of the statistical docking algorithm AutoDock to be used for the exploration of conformational space around the heme target. A theoretical P. falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) enzyme model, constructed by the Biochemistry Department at Rhodes University, provided the ideal model to validate the AutoDock program. The protein model was accordingly subjected to rigorous docking simulations with over 30 different ligand molecules using the AutoDock algorithm which allowed for the docking algorithm’s limitations to be ascertained and improved upon. This investigation facilitated the successful validation of the protein model, which can now be used for the rational design of new PfDXR-inhibiting anti-plasmodial compounds, as well as enabling us to propose an improvement of the docking algorithm for application to the heme systems. The second case study was used to investigate the applicability of an ab initio molecular dynamics algorithm for simulation of bond breaking/forming events between the marine compounds and their heme target. This validation involved the exploration of intermolecular interactions in a naturally occurring nonoligomeric zipper using the Car-Parrinello Molecular Dynamics (CPMD) method. This study allowed us to propose a model for the intermolecular forces responsible for zipper self-assembly and showcased the CPMD method’s abilities to simulate and predict bond forming/breaking events. Data from the computational analyses suggested that the interactions between marine isonitriles, isocyanates and isothiocyanates occur through bond-less electrostatic attractions rather than through formal intermolecular bonds as had been previously suggested. Accordingly, a simple bicyclic tertiary isonitrile (5.14) was synthesized using Kitano et al’s relatively underutilized isonitrile synthetic method for the conversion of tertiary alcohols to their corresponding isonitriles. This compound’s potential for heme detoxification inhibition was then explored in vitro via the pyridine-hemochrome assay. The assay data suggested that the synthesized isonitrile was capable of inhibiting heme polymerization in a similar fashion to the known inhibitor chloroquine. Attempts to synthesize tricyclic analogues of 5.14 were unsuccessful and highlighted the limitation of Kitano et al’s isonitrile synthetic methodology.
- Full Text:
- Date Issued: 2011
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