Falcipains as malarial drug targets
- Authors: Kanzi, Aquillah Mumo
- Date: 2013
- Subjects: Malaria Malaria -- Chemotherapy Plasmodium falciparum Antimalarials -- Development Cysteine proteinases Cysteine proteinases -- Inhibitors Papain Drug development Bioinformatics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3897 , http://hdl.handle.net/10962/d1003842
- Description: Malaria is an infectious disease caused by parasites of the Plasmodium genus with mortality rates of more than a million annually, hence a major global public health concern. Plasmodium falciparum (P. falciparum) accounts for over 90% of malaria incidence. Increased resistance to antimalarial drugs by the Plasmodium parasite, coupled with the lack of an effective malaria vaccine necessitates the urgent need for new research avenues to develop novel and more potent antimalarial drugs. This study focused on falcipains, a group of P. falciparum cysteine proteases that belong to the clan CA and papain family C1, that have emerged as potential drug targets due to their involvement in a range of crucial functions in the P. falciparum life cycle. Recently, falcipain-2 has been validated as a drug target but little is known of its Plasmodium orthologs. Currently, there are several falcipain inhibitors that have been identified, most of which are peptide based but none has proceeded to drug development due to associated poor pharmacological profiles and susceptibility to degradation by host cysteine proteases. Non-peptides inhibitors have been shown to be more stable in vivo but limited information exists. In vivo studies on falcipain-2 and falcipain-3 inhibitors have also been complicated by varying outcomes, thus a good understanding of the structural variations of falcipain Plasmodium orthologs at the active site could go a long way to ease in vivo results interpretation and effective inhibitor design. In this study, we use bioinformatics approaches to perform comparative sequence and structural analysis and molecular docking to characterize protein-inhibitor interactions of falcipain homologs at the active site. Known FP-2 and FP-3 small molecule nonpeptide inhibitors were used to identify residue variations and their effect on inhibitor binding. This was done with the aim of screening a collection of selected non-peptide compounds of South African natural origin to identify possible new inhibitor leads. Natural compounds with high binding affinities across all Plasmodium orthologs were identified. These compounds were then used to search the ZINC database for similar compounds which could have better binding affinities across all selected falcipain homologs. Compounds with high binding affinities across all Plasmodium orthologs were found.
- Full Text:
- Authors: Kanzi, Aquillah Mumo
- Date: 2013
- Subjects: Malaria Malaria -- Chemotherapy Plasmodium falciparum Antimalarials -- Development Cysteine proteinases Cysteine proteinases -- Inhibitors Papain Drug development Bioinformatics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3897 , http://hdl.handle.net/10962/d1003842
- Description: Malaria is an infectious disease caused by parasites of the Plasmodium genus with mortality rates of more than a million annually, hence a major global public health concern. Plasmodium falciparum (P. falciparum) accounts for over 90% of malaria incidence. Increased resistance to antimalarial drugs by the Plasmodium parasite, coupled with the lack of an effective malaria vaccine necessitates the urgent need for new research avenues to develop novel and more potent antimalarial drugs. This study focused on falcipains, a group of P. falciparum cysteine proteases that belong to the clan CA and papain family C1, that have emerged as potential drug targets due to their involvement in a range of crucial functions in the P. falciparum life cycle. Recently, falcipain-2 has been validated as a drug target but little is known of its Plasmodium orthologs. Currently, there are several falcipain inhibitors that have been identified, most of which are peptide based but none has proceeded to drug development due to associated poor pharmacological profiles and susceptibility to degradation by host cysteine proteases. Non-peptides inhibitors have been shown to be more stable in vivo but limited information exists. In vivo studies on falcipain-2 and falcipain-3 inhibitors have also been complicated by varying outcomes, thus a good understanding of the structural variations of falcipain Plasmodium orthologs at the active site could go a long way to ease in vivo results interpretation and effective inhibitor design. In this study, we use bioinformatics approaches to perform comparative sequence and structural analysis and molecular docking to characterize protein-inhibitor interactions of falcipain homologs at the active site. Known FP-2 and FP-3 small molecule nonpeptide inhibitors were used to identify residue variations and their effect on inhibitor binding. This was done with the aim of screening a collection of selected non-peptide compounds of South African natural origin to identify possible new inhibitor leads. Natural compounds with high binding affinities across all Plasmodium orthologs were identified. These compounds were then used to search the ZINC database for similar compounds which could have better binding affinities across all selected falcipain homologs. Compounds with high binding affinities across all Plasmodium orthologs were found.
- Full Text:
In-silico analysis of Plasmodium falciparum Hop protein and its interactions with Hsp70 and Hsp90
- Authors: Clitheroe, Crystal-Leigh
- Date: 2013
- Subjects: Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3896 , http://hdl.handle.net/10962/d1003819 , Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Description: A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.
- Full Text:
- Authors: Clitheroe, Crystal-Leigh
- Date: 2013
- Subjects: Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3896 , http://hdl.handle.net/10962/d1003819 , Plasmodium falciparum , Heat shock proteins , Molecular chaperones , Homology (Biology) , Protein-protein interactions , Malaria -- Chemotherapy
- Description: A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.
- Full Text:
Sequence and structural investigation of the nonribosomal peptide synthetases of Bacillus atrophaeus UCMB 5137(63Z)
- Authors: Ryan, Candice Nancy
- Date: 2013 , 2013-04-19
- Subjects: Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3891 , http://hdl.handle.net/10962/d1003057 , Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Description: Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Authors: Ryan, Candice Nancy
- Date: 2013 , 2013-04-19
- Subjects: Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3891 , http://hdl.handle.net/10962/d1003057 , Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Description: Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
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