Evaluation of an NADPH-dependent assay for inhibition screening of Salmonella enterica DOXP Reguctoisomerase for identification of novel drug hit compounds
- Authors: Ngcongco, Khanyisile
- Date: 2020
- Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167132 , vital:41440
- Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
- Full Text:
- Authors: Ngcongco, Khanyisile
- Date: 2020
- Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167132 , vital:41440
- Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
- Full Text:
Exploring Quinolinyl-Thiazolidinedione hybrid compounds as potential anti-tubercular agents
- Authors: Mtshare, Thanduxolo Elihle
- Date: 2020
- Subjects: Quinoline , Mycobacterium tuberculosis , Tuberculosis -- Chemotherapy , Plasmodium falciparum , Thiazolidinedione
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/143314 , vital:38232
- Description: Tuberculosis (TB) is an infectious disease caused by the pathogen, Mycobacterium tuberculosis. According to the World Health Organization, TB is the ninth leading cause of death worldwide ranking above HIV/AIDS. This high mortality rate of TB begs the questions about the efficiency of the current therapy and raises an urgent need to create novel anti-tuberculosis agents which will aid in curbing this burden. Quinoline containing compounds have remarkable biological activities across a wide spectrum of diseases including anti-tuberculosis. On the other hand, thiazolidinedione containing compounds possess a broad spectrum of biological properties. In this study, we rationally designed compounds containing these pharmacophoric units and investigated them for their potential biological activity against Mycobacterium tuberculosis. Considering antimalarial activity of quinoline-based compounds, the compounds achieved were also cross-screened for their activity against the Plasmodium falciparum parasite, a causative agent of malaria. In all the synthesized compounds, compound 2.6a, 2.6b and 2.7b emerged as most active compounds against the H37Rv strain with MIC₉₀ values ranging in between of 1.08 – 17.1 μM. In addition, none of the compounds showed any inhibitory activities against the 3D7 strain of P. falciparum parasite. All the compounds prepared in this study showed no significant human cytotoxic effects as measured by HeLa cell line.
- Full Text:
- Authors: Mtshare, Thanduxolo Elihle
- Date: 2020
- Subjects: Quinoline , Mycobacterium tuberculosis , Tuberculosis -- Chemotherapy , Plasmodium falciparum , Thiazolidinedione
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/143314 , vital:38232
- Description: Tuberculosis (TB) is an infectious disease caused by the pathogen, Mycobacterium tuberculosis. According to the World Health Organization, TB is the ninth leading cause of death worldwide ranking above HIV/AIDS. This high mortality rate of TB begs the questions about the efficiency of the current therapy and raises an urgent need to create novel anti-tuberculosis agents which will aid in curbing this burden. Quinoline containing compounds have remarkable biological activities across a wide spectrum of diseases including anti-tuberculosis. On the other hand, thiazolidinedione containing compounds possess a broad spectrum of biological properties. In this study, we rationally designed compounds containing these pharmacophoric units and investigated them for their potential biological activity against Mycobacterium tuberculosis. Considering antimalarial activity of quinoline-based compounds, the compounds achieved were also cross-screened for their activity against the Plasmodium falciparum parasite, a causative agent of malaria. In all the synthesized compounds, compound 2.6a, 2.6b and 2.7b emerged as most active compounds against the H37Rv strain with MIC₉₀ values ranging in between of 1.08 – 17.1 μM. In addition, none of the compounds showed any inhibitory activities against the 3D7 strain of P. falciparum parasite. All the compounds prepared in this study showed no significant human cytotoxic effects as measured by HeLa cell line.
- Full Text:
Investigating assay formats for screening malaria Hsp90-Hop interaction inhibitors
- Authors: Derry, Leigh-Anne Tracy Kim
- Date: 2019
- Subjects: Antimalarials , Heat shock proteins , Drug interactions , Drug resistance , Plasmodium falciparum , High throughput screening (Drug development) , Bioluminescence resonance energy transfer (BRET) , Fluorescence resonance energy transfer (FRET)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63345 , vital:28395
- Description: Although significant gains have been made in the combat against malaria in the last decade, the persistent threat of drug and insecticide resistance continues to motivate the search for new classes of antimalarial drug compounds and targets. Due to their predominance in cellular reactions, protein-protein interactions (P-PIs) are emerging as a promising general target class for therapeutic development. The P-PI which is the focus of this project is the interaction between the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Hsp70/Hsp90 organising protein (Hop). Hop binds to Hsp70 and Hsp90 and facilitates the transfer of client proteins (proteins undergoing folding) from the former to the latter and also regulates nucleotide exchange on Hsp90. Due to its role in correcting protein misfolding during cell stress, Hsp90 is being pursued as a cancer drug target and compounds that inhibit its ATPase activity have entered clinical trials. However, it has been proposed that inhibiting the interaction between Hsp90 and Hop may be alternative approach for inhibiting Hsp90 function for cancer therapy. The malaria parasite Plasmodium falciparum experiences temperature fluctuations during vector-host transitions and febrile episodes and cell stress due to rapid growth and immune responses. Hence, it also depends on chaperones, including PfHsp90, to maintain protein functionality and pathogenesis, demonstrated inter alia by the sensitivity of parasites to Hsp90 inhibitors. In addition, PfHsp90 exists as a complex with the malarial Hop homologue, PfHop, in parasite lysates. Consequently, the purpose of this study was to explore P-PI assay formats that can confirm the interaction of PfHsp90 and PfHop and can be used to identify inhibitors of the interaction, preferably in a medium- to high-throughput screening mode. As a first approach, cell-based bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays were performed in HeLa cells. To facilitate this, expression plasmid constructs containing coding sequences of P. falciparum and mammalian Hsp90 and Hop and their interacting domains (Hsp90 C-domain and Hop TPR2A domain) fused to the BRET and FRET reporter proteins – yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and Renilla luciferase (Rluc) - were prepared and used for HeLa cell transient transfections. The FRET assay produced positive interaction signals for the full-length P. falciparum and mammalian Hsp90-Hop interactions. However, C-domain-TPR2A domain interactions were not detected, no interactions could be demonstrated with the BRET assay and western blotting experiments failed to detect expression of all the interaction partners in transiently transfected HeLa cells. Consequently, an alternative in vitro FRET assay format using recombinant proteins was investigated. Expression constructs for the P. falciparum and mammalian C-domains and TPR2A domains fused respectively to YFP and CFP were prepared and the corresponding fusion proteins expressed and purified from E. coli. No interaction was found with the mammalian interaction partners, but interaction of the P. falciparum C-domain and TPR2A domain was consistently detected with a robust Z’ factor value of 0.54. A peptide corresponding to the PfTPR2A domain sequence primarily responsible for Hsp90 binding (based on a human TPR2A peptide described by Horibe et al., 2011) was designed and showed dose-dependent inhibition of the interaction, with 53.7% inhibition at 100 μM. The components of the assay are limited to the purified recombinant proteins, requires minimal liquid steps and may thus be a useful primary screening format for identifying inhibitors of P. falciparum Hsp90-Hop interaction.
- Full Text:
- Authors: Derry, Leigh-Anne Tracy Kim
- Date: 2019
- Subjects: Antimalarials , Heat shock proteins , Drug interactions , Drug resistance , Plasmodium falciparum , High throughput screening (Drug development) , Bioluminescence resonance energy transfer (BRET) , Fluorescence resonance energy transfer (FRET)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63345 , vital:28395
- Description: Although significant gains have been made in the combat against malaria in the last decade, the persistent threat of drug and insecticide resistance continues to motivate the search for new classes of antimalarial drug compounds and targets. Due to their predominance in cellular reactions, protein-protein interactions (P-PIs) are emerging as a promising general target class for therapeutic development. The P-PI which is the focus of this project is the interaction between the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Hsp70/Hsp90 organising protein (Hop). Hop binds to Hsp70 and Hsp90 and facilitates the transfer of client proteins (proteins undergoing folding) from the former to the latter and also regulates nucleotide exchange on Hsp90. Due to its role in correcting protein misfolding during cell stress, Hsp90 is being pursued as a cancer drug target and compounds that inhibit its ATPase activity have entered clinical trials. However, it has been proposed that inhibiting the interaction between Hsp90 and Hop may be alternative approach for inhibiting Hsp90 function for cancer therapy. The malaria parasite Plasmodium falciparum experiences temperature fluctuations during vector-host transitions and febrile episodes and cell stress due to rapid growth and immune responses. Hence, it also depends on chaperones, including PfHsp90, to maintain protein functionality and pathogenesis, demonstrated inter alia by the sensitivity of parasites to Hsp90 inhibitors. In addition, PfHsp90 exists as a complex with the malarial Hop homologue, PfHop, in parasite lysates. Consequently, the purpose of this study was to explore P-PI assay formats that can confirm the interaction of PfHsp90 and PfHop and can be used to identify inhibitors of the interaction, preferably in a medium- to high-throughput screening mode. As a first approach, cell-based bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays were performed in HeLa cells. To facilitate this, expression plasmid constructs containing coding sequences of P. falciparum and mammalian Hsp90 and Hop and their interacting domains (Hsp90 C-domain and Hop TPR2A domain) fused to the BRET and FRET reporter proteins – yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and Renilla luciferase (Rluc) - were prepared and used for HeLa cell transient transfections. The FRET assay produced positive interaction signals for the full-length P. falciparum and mammalian Hsp90-Hop interactions. However, C-domain-TPR2A domain interactions were not detected, no interactions could be demonstrated with the BRET assay and western blotting experiments failed to detect expression of all the interaction partners in transiently transfected HeLa cells. Consequently, an alternative in vitro FRET assay format using recombinant proteins was investigated. Expression constructs for the P. falciparum and mammalian C-domains and TPR2A domains fused respectively to YFP and CFP were prepared and the corresponding fusion proteins expressed and purified from E. coli. No interaction was found with the mammalian interaction partners, but interaction of the P. falciparum C-domain and TPR2A domain was consistently detected with a robust Z’ factor value of 0.54. A peptide corresponding to the PfTPR2A domain sequence primarily responsible for Hsp90 binding (based on a human TPR2A peptide described by Horibe et al., 2011) was designed and showed dose-dependent inhibition of the interaction, with 53.7% inhibition at 100 μM. The components of the assay are limited to the purified recombinant proteins, requires minimal liquid steps and may thus be a useful primary screening format for identifying inhibitors of P. falciparum Hsp90-Hop interaction.
- Full Text:
Development and optimisation of a novel Plasmodium falciparum Hsp90-Hop interaction assay
- Authors: Wambua, Lynn
- Date: 2018
- Subjects: Plasmodium falciparum , Molecular chaperones , Heat shock proteins , Protein-protein interactions , Antimalarials
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62626 , vital:28216
- Description: Protein-protein interactions are involved in a range of disease processes and thus have become the focus of many drug discovery programs. Widespread drug resistance to all currently used antimalarial drugs drives the search for alternative drug targets with novel mechanisms of action that offer new therapeutic options. Molecular chaperones such as heat shock proteins facilitate protein folding, play a role in protein trafficking and prevent protein misfolding in cells under stress. Heat shock protein 90 (Hsp90) is a well-studied chaperone that has been the focus of cancer drug development with moderate success. In Plasmodium falciparum (P. falciparum), heat shock proteins are thought to play a vital role in parasite survival of the physiologically diverse habitats of the parasite lifecycle and because Hsp90 is prominently expressed in P. falciparum, the chaperone is considered a potentially ideal drug target. Hsp90 function in cells is regulated by interactions with co-chaperones, which includes Heat shock protein 70-Heat shock protein 90 organising protein (Hop). As opposed to directly inhibiting Hsp90 activity, targeting Hsp90 interaction with Hop has recently been suggested as an alternative method of Hsp90 inhibition that has not been explored in P. falciparum. The aim of this research project was to demonstrate PfHsp90 and PfHop robustly interact in vitro and to facilitate high-throughput screening of PfHsp90-PfHop inhibitors by developing and optimising a novel plate capture Hsp90-Hop interaction assay. To establish the assay, the respective domains of the proteins that mediate Hsp90-Hop interaction were used (Hsp90 C- terminal domain and Hop TPR2A domain). The human Hsp90 C-terminal domain and glutathione-S-transferase (GST) coding sequences were cloned into pET-28a(+) and murine and P. falciparum TPR2A sequences into pGEX-4T-1 plasmids to enable expression of histidine-tagged and GST fusion proteins, respectively, in Escherichia coli. The P. falciparum Hsp90 C-terminal domain sequence cloned into pET-28a(+) was supplied by GenScript. The constructs were transformed into T7 Express lysYcompetent E. coli cells and subsequent small- scale expression studies showed the recombinant proteins were expressed in a soluble form allowing for subsequent protein purification. Purification of the recombinant proteins was achieved using nickel-NTA and glutathione affinity chromatography for the His-tagged (Hsp90 C-terminal domains and GST) and GST fusion proteins (TPR2A domains), respectively. The purified proteins were used to establish and optimise mammalian and P. falciparum Hsp90- Hop interaction assays on nickel-coated plates by immobilising the His-tagged C-terminal domains on the plates and detecting the binding of the GST-TPR2A domains using a colorimetric GST enzyme assay. Z’-factor values above 0.5 were observed for both assays indicating good separation between the protein interaction signals and negative control background signals, although relatively high background signals were observed for the mammalian interaction due to non-specific binding of murine TPR2A to the plate. Designed human and P. falciparum TPR peptides were observed to be effective inhibitors of the mammalian and P. falciparum interactions, demonstrating the assay’s ability to respond to inhibitor compounds. Comparison of assay performance using GST assay kit reagents and lab- prepared reagents showed the assay was more efficient using lab-prepared reagents, however, lower GST signals were observed when comparing assay performance using a custom prepared Ni-NTA plate to a purchased Ni-NTA plate. The Hsp90-Hop interaction assays were also performed using an alternative assay format in which the GST-TPR2A fusion proteins were immobilised on glutathione-coated plates and binding of the His-tagged C-terminal domains detected with a nickel-horseradish peroxidase (HRP) conjugate and a colorimetric HRP substrate. The assay showed higher interaction signals for the P. falciparum proteins but comparatively low signals for the mammalian proteins. Z’-factor values for the assay were above 0.8 for both protein sets, suggesting this assay format is superior to the GST assay. However, further optimisation of this assay format is required. This study demonstrated direct binding of PfHsp90-PfHop in vitro and established a novel and robust PfHsp90-PfHop interaction assay format that can be used in future screening campaigns.
- Full Text:
- Authors: Wambua, Lynn
- Date: 2018
- Subjects: Plasmodium falciparum , Molecular chaperones , Heat shock proteins , Protein-protein interactions , Antimalarials
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62626 , vital:28216
- Description: Protein-protein interactions are involved in a range of disease processes and thus have become the focus of many drug discovery programs. Widespread drug resistance to all currently used antimalarial drugs drives the search for alternative drug targets with novel mechanisms of action that offer new therapeutic options. Molecular chaperones such as heat shock proteins facilitate protein folding, play a role in protein trafficking and prevent protein misfolding in cells under stress. Heat shock protein 90 (Hsp90) is a well-studied chaperone that has been the focus of cancer drug development with moderate success. In Plasmodium falciparum (P. falciparum), heat shock proteins are thought to play a vital role in parasite survival of the physiologically diverse habitats of the parasite lifecycle and because Hsp90 is prominently expressed in P. falciparum, the chaperone is considered a potentially ideal drug target. Hsp90 function in cells is regulated by interactions with co-chaperones, which includes Heat shock protein 70-Heat shock protein 90 organising protein (Hop). As opposed to directly inhibiting Hsp90 activity, targeting Hsp90 interaction with Hop has recently been suggested as an alternative method of Hsp90 inhibition that has not been explored in P. falciparum. The aim of this research project was to demonstrate PfHsp90 and PfHop robustly interact in vitro and to facilitate high-throughput screening of PfHsp90-PfHop inhibitors by developing and optimising a novel plate capture Hsp90-Hop interaction assay. To establish the assay, the respective domains of the proteins that mediate Hsp90-Hop interaction were used (Hsp90 C- terminal domain and Hop TPR2A domain). The human Hsp90 C-terminal domain and glutathione-S-transferase (GST) coding sequences were cloned into pET-28a(+) and murine and P. falciparum TPR2A sequences into pGEX-4T-1 plasmids to enable expression of histidine-tagged and GST fusion proteins, respectively, in Escherichia coli. The P. falciparum Hsp90 C-terminal domain sequence cloned into pET-28a(+) was supplied by GenScript. The constructs were transformed into T7 Express lysYcompetent E. coli cells and subsequent small- scale expression studies showed the recombinant proteins were expressed in a soluble form allowing for subsequent protein purification. Purification of the recombinant proteins was achieved using nickel-NTA and glutathione affinity chromatography for the His-tagged (Hsp90 C-terminal domains and GST) and GST fusion proteins (TPR2A domains), respectively. The purified proteins were used to establish and optimise mammalian and P. falciparum Hsp90- Hop interaction assays on nickel-coated plates by immobilising the His-tagged C-terminal domains on the plates and detecting the binding of the GST-TPR2A domains using a colorimetric GST enzyme assay. Z’-factor values above 0.5 were observed for both assays indicating good separation between the protein interaction signals and negative control background signals, although relatively high background signals were observed for the mammalian interaction due to non-specific binding of murine TPR2A to the plate. Designed human and P. falciparum TPR peptides were observed to be effective inhibitors of the mammalian and P. falciparum interactions, demonstrating the assay’s ability to respond to inhibitor compounds. Comparison of assay performance using GST assay kit reagents and lab- prepared reagents showed the assay was more efficient using lab-prepared reagents, however, lower GST signals were observed when comparing assay performance using a custom prepared Ni-NTA plate to a purchased Ni-NTA plate. The Hsp90-Hop interaction assays were also performed using an alternative assay format in which the GST-TPR2A fusion proteins were immobilised on glutathione-coated plates and binding of the His-tagged C-terminal domains detected with a nickel-horseradish peroxidase (HRP) conjugate and a colorimetric HRP substrate. The assay showed higher interaction signals for the P. falciparum proteins but comparatively low signals for the mammalian proteins. Z’-factor values for the assay were above 0.8 for both protein sets, suggesting this assay format is superior to the GST assay. However, further optimisation of this assay format is required. This study demonstrated direct binding of PfHsp90-PfHop in vitro and established a novel and robust PfHsp90-PfHop interaction assay format that can be used in future screening campaigns.
- Full Text:
Synthesis, characterisation and evaluation of novel ferrocene-thiazole derivatives as antiplasmodial agents
- Authors: Hakizimana, Emmanuel Victor
- Date: 2017
- Subjects: Plasmodium , Malaria -- Chemotherapy , Plasmodium falciparum , Plasmodium -- Inhibitors , Drug resistance in microorganisms , Thiaszoles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/5304 , vital:20807
- Description: Malaria is mosquito-transmitted disease which continues to pose threat to humanity, despite the efforts undertaken by the scientific community, government entities and international organizations. The major problem is that Plasmodium species have developed resistance against available drugs. In order to counter this problem, antimalarial drugs that are efficacious and with novel mode of action are of great necessity. Thiazole derivatives, in particular aminomethylthiazole analogues, have been shown to exhibit promising antimalarial activity against Plasmodium falciparum strains. Previous studies reported the hit compound MMV010539, which showed good antimalarial activity against both K1 (CQ and multidrug resistant strains) and NF54 (CQ sensitive strain). In this study, MMV010539 was deemed to be as an attractive compound to generate novel analogues by addition of ferrocenyl organometallic unit. The ferrocene based compounds have shown biological activity; and with ferroquine currently in clinical trials there has been increasing research into identifying new ferrocenyl-containing molecules as potential antimalarial agents. Herein, thiazole ferrocene based molecules 3.22a-e were synthesised in low to good yields. Their structural identities were confirmed using conventional spectroscopic techniques (¹H and ¹³C NMR, FT-IR spectroscopy and mass spectrometry). The cell cytotoxicity assay of all final compounds confirmed that all ferrocene-thiazole blends 3.22a-e were non-toxic against HeLa cell lines. However, the in vitro biological assay revealed that despite the absence of cell cytotoxicity these compounds poorly inhibited the growth of Plasmodium falciparum parasite. As the aim was to expand further the structure-activity relationship (SAR) of MMV010539, this study confirmed the previous findings that there is a limited structural modification that could be accommodated as indicated in Figure 3.3 (Panel C). Moreover, the combination of ferrocenyl moiety and various alkylamines resulted in compounds with poor antiplasmodial potency, further suggesting that the free amine (Panel A, Figure 3.3) is important for activity.
- Full Text:
- Authors: Hakizimana, Emmanuel Victor
- Date: 2017
- Subjects: Plasmodium , Malaria -- Chemotherapy , Plasmodium falciparum , Plasmodium -- Inhibitors , Drug resistance in microorganisms , Thiaszoles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/5304 , vital:20807
- Description: Malaria is mosquito-transmitted disease which continues to pose threat to humanity, despite the efforts undertaken by the scientific community, government entities and international organizations. The major problem is that Plasmodium species have developed resistance against available drugs. In order to counter this problem, antimalarial drugs that are efficacious and with novel mode of action are of great necessity. Thiazole derivatives, in particular aminomethylthiazole analogues, have been shown to exhibit promising antimalarial activity against Plasmodium falciparum strains. Previous studies reported the hit compound MMV010539, which showed good antimalarial activity against both K1 (CQ and multidrug resistant strains) and NF54 (CQ sensitive strain). In this study, MMV010539 was deemed to be as an attractive compound to generate novel analogues by addition of ferrocenyl organometallic unit. The ferrocene based compounds have shown biological activity; and with ferroquine currently in clinical trials there has been increasing research into identifying new ferrocenyl-containing molecules as potential antimalarial agents. Herein, thiazole ferrocene based molecules 3.22a-e were synthesised in low to good yields. Their structural identities were confirmed using conventional spectroscopic techniques (¹H and ¹³C NMR, FT-IR spectroscopy and mass spectrometry). The cell cytotoxicity assay of all final compounds confirmed that all ferrocene-thiazole blends 3.22a-e were non-toxic against HeLa cell lines. However, the in vitro biological assay revealed that despite the absence of cell cytotoxicity these compounds poorly inhibited the growth of Plasmodium falciparum parasite. As the aim was to expand further the structure-activity relationship (SAR) of MMV010539, this study confirmed the previous findings that there is a limited structural modification that could be accommodated as indicated in Figure 3.3 (Panel C). Moreover, the combination of ferrocenyl moiety and various alkylamines resulted in compounds with poor antiplasmodial potency, further suggesting that the free amine (Panel A, Figure 3.3) is important for activity.
- Full Text:
Synthesis, characterisation and evaluation of novel ferrocene-thiazole derivatives as antiplasmodial agents
- Authors: Hakizimana, Emmanuel Victor
- Date: 2017
- Subjects: Plasmodium , Malaria -- Chemotherapy , Plasmodium falciparum , Plasmodium -- Inhibitors , Drug resistance in microorganisms , Thiaszoles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/96068 , vital:31232
- Description: Malaria is mosquito-transmitted disease which continues to pose threat to humanity, despite the efforts undertaken by the scientific community, government entities and international organizations. The major problem is that Plasmodium species have developed resistance against available drugs. In order to counter this problem, antimalarial drugs that are efficacious and with novel mode of action are of great necessity. Thiazole derivatives, in particular aminomethylthiazole analogues, have been shown to exhibit promising antimalarial activity against Plasmodium falciparum strains. Previous studies reported the hit compound MMV010539, which showed good antimalarial activity against both K1 (CQ and multidrug resistant strains) and NF54 (CQ sensitive strain). In this study, MMV010539 was deemed to be as an attractive compound to generate novel analogues by addition of ferrocenyl organometallic unit. The ferrocene based compounds have shown biological activity; and with ferroquine currently in clinical trials there has been increasing research into identifying new ferrocenyl-containing molecules as potential antimalarial agents. Herein, thiazole ferrocene based molecules 3.22a-e were synthesised in low to good yields. Their structural identities were confirmed using conventional spectroscopic techniques (¹H and ¹³C NMR, FT-IR spectroscopy and mass spectrometry). The cell cytotoxicity assay of all final compounds confirmed that all ferrocene-thiazole blends 3.22a-e were non-toxic against HeLa cell lines. However, the in vitro biological assay revealed that despite the absence of cell cytotoxicity these compounds poorly inhibited the growth of Plasmodium falciparum parasite. As the aim was to expand further the structure-activity relationship (SAR) of MMV010539, this study confirmed the previous findings that there is a limited structural modification that could be accommodated as indicated in Figure 3.3 (Panel C). Moreover, the combination of ferrocenyl moiety and various alkylamines resulted in compounds with poor antiplasmodial potency, further suggesting that the free amine (Panel A, Figure 3.3) is important for activity.
- Full Text:
- Authors: Hakizimana, Emmanuel Victor
- Date: 2017
- Subjects: Plasmodium , Malaria -- Chemotherapy , Plasmodium falciparum , Plasmodium -- Inhibitors , Drug resistance in microorganisms , Thiaszoles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/96068 , vital:31232
- Description: Malaria is mosquito-transmitted disease which continues to pose threat to humanity, despite the efforts undertaken by the scientific community, government entities and international organizations. The major problem is that Plasmodium species have developed resistance against available drugs. In order to counter this problem, antimalarial drugs that are efficacious and with novel mode of action are of great necessity. Thiazole derivatives, in particular aminomethylthiazole analogues, have been shown to exhibit promising antimalarial activity against Plasmodium falciparum strains. Previous studies reported the hit compound MMV010539, which showed good antimalarial activity against both K1 (CQ and multidrug resistant strains) and NF54 (CQ sensitive strain). In this study, MMV010539 was deemed to be as an attractive compound to generate novel analogues by addition of ferrocenyl organometallic unit. The ferrocene based compounds have shown biological activity; and with ferroquine currently in clinical trials there has been increasing research into identifying new ferrocenyl-containing molecules as potential antimalarial agents. Herein, thiazole ferrocene based molecules 3.22a-e were synthesised in low to good yields. Their structural identities were confirmed using conventional spectroscopic techniques (¹H and ¹³C NMR, FT-IR spectroscopy and mass spectrometry). The cell cytotoxicity assay of all final compounds confirmed that all ferrocene-thiazole blends 3.22a-e were non-toxic against HeLa cell lines. However, the in vitro biological assay revealed that despite the absence of cell cytotoxicity these compounds poorly inhibited the growth of Plasmodium falciparum parasite. As the aim was to expand further the structure-activity relationship (SAR) of MMV010539, this study confirmed the previous findings that there is a limited structural modification that could be accommodated as indicated in Figure 3.3 (Panel C). Moreover, the combination of ferrocenyl moiety and various alkylamines resulted in compounds with poor antiplasmodial potency, further suggesting that the free amine (Panel A, Figure 3.3) is important for activity.
- Full Text:
Development of a high-throughput bioassay to determine the rate of antimalarial drug action using fluorescent vitality probes
- Authors: Laming, Dustin
- Date: 2016
- Subjects: Malaria -- Africa , Plasmodium falciparum , Drug development , Fluorescence
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64434 , vital:28542
- Description: Malaria is one of the most prevalent diseases in Africa and the Plasmodium falciparum species is widely accepted as the most virulent, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials there has been little progress towards a proven vaccine. Pending a long term solution, endemic countries rely heavily on the development of innovative drugs with acute efficacy coupled with rapids mode of action. Until recently the rate of drug action has been measured by light microscopic examination of parasite morphology using blood slides of drug treated parasite cultures at regular time intervals. This technique is tedious and, most importantly, subject to interpretation with regards to distinguishing between viable and comprised parasite cells, thus making it impossible to objectively quantitate the rate of drug action. This study aimed to develop a series of bioassays using the calcein-acetoxymethyl and propidium iodide vitality probes which would allow the rate of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other. A novel bioassay using these fluorescent vitality probes coupled with fluorescence microscopy was developed and optimized and allowed the rate of drug action on malaria parasites to be assessed i) rapidly (in relation to current assay techniques) and ii) in a semi-quantitative manner. Extrapolation to flow cytometry for improved quantification provided favourable rankings of drug killing rates in the pilot study, however, requires further development to increase throughput and approach the ultimate goal of producing a medium-throughput assay for rapidly assessing the rate of action of antimalarial drugs. Attempts to adapt the assay for use in a multiwell plate reader, as well as using ATP measurements as an indication of parasite vitality after drug treatment, was met with erratic results. The viability probes assay as it stands represents an improvement on other assay formats in terms of rapidity and quantification of live/compromised parasites in cultures.
- Full Text:
- Authors: Laming, Dustin
- Date: 2016
- Subjects: Malaria -- Africa , Plasmodium falciparum , Drug development , Fluorescence
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64434 , vital:28542
- Description: Malaria is one of the most prevalent diseases in Africa and the Plasmodium falciparum species is widely accepted as the most virulent, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials there has been little progress towards a proven vaccine. Pending a long term solution, endemic countries rely heavily on the development of innovative drugs with acute efficacy coupled with rapids mode of action. Until recently the rate of drug action has been measured by light microscopic examination of parasite morphology using blood slides of drug treated parasite cultures at regular time intervals. This technique is tedious and, most importantly, subject to interpretation with regards to distinguishing between viable and comprised parasite cells, thus making it impossible to objectively quantitate the rate of drug action. This study aimed to develop a series of bioassays using the calcein-acetoxymethyl and propidium iodide vitality probes which would allow the rate of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other. A novel bioassay using these fluorescent vitality probes coupled with fluorescence microscopy was developed and optimized and allowed the rate of drug action on malaria parasites to be assessed i) rapidly (in relation to current assay techniques) and ii) in a semi-quantitative manner. Extrapolation to flow cytometry for improved quantification provided favourable rankings of drug killing rates in the pilot study, however, requires further development to increase throughput and approach the ultimate goal of producing a medium-throughput assay for rapidly assessing the rate of action of antimalarial drugs. Attempts to adapt the assay for use in a multiwell plate reader, as well as using ATP measurements as an indication of parasite vitality after drug treatment, was met with erratic results. The viability probes assay as it stands represents an improvement on other assay formats in terms of rapidity and quantification of live/compromised parasites in cultures.
- Full Text:
Synthesis of silver nanoparticles and their role against human and Plasmodium falciparum leucine aminopeptidase
- Authors: Mnkandhla, Dumisani
- Date: 2015
- Subjects: Silver , Nanoparticles , Plasmodium falciparum , Leucine aminopeptidase , Antimalarials , Nanotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4150 , http://hdl.handle.net/10962/d1017911
- Description: Antimalarial drug discovery remains a challenging endeavour as malaria parasites continue to develop resistance to drugs, including those which are currently the last line of defence against the disease. Plasmodium falciparum is the most virulent of the malaria parasites and it delivers its deadliest impact during the erythrocytic stages of the parasite’s life cycle; a stage characterised by elevated catabolism of haemoglobin and anabolism of parasite proteins. The present study investigates the use of nanotechnology in the form of metallic silver nanoparticles (AgNPs) against P. falciparum leucine aminopeptidase (PfLAP), a validated biomedical target involved in haemoglobin metabolism. AgNPs were also tested against the human homolog cytosolic Homo sapiens leucine aminopeptidase (HsLAP) to ascertain their selective abilities. PfLAP and HsLAP were successfully expressed in Escherichia coli BL21(DE3) cells. PfLAP showed optimal thermal stability at 25 °C and optimal pH stability at pH 8.0 with a Km of 42.7 mM towards leucine-p-nitroanilide (LpNA) and a Vmax of 59.9 μmol.ml⁻¹.min⁻¹. HsLAP was optimally stable at 37 °C and at pH 7.0 with a Km of 16.7 mM and a Vmax of 17.2 μmol.ml⁻¹.min⁻¹. Both enzymes exhibited optimal activity in the presence of 2 mM Mn²⁺. On interaction with polyvinylpyrrolidone (PVP) stabilised AgNPs, both enzymes were inhibited to differing extents with PfLAP losing three fold of its catalytic efficiency relative to HsLAP. These results show the ability of AgNPs to selectively inhibit PfLAP whilst having much lesser effects on its human homolog. With the use of available targeting techniques, the present study shows the potential use of nanotechnology based approaches as “silver bullets” that can target PfLAP without adversely affecting the host. However further research needs to be conducted to better understand the mechanisms of AgNP action, drug targeting and the health and safety issues associated with nanotechnology use.
- Full Text:
- Authors: Mnkandhla, Dumisani
- Date: 2015
- Subjects: Silver , Nanoparticles , Plasmodium falciparum , Leucine aminopeptidase , Antimalarials , Nanotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4150 , http://hdl.handle.net/10962/d1017911
- Description: Antimalarial drug discovery remains a challenging endeavour as malaria parasites continue to develop resistance to drugs, including those which are currently the last line of defence against the disease. Plasmodium falciparum is the most virulent of the malaria parasites and it delivers its deadliest impact during the erythrocytic stages of the parasite’s life cycle; a stage characterised by elevated catabolism of haemoglobin and anabolism of parasite proteins. The present study investigates the use of nanotechnology in the form of metallic silver nanoparticles (AgNPs) against P. falciparum leucine aminopeptidase (PfLAP), a validated biomedical target involved in haemoglobin metabolism. AgNPs were also tested against the human homolog cytosolic Homo sapiens leucine aminopeptidase (HsLAP) to ascertain their selective abilities. PfLAP and HsLAP were successfully expressed in Escherichia coli BL21(DE3) cells. PfLAP showed optimal thermal stability at 25 °C and optimal pH stability at pH 8.0 with a Km of 42.7 mM towards leucine-p-nitroanilide (LpNA) and a Vmax of 59.9 μmol.ml⁻¹.min⁻¹. HsLAP was optimally stable at 37 °C and at pH 7.0 with a Km of 16.7 mM and a Vmax of 17.2 μmol.ml⁻¹.min⁻¹. Both enzymes exhibited optimal activity in the presence of 2 mM Mn²⁺. On interaction with polyvinylpyrrolidone (PVP) stabilised AgNPs, both enzymes were inhibited to differing extents with PfLAP losing three fold of its catalytic efficiency relative to HsLAP. These results show the ability of AgNPs to selectively inhibit PfLAP whilst having much lesser effects on its human homolog. With the use of available targeting techniques, the present study shows the potential use of nanotechnology based approaches as “silver bullets” that can target PfLAP without adversely affecting the host. However further research needs to be conducted to better understand the mechanisms of AgNP action, drug targeting and the health and safety issues associated with nanotechnology use.
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Synthesis of silver nanoparticles and their role against a thiazolekinase enzyme from Plasmodium falciparum
- Yao, Jia
- Authors: Yao, Jia
- Date: 2014
- Subjects: Silver , Nanoparticles , Thiazoles , Plasmodium falciparum , Antimalarials , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4168 , http://hdl.handle.net/10962/d1020894
- Description: Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.
- Full Text:
- Authors: Yao, Jia
- Date: 2014
- Subjects: Silver , Nanoparticles , Thiazoles , Plasmodium falciparum , Antimalarials , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4168 , http://hdl.handle.net/10962/d1020894
- Description: Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.
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The interaction of silver nanoparticles with triosephosphate isomerase from human and malarial parasite (Plasmodium falciparum) : a comparative study
- De Moor, Warren Ralph Josephus
- Authors: De Moor, Warren Ralph Josephus
- Date: 2014
- Subjects: Silver , Nanoparticles , Triose-phosphate isomerase , Plasmodium falciparum , Nanotechnology , Antimalarials , Povidone
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4169 , http://hdl.handle.net/10962/d1020895
- Description: The advent of advanced modern nanotechnology techniques offers new and exciting opportunities to develop novel nanotech-derived antimalarial nanodrugs with enhanced selective and targeting abilities that allow for lower effective drug dosages, longer drug persistence and reduced drug degradation within the body. Using a nanodrug approach also has the advantage of avoiding drug resistance problems that plague reconfigured versions of already-existing antimalarial drugs. In this study recombinant triosephosphate isomerase enzymes from Plasmodium falciparum (PfTIM) and Humans (hTIM) were recombinantly expressed, purified and characterised. PfTIM was shown to have optimal pH stability at pH 5.0-5.5 and thermal stability at 25°C with Km 4.34 mM and Vmax 0.876 μmol.ml⁻ₑmin⁻ₑ. For hTIM, these parameters were as follows: pH optima of 6.5-7.0; temperature optima of 30°C, with Km 2.27 mM and Vmax 0.714 μmol.ml⁻ₑmin⁻ₑ. Recombinant TIM enzymes were subjected to inhibition studies using polyvinylpyrrolidone (PVP) stabilised silver nanoparticles (AgNPs) of 4-12 nm in diameter. These studies showed that the AgNPs were able to selectively inhibit PfTIM over hTIM with an 8-fold greater decrease in enzymatic efficiency (Kcat/Km) observed for PfTIM, as compared to hTIM, for kinetics tests done using 0.06 μM of AgNPs. Complete inhibition of PfTIM under optimal conditions was achieved using 0.25 μM AgNPs after 45 minutes while hTIM maintained approximately 31% of its activity at this AgNP concentration. The above results indicate that selective enzymatic targeting of the important, key metabolic enzyme TIM, can be achieved using nanotechnology-derived nanodrugs. It was demonstrated that the key structural differences, between the two enzyme variants, were significant enough to create unique characteristics for each TIM variant, thereby allowing for selective enzyme targeting using AgNPs. If these AgNPs could be coupled with a nanotechnology-derived, targeted localization mechanism – possibly using apoferritin to deliver the AgNPs to infected erythrocytes (Burns and Pollock, 2008) – then such an approach could offer new opportunities for the development of viable antimalarial nanodrugs. For this to be achieved further research into several key areas will be required, including nanoparticle toxicity, drug localization and testing the lethality of the system on live parasite cultures.
- Full Text:
- Authors: De Moor, Warren Ralph Josephus
- Date: 2014
- Subjects: Silver , Nanoparticles , Triose-phosphate isomerase , Plasmodium falciparum , Nanotechnology , Antimalarials , Povidone
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4169 , http://hdl.handle.net/10962/d1020895
- Description: The advent of advanced modern nanotechnology techniques offers new and exciting opportunities to develop novel nanotech-derived antimalarial nanodrugs with enhanced selective and targeting abilities that allow for lower effective drug dosages, longer drug persistence and reduced drug degradation within the body. Using a nanodrug approach also has the advantage of avoiding drug resistance problems that plague reconfigured versions of already-existing antimalarial drugs. In this study recombinant triosephosphate isomerase enzymes from Plasmodium falciparum (PfTIM) and Humans (hTIM) were recombinantly expressed, purified and characterised. PfTIM was shown to have optimal pH stability at pH 5.0-5.5 and thermal stability at 25°C with Km 4.34 mM and Vmax 0.876 μmol.ml⁻ₑmin⁻ₑ. For hTIM, these parameters were as follows: pH optima of 6.5-7.0; temperature optima of 30°C, with Km 2.27 mM and Vmax 0.714 μmol.ml⁻ₑmin⁻ₑ. Recombinant TIM enzymes were subjected to inhibition studies using polyvinylpyrrolidone (PVP) stabilised silver nanoparticles (AgNPs) of 4-12 nm in diameter. These studies showed that the AgNPs were able to selectively inhibit PfTIM over hTIM with an 8-fold greater decrease in enzymatic efficiency (Kcat/Km) observed for PfTIM, as compared to hTIM, for kinetics tests done using 0.06 μM of AgNPs. Complete inhibition of PfTIM under optimal conditions was achieved using 0.25 μM AgNPs after 45 minutes while hTIM maintained approximately 31% of its activity at this AgNP concentration. The above results indicate that selective enzymatic targeting of the important, key metabolic enzyme TIM, can be achieved using nanotechnology-derived nanodrugs. It was demonstrated that the key structural differences, between the two enzyme variants, were significant enough to create unique characteristics for each TIM variant, thereby allowing for selective enzyme targeting using AgNPs. If these AgNPs could be coupled with a nanotechnology-derived, targeted localization mechanism – possibly using apoferritin to deliver the AgNPs to infected erythrocytes (Burns and Pollock, 2008) – then such an approach could offer new opportunities for the development of viable antimalarial nanodrugs. For this to be achieved further research into several key areas will be required, including nanoparticle toxicity, drug localization and testing the lethality of the system on live parasite cultures.
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Characterisation of a plasmodium falciparum type II Hsp40 chaperone exported to the cytosol of infected erythrocytes
- Maphumulo, Philile Nompumelelo
- Authors: Maphumulo, Philile Nompumelelo
- Date: 2013
- Subjects: Erythrocytes , Heat shock proteins , Plasmodium falciparum , Molecular chaperones , Malaria -- Prevention -- Research , Protein folding , Proteins -- Analysis , Malaria -- Immunological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4128 , http://hdl.handle.net/10962/d1015681
- Description: Heat Shock 40 kDa proteins (Hsp40s) partner with heat shock 70 kDa proteins (Hsp70s) in facilitating, among other chaperone activities; correct protein transport, productive protein folding and assembly within the cells; under both normal and stressful conditions. Hsp40 proteins regulate the ATPase activity of Hsp70 through interaction with the J-domain. Plasmodium falciparum Hsp70s (PfHsp70s) do not contain a Plasmodium export element (PEXEL) sequence although PfHsp70-1 and PfHsp70-3 have been located outside of the parasitophorous vacuole. Studies reveal that a type I P. falciparum (PfHsp40) chaperone (PF14_0359) stimulates the rate of ATP hydrolysis of the cytosolic PfHsp70 (PfHsp70-1) and that of human Hsp70A1A. PFE0055c is a PEXEL-bearing type II Hsp40 that is exported into the cytosol of P. falciparum-infected erythrocytes; where it potentially interacts with human Hsp70. Studies reveal that PFE0055c associates with structures found in the erythrocyte cytosol termed “J-dots” which are believed to be involved in trafficking parasite-encoded proteins through the erythrocyte cytosol. If P. falciparum exports PFE0055c into the host cytosol, it may be proposed that it interacts with human Hsp70, making it a possible drug target. The effect of PFE0055c on the ATPase activity of human Hsp70A1A has not been previously characterised. Central to this study was bioinformatic analysis and biochemical characterisation PFE0055c using an in vitro (ATPase assay) approach. Structural domains that classify PFE0055c as a type II Hsp40 were identified with similarity to two other exported type II PfHsp40s. Plasmids encoding the hexahistidine-tagged versions of PFE0055c and human Hsp70A1A were used for the expression and purification of these proteins from Escherichia coli. Purification was achieved using nickel affinity chromatography. The urea-denaturing method was used to obtain the purified PFE0055c whilst human Hsp70A1A was purified using the native method. PFE0055c could stimulate the ATPase activity of alfalfa Hsp70, although such was not the case for human Hsp70A1A in vitro.
- Full Text:
- Authors: Maphumulo, Philile Nompumelelo
- Date: 2013
- Subjects: Erythrocytes , Heat shock proteins , Plasmodium falciparum , Molecular chaperones , Malaria -- Prevention -- Research , Protein folding , Proteins -- Analysis , Malaria -- Immunological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4128 , http://hdl.handle.net/10962/d1015681
- Description: Heat Shock 40 kDa proteins (Hsp40s) partner with heat shock 70 kDa proteins (Hsp70s) in facilitating, among other chaperone activities; correct protein transport, productive protein folding and assembly within the cells; under both normal and stressful conditions. Hsp40 proteins regulate the ATPase activity of Hsp70 through interaction with the J-domain. Plasmodium falciparum Hsp70s (PfHsp70s) do not contain a Plasmodium export element (PEXEL) sequence although PfHsp70-1 and PfHsp70-3 have been located outside of the parasitophorous vacuole. Studies reveal that a type I P. falciparum (PfHsp40) chaperone (PF14_0359) stimulates the rate of ATP hydrolysis of the cytosolic PfHsp70 (PfHsp70-1) and that of human Hsp70A1A. PFE0055c is a PEXEL-bearing type II Hsp40 that is exported into the cytosol of P. falciparum-infected erythrocytes; where it potentially interacts with human Hsp70. Studies reveal that PFE0055c associates with structures found in the erythrocyte cytosol termed “J-dots” which are believed to be involved in trafficking parasite-encoded proteins through the erythrocyte cytosol. If P. falciparum exports PFE0055c into the host cytosol, it may be proposed that it interacts with human Hsp70, making it a possible drug target. The effect of PFE0055c on the ATPase activity of human Hsp70A1A has not been previously characterised. Central to this study was bioinformatic analysis and biochemical characterisation PFE0055c using an in vitro (ATPase assay) approach. Structural domains that classify PFE0055c as a type II Hsp40 were identified with similarity to two other exported type II PfHsp40s. Plasmids encoding the hexahistidine-tagged versions of PFE0055c and human Hsp70A1A were used for the expression and purification of these proteins from Escherichia coli. Purification was achieved using nickel affinity chromatography. The urea-denaturing method was used to obtain the purified PFE0055c whilst human Hsp70A1A was purified using the native method. PFE0055c could stimulate the ATPase activity of alfalfa Hsp70, although such was not the case for human Hsp70A1A in vitro.
- 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:
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:
- 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:
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:
- 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:
Biochemical characterization of plasmodium falciparum heat shock protein 70
- Matambo, Tonderayi Sylvester
- Authors: Matambo, Tonderayi Sylvester
- Date: 2004
- Subjects: Plasmodium falciparum , Malaria -- Prevention , Protein folding , Proteins -- Purification , Heat shock proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4134 , http://hdl.handle.net/10962/d1015767
- Description: Plamodium falciparum heat shock protein (PfHsp70) is believed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. Bioinformatic analysis reveal that PfHsp70 consists of the three canonical Hsp70 domains; an ATPase domain of 45 kDa, Substrate binding domain of 15 kDa and a C-terminal domain of 10 kDa. At the C-terminus there is a GGMP repeat motif that is commonly found in Hsp70s of parasitic origins. Plasmodium falciparum genome is 80% A-T rich, making it difficult to recombinantly express its proteins in Escherhia coli (E. coli) as a result of rare codon usage. In this study we carried out experiments to improve expression in E. coli by inserting the PfHsp70 coding region into the pQE30 expression vector. However multiple bands were detected by Western analysis, probably due to the presence of rare codons. The RIG plasmid, which encodes tRNAs for rare codons in particular Arg (AGA/AGG), Ile (AUA) and Gly (GGA) was engineered into the E. coli strain resulting in production of full length PfHsp70. Purification was achieved through Ni²⁺ Chelating sepharose under denaturing conditions. PfHsp70 was found to have a very low basal ATPase activity of 0.262 ± 0.05 nmoles/min/mg of protein. In the presence of reduced and carboxymethylated lactalbumin (RCMLA) a 11-fold increase in ATPase activity was noted whereas in the presence of both RCMLA and Trypanosoma cruzi DnaJ (Tcj2) a 16-fold was achieved. For ATP hydrolysis kcat value of 0.003 min⁻¹ was obtained whereas for ADP release a greater kcat value of 0.8 min⁻¹ was obtained. These results indicated that rate of ATP hydrolysis maybe the rate-determining step in the ATPase cycle of PfHsp70.
- Full Text:
- Authors: Matambo, Tonderayi Sylvester
- Date: 2004
- Subjects: Plasmodium falciparum , Malaria -- Prevention , Protein folding , Proteins -- Purification , Heat shock proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4134 , http://hdl.handle.net/10962/d1015767
- Description: Plamodium falciparum heat shock protein (PfHsp70) is believed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. Bioinformatic analysis reveal that PfHsp70 consists of the three canonical Hsp70 domains; an ATPase domain of 45 kDa, Substrate binding domain of 15 kDa and a C-terminal domain of 10 kDa. At the C-terminus there is a GGMP repeat motif that is commonly found in Hsp70s of parasitic origins. Plasmodium falciparum genome is 80% A-T rich, making it difficult to recombinantly express its proteins in Escherhia coli (E. coli) as a result of rare codon usage. In this study we carried out experiments to improve expression in E. coli by inserting the PfHsp70 coding region into the pQE30 expression vector. However multiple bands were detected by Western analysis, probably due to the presence of rare codons. The RIG plasmid, which encodes tRNAs for rare codons in particular Arg (AGA/AGG), Ile (AUA) and Gly (GGA) was engineered into the E. coli strain resulting in production of full length PfHsp70. Purification was achieved through Ni²⁺ Chelating sepharose under denaturing conditions. PfHsp70 was found to have a very low basal ATPase activity of 0.262 ± 0.05 nmoles/min/mg of protein. In the presence of reduced and carboxymethylated lactalbumin (RCMLA) a 11-fold increase in ATPase activity was noted whereas in the presence of both RCMLA and Trypanosoma cruzi DnaJ (Tcj2) a 16-fold was achieved. For ATP hydrolysis kcat value of 0.003 min⁻¹ was obtained whereas for ADP release a greater kcat value of 0.8 min⁻¹ was obtained. These results indicated that rate of ATP hydrolysis maybe the rate-determining step in the ATPase cycle of PfHsp70.
- Full Text:
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