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:
- Date Issued: 2020
- 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:
- Date Issued: 2020
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:
- Date Issued: 2019
- 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:
- Date Issued: 2019
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:
- Date Issued: 2018
- 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:
- Date Issued: 2018
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:
- Date Issued: 2016
- 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:
- Date Issued: 2016
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