A novel, improved throughput bioassay for determining the delative speed of antimalarial drug action using fluorescent vitality probes
- Authors: Laming, Dustin
- Date: 2020
- Subjects: Plasmodium falciparum , Malaria -- Treatment -- Africa , Antimalarials , Malaria vaccine
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/139902 , vital:37810
- Description: Malaria is one of the most prevalent diseases in Africa and Plasmodium falciparum is widely accepted as the most virulent of the malaria parasite species, 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 that are not only efficacious but are also quick acting. Traditional methods of evaluating antimalarial killing speeds via morphological assessments are inherently flawed by tedious, subjective interpretations of the heterogenous parasite morphology encountered in routine parasite culture conditions. This has led to the introduction of alternative assay formats to determine how rapidly compounds act on parasites in vitro: a parasite reduction ratio (PRR) assay that measures the recovery of parasite cultures from drug exposure; determining the shift in IC50 values of compounds when dose-response assays are carried out for different time periods; a bioluminescence relative rate of kill (BRRoK) assay that compares the extent to which compounds reduce firefly luciferase activity in transgenic parasites. Recent whole cell in vitro screening efforts have resulted in the generation of chemically diverse compound libraries such as the Medicines for Malaria Venture’s Pathogen Box, which houses 125 novel compounds with in vitro antiplasmodial activity. Assessing the relative killing speeds of these compounds would aid prioritizing fast-acting compounds that can be exploited as starting points for further development. This study aimed to develop a bioassay using the calcein-acetoxymethyl and propidium iodide fluorescent vitality probes, which would allow the relative speed of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other using a quantitative, improved throughput approach. Initially applied to human (HeLa) cells, the assay was used to compare the relative speeds of action of 3 potential anti-cancer compounds by fluorescence microscopy. Subsequently adapted to P. falciparum, the assay was able to rank the relative speeds of action of standard antimalarials by fluorescence microscopy and two flow cytometry formats. Application of a multiwell flow cytometer increased throughput and enabled the assessment of experimental compounds, which included a set of artemisinin analogs and 125 antimalarial compounds in the MMV Pathogen Box. The latter culminated in the identification of five rapidly parasiticidal compounds in relation to the other compounds in the library, which may act as benchmark references for future studies and form the basis of the next generation of fast acting antimalarials that could be used to combat modern, resistant malaria.
- Full Text:
- Date Issued: 2020
- Authors: Laming, Dustin
- Date: 2020
- Subjects: Plasmodium falciparum , Malaria -- Treatment -- Africa , Antimalarials , Malaria vaccine
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/139902 , vital:37810
- Description: Malaria is one of the most prevalent diseases in Africa and Plasmodium falciparum is widely accepted as the most virulent of the malaria parasite species, 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 that are not only efficacious but are also quick acting. Traditional methods of evaluating antimalarial killing speeds via morphological assessments are inherently flawed by tedious, subjective interpretations of the heterogenous parasite morphology encountered in routine parasite culture conditions. This has led to the introduction of alternative assay formats to determine how rapidly compounds act on parasites in vitro: a parasite reduction ratio (PRR) assay that measures the recovery of parasite cultures from drug exposure; determining the shift in IC50 values of compounds when dose-response assays are carried out for different time periods; a bioluminescence relative rate of kill (BRRoK) assay that compares the extent to which compounds reduce firefly luciferase activity in transgenic parasites. Recent whole cell in vitro screening efforts have resulted in the generation of chemically diverse compound libraries such as the Medicines for Malaria Venture’s Pathogen Box, which houses 125 novel compounds with in vitro antiplasmodial activity. Assessing the relative killing speeds of these compounds would aid prioritizing fast-acting compounds that can be exploited as starting points for further development. This study aimed to develop a bioassay using the calcein-acetoxymethyl and propidium iodide fluorescent vitality probes, which would allow the relative speed of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other using a quantitative, improved throughput approach. Initially applied to human (HeLa) cells, the assay was used to compare the relative speeds of action of 3 potential anti-cancer compounds by fluorescence microscopy. Subsequently adapted to P. falciparum, the assay was able to rank the relative speeds of action of standard antimalarials by fluorescence microscopy and two flow cytometry formats. Application of a multiwell flow cytometer increased throughput and enabled the assessment of experimental compounds, which included a set of artemisinin analogs and 125 antimalarial compounds in the MMV Pathogen Box. The latter culminated in the identification of five rapidly parasiticidal compounds in relation to the other compounds in the library, which may act as benchmark references for future studies and form the basis of the next generation of fast acting antimalarials that could be used to combat modern, resistant malaria.
- Full Text:
- Date Issued: 2020
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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