A medicinal chemistry study in nitrogen containing heterocycles
- Authors: Lunga, Mayibongwe Junior
- Date: 2018
- Subjects: Indole , Tetrazoles , Antimalarials , Heat shock proteins , Plasmodium falciparum
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63521 , vital:28430
- Description: Heterocyclic structures have found extensive utility in the field of medicinal chemistry, as prominent regions of pharmacophores resulting in numerous drug treatments for many diseases. Accordingly, in this project we explored the respective antimalarial and anticancer activity exhibited by compounds featuring nitrogen containing indole and tetrazole heterocycles respectively. This thesis therefore comprises of two distinct parts. Part 1. Following the development of resistance towards traditional antimalarial therapy such as chloroquine and emerging resistance towards artemisinin combination therapies, the WHO reported the urgent need for new, effective drugs and identification of new drug targets to combat the Plasmodium falciparum parasite. In 2015 the parasite was the cause of 429 000 deaths, the majority occurring in the sub-Saharan region of Africa. This highlights the failing effectiveness of vector control strategies, reiterating the need to develop alternative control and treatment strategies. In response to this need we wanted to expand and further describe the SAR of the indole based series, indolyl-3-ethanone-α- thioethers, previously synthesized in our laboratory. These compounds were found to exhibit antimalarial activity with compounds 2.26 and 2.27 exhibiting activity against P. falciparum 3D7 in the nanomolar range. Based on these compounds we synthesized compounds 3.21 and 3.24 – 3.32 following a three step reaction pathway. Our results in this study, indicate that compound 3.28, a pnitrothiophenol analogue of 2.27 was the most active of the compounds we synthesized and furthermore was superior in activity against Plasmodium compared to 2.27. This result indicated that the presence of p-NO2 is important in enhancing anti-plasmodial activity. Comparing compounds 3.25 and 3.26 with an oxygen on the ether bridge to compounds 3.29 and 3.30 with a sulfur, we observed an increase in hydrophilicity coupled to a decrease in anti-plasmodial activity in the compounds, thus, highlighting the importance of sulfur for enhanced activity. Furthermore, we investigated bioisosteric replacement of the 5-chloro substituent present in hit compounds 2.27 and 3.28, with an electron withdrawing nitrile (3.27) and electron donating methyl (3.29) and methoxy (3.31) substituents. These substituents decreased anti-plasmodial activity, confirming that a chlorine substituent is optimal for biological activity. This study furthered our understanding of the SAR of indolyl-3-ethanone-α- thioethers for the development of potent anti-plasmodial lead compounds. Part 2. Triple negative breast cancer (TNBC), which disproportionately affects women of sub-Saharan Africa, is unresponsive to hormone-based therapies. This emergence presents a population of patients devoid of effective drug treatment, signaling the urgent need to develop new effective therapies with novel drug targets. Therefore, we identified our target in TNBC cells as the protein-protein interaction between the co-chaperones HOP and HSP90. We reasoned that a disruption of this interaction would ultimately result in cancer cell death via the degradation of essential oncogenic client proteins. Following a fragment screening campaign, which identified several acid and tetrazole containing hits (4.56 – 4.58) which bound to HOP, with low anticancer activity, we sought to develop synthetic methodology to elaborate our fragment hits synthesizing tetrazole containing fragments to target TNBC cell lines. We therefore proceeded to synthesize a range of multi substituted fragments (4.59 – 4.63), utilizing a nitrile (4.66) to access tetrazoles via 1,3-cycloaddition and an acid by nitrile hydrolysis. We successfully synthesized the tetrazole and acid fragments which are currently undergoing characterization for activity against TNBC. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Date Issued: 2018
- Authors: Lunga, Mayibongwe Junior
- Date: 2018
- Subjects: Indole , Tetrazoles , Antimalarials , Heat shock proteins , Plasmodium falciparum
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63521 , vital:28430
- Description: Heterocyclic structures have found extensive utility in the field of medicinal chemistry, as prominent regions of pharmacophores resulting in numerous drug treatments for many diseases. Accordingly, in this project we explored the respective antimalarial and anticancer activity exhibited by compounds featuring nitrogen containing indole and tetrazole heterocycles respectively. This thesis therefore comprises of two distinct parts. Part 1. Following the development of resistance towards traditional antimalarial therapy such as chloroquine and emerging resistance towards artemisinin combination therapies, the WHO reported the urgent need for new, effective drugs and identification of new drug targets to combat the Plasmodium falciparum parasite. In 2015 the parasite was the cause of 429 000 deaths, the majority occurring in the sub-Saharan region of Africa. This highlights the failing effectiveness of vector control strategies, reiterating the need to develop alternative control and treatment strategies. In response to this need we wanted to expand and further describe the SAR of the indole based series, indolyl-3-ethanone-α- thioethers, previously synthesized in our laboratory. These compounds were found to exhibit antimalarial activity with compounds 2.26 and 2.27 exhibiting activity against P. falciparum 3D7 in the nanomolar range. Based on these compounds we synthesized compounds 3.21 and 3.24 – 3.32 following a three step reaction pathway. Our results in this study, indicate that compound 3.28, a pnitrothiophenol analogue of 2.27 was the most active of the compounds we synthesized and furthermore was superior in activity against Plasmodium compared to 2.27. This result indicated that the presence of p-NO2 is important in enhancing anti-plasmodial activity. Comparing compounds 3.25 and 3.26 with an oxygen on the ether bridge to compounds 3.29 and 3.30 with a sulfur, we observed an increase in hydrophilicity coupled to a decrease in anti-plasmodial activity in the compounds, thus, highlighting the importance of sulfur for enhanced activity. Furthermore, we investigated bioisosteric replacement of the 5-chloro substituent present in hit compounds 2.27 and 3.28, with an electron withdrawing nitrile (3.27) and electron donating methyl (3.29) and methoxy (3.31) substituents. These substituents decreased anti-plasmodial activity, confirming that a chlorine substituent is optimal for biological activity. This study furthered our understanding of the SAR of indolyl-3-ethanone-α- thioethers for the development of potent anti-plasmodial lead compounds. Part 2. Triple negative breast cancer (TNBC), which disproportionately affects women of sub-Saharan Africa, is unresponsive to hormone-based therapies. This emergence presents a population of patients devoid of effective drug treatment, signaling the urgent need to develop new effective therapies with novel drug targets. Therefore, we identified our target in TNBC cells as the protein-protein interaction between the co-chaperones HOP and HSP90. We reasoned that a disruption of this interaction would ultimately result in cancer cell death via the degradation of essential oncogenic client proteins. Following a fragment screening campaign, which identified several acid and tetrazole containing hits (4.56 – 4.58) which bound to HOP, with low anticancer activity, we sought to develop synthetic methodology to elaborate our fragment hits synthesizing tetrazole containing fragments to target TNBC cell lines. We therefore proceeded to synthesize a range of multi substituted fragments (4.59 – 4.63), utilizing a nitrile (4.66) to access tetrazoles via 1,3-cycloaddition and an acid by nitrile hydrolysis. We successfully synthesized the tetrazole and acid fragments which are currently undergoing characterization for activity against TNBC. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Date Issued: 2018
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
An in-silico study of the type II NADH: Quinone Oxidoreductase (ndh2). A new anti-malaria drug target
- Authors: Baye, Bertha Cinthia
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium , Molecular dynamics , Computer simulation , Quinone , Antimalarials , Molecules Models , Docking , Drugs Computer-aided design
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365633 , vital:65767 , DOI https://doi.org/10.21504/10962/365633
- Description: Malaria is caused by Plasmodium parasites, spread to people through the bites of infected female Anopheles mosquitoes. This study focuses on all 5 (Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax) parasites that cause malaria in humans. Africa is a developing continent, and it is the most affected with an estimation of 90% of more than 400 000 malaria-related deaths reported by the World Health Organization (WHO) report in 2020, in which 61% of that number are children under the ages of five. Malaria resistance was initially observed in early 1986 and with the progression of time anti-malarial drug resistance has only increased. As a result, there is a need to study the malarial proteins mechanism of action and identify alternative treatment strategies for this disease. Type II NADH: quinone oxidoreductase (NDH2) is a monotopic protein that catalyses the electron transfer from NADH to quinone via FAD without a proton-pumping activity, and functions as an initial enzyme, either in addition to or as an alternative to proton-pumping NADH dehydrogenase (complex I) in the respiratory chain of bacteria, archaea, and fungal and plant mitochondrial. The structures for the Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax were modelled from the crystal structure of Plasmodium falciparum (5JWA). Compounds from the South African natural compounds database (SANCDB) were docked against both the NDH2 crystal structure and modelled structures. By performing in silico screening the study aimed to find potential compounds that might interrupt the electron transfer to quinone therefore disturbing the enzyme‟s function and thereby possibly eliminating the plasmodium parasite. CHARMM-GUI was used to create the membrane (since this work is with membrane-bound proteins) and to orient the protein on the membrane using OPM server guidelines, the interface produced GROMACS topology files that were used in molecular dynamics simulations. Molecular dynamics simulations were performed in the Centre for high performance computing (CHPC) cluster under the CHEM0802 project and the trajectories produced were further analysed. In this work not only were hit compounds from SANCDB identified, but also differences in behaviour across species and in the presence or absence of the membrane were described. This highlights the need to include the correct protein environment when studying these systems. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Baye, Bertha Cinthia
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium , Molecular dynamics , Computer simulation , Quinone , Antimalarials , Molecules Models , Docking , Drugs Computer-aided design
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365633 , vital:65767 , DOI https://doi.org/10.21504/10962/365633
- Description: Malaria is caused by Plasmodium parasites, spread to people through the bites of infected female Anopheles mosquitoes. This study focuses on all 5 (Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax) parasites that cause malaria in humans. Africa is a developing continent, and it is the most affected with an estimation of 90% of more than 400 000 malaria-related deaths reported by the World Health Organization (WHO) report in 2020, in which 61% of that number are children under the ages of five. Malaria resistance was initially observed in early 1986 and with the progression of time anti-malarial drug resistance has only increased. As a result, there is a need to study the malarial proteins mechanism of action and identify alternative treatment strategies for this disease. Type II NADH: quinone oxidoreductase (NDH2) is a monotopic protein that catalyses the electron transfer from NADH to quinone via FAD without a proton-pumping activity, and functions as an initial enzyme, either in addition to or as an alternative to proton-pumping NADH dehydrogenase (complex I) in the respiratory chain of bacteria, archaea, and fungal and plant mitochondrial. The structures for the Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax were modelled from the crystal structure of Plasmodium falciparum (5JWA). Compounds from the South African natural compounds database (SANCDB) were docked against both the NDH2 crystal structure and modelled structures. By performing in silico screening the study aimed to find potential compounds that might interrupt the electron transfer to quinone therefore disturbing the enzyme‟s function and thereby possibly eliminating the plasmodium parasite. CHARMM-GUI was used to create the membrane (since this work is with membrane-bound proteins) and to orient the protein on the membrane using OPM server guidelines, the interface produced GROMACS topology files that were used in molecular dynamics simulations. Molecular dynamics simulations were performed in the Centre for high performance computing (CHPC) cluster under the CHEM0802 project and the trajectories produced were further analysed. In this work not only were hit compounds from SANCDB identified, but also differences in behaviour across species and in the presence or absence of the membrane were described. This highlights the need to include the correct protein environment when studying these systems. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Antimalarial activity of quinoline thiosemicarbazones: synthesis and antiplasmodial evaluation
- Nqeno, Lukhanyiso Khanyisile
- Authors: Nqeno, Lukhanyiso Khanyisile
- Date: 2022-04-06
- Subjects: Antimalarials , Quinoline , Thiosemicarbazones , Malaria Chemotherapy , Plasmodium falciparum , Malaria Africa, Sub-Saharan , Iron chelates Therapeutic use
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/291292 , vital:56841
- Description: Africa is one of the regions that is most affected by malaria, as 90% of all malaria deaths occur in sub-saharan Africa. Malaria is a life threatening disease responsible for an estimated 800000 deaths each year, the majority of these deaths occurred in children under the age of five. The disease is a mosquito-borne, and it is transmitted to humans by the female Anopheles mosquito. The parasite responsible for this disease belong to the Plasmodium genus with Plasmodium falciparum causing the most severe cases of the disease in humans. The most widely available anti-malarials were designed to specifically target the pathogenic blood stage in humans, however, in order to completely eradicate malaria there is a need for the development of medicines that not only target the pathogenic blood stage of the parasite but also block parasite transmission and eliminate asymptomatic and cryptic hepatic forms of the parasite. Iron chelators have recently gained importance as potent antimalarials, to cause infection nearly all protozoa obtain growth essential iron from their hosts. Iron is required for the development of the parasite. Deprivation of utilizable iron by chelation is a proficient approach to arrest parasite growth and associated infection. Thiosemicarbazones are known iron chelating agents by bonding through the sulfur and azomethine nitrogen atoms. This study is aimed at the identification of thiosemicarbazone based derivatives as possible antimalarial agents. Due to their iron chelation abilities there has been increasing interest in the investigation of thiosemicarbazones as possible antimalarials. During the course of this project, several thiosemicarbazone derivatives were synthesized and their structure confirmed using routine analytical techniques (NMR, FTIR, and HRMS). The synthesized compounds were evaluated in vitro against the chloroquine sensitive strain (3D7) of P. falciparum for antimarial activity. The compounds were also evaluated agsinst Hela cells for overt cytotoxicity. The compounds generally showed poor antimalarial activity. One compound (LKN11) was identified to possess intrinsic and moderate antimalarial activity of 6.6 μM. The compounds were generally not cytotoxic against Hela cell at concentrations of up to 20 μM, with only compound LKN10 showing modest cytotoxic activity of 9.5 μM. This research went on to identify two thiosemicarbazone based derivatives which had a significant effect on HeLa and pLDH cells. , Thesis (MSc) -- Faculty of Science, Chemistry, 2022
- Full Text:
- Date Issued: 2022-04-06
- Authors: Nqeno, Lukhanyiso Khanyisile
- Date: 2022-04-06
- Subjects: Antimalarials , Quinoline , Thiosemicarbazones , Malaria Chemotherapy , Plasmodium falciparum , Malaria Africa, Sub-Saharan , Iron chelates Therapeutic use
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/291292 , vital:56841
- Description: Africa is one of the regions that is most affected by malaria, as 90% of all malaria deaths occur in sub-saharan Africa. Malaria is a life threatening disease responsible for an estimated 800000 deaths each year, the majority of these deaths occurred in children under the age of five. The disease is a mosquito-borne, and it is transmitted to humans by the female Anopheles mosquito. The parasite responsible for this disease belong to the Plasmodium genus with Plasmodium falciparum causing the most severe cases of the disease in humans. The most widely available anti-malarials were designed to specifically target the pathogenic blood stage in humans, however, in order to completely eradicate malaria there is a need for the development of medicines that not only target the pathogenic blood stage of the parasite but also block parasite transmission and eliminate asymptomatic and cryptic hepatic forms of the parasite. Iron chelators have recently gained importance as potent antimalarials, to cause infection nearly all protozoa obtain growth essential iron from their hosts. Iron is required for the development of the parasite. Deprivation of utilizable iron by chelation is a proficient approach to arrest parasite growth and associated infection. Thiosemicarbazones are known iron chelating agents by bonding through the sulfur and azomethine nitrogen atoms. This study is aimed at the identification of thiosemicarbazone based derivatives as possible antimalarial agents. Due to their iron chelation abilities there has been increasing interest in the investigation of thiosemicarbazones as possible antimalarials. During the course of this project, several thiosemicarbazone derivatives were synthesized and their structure confirmed using routine analytical techniques (NMR, FTIR, and HRMS). The synthesized compounds were evaluated in vitro against the chloroquine sensitive strain (3D7) of P. falciparum for antimarial activity. The compounds were also evaluated agsinst Hela cells for overt cytotoxicity. The compounds generally showed poor antimalarial activity. One compound (LKN11) was identified to possess intrinsic and moderate antimalarial activity of 6.6 μM. The compounds were generally not cytotoxic against Hela cell at concentrations of up to 20 μM, with only compound LKN10 showing modest cytotoxic activity of 9.5 μM. This research went on to identify two thiosemicarbazone based derivatives which had a significant effect on HeLa and pLDH cells. , Thesis (MSc) -- Faculty of Science, Chemistry, 2022
- Full Text:
- Date Issued: 2022-04-06
Antimalarial secondary metabolites from Morinda lucida
- Authors: Chithambo, Bertha
- Date: 2017
- Subjects: Botanical chemistry , Anthraquinones , Antimalarials , Rubiaceae -- Therapeutic use , Malaria -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/45730 , vital:25535
- Description: Antimalarial activities of secondary metabolites from Morinda lucida (Rubiaceae), were investigated. Even though M. lucida is traditionally used to treat malaria, diabetes, jaundice, hypertension, dysentery and many other diseases, the compounds in this plant have not yet been fully investigated and characterised. Most of the studies that have been done on this plant focused on the medicinal properties of the crude extracts but have not gone further to isolate and characterise the compounds. In this study, the methanol - dichloromethane crude extract from the bark of M. lucida was fractionated into fractions 1-8. Fractions 2-5 were purified in order to isolate active secondary metabolites. The isolated pure compounds were characterised and identified. An in vitro antimalarial assay was carried out on the crude extract, fractions, pure compounds and solutions made from different combinations of pure compounds using the parasite lactate dehydrogenase (pLDH) assay. An IC50 done on the methanolic crude extract gave a value of 25 µg/mL. The % cell viability for the crude extract in cell toxicity assay remained at 100%. Each of the pure compounds tested had very little activity. Their activities were increased when samples from the different compounds were mixed. One of these mixtures reduced malaria viability to about 22 % at 20 µM and gave an IC50 value of 17 µM. Antibacterial assays were also carried out on the crude extract and fractions. Fractions 2 and 3 were relatively active (MIC values ranging between 125-1000 µg/mL) against M. cattarhalis and E. faecalis. Fraction 2 was also the most active on S. typhimurium and S. aureus (MIC value of 1000 µg/mL) compared with the other fractions. This same fraction also showed some activity against M. tuberculosis with MIC90 and MIC99 values of 40.9 and 46.3 µg/mL respectively in an anti-tuberculosis assay.The following compounds, comprising of iridoids (asperuloside and asperulosidic acid), terpenoids (stigmasterol, P-sitosterol, campesterol, lanosterol and cycloartenol) and anthraquinones [5,15-O-dimethylmorindol, 1,7-dihydroxy-2-methoxy-5-(methoxymethyl) anthraquinone and 1,6-dihydroxy-2-methoxy-5-(methoxymethyl)anthraquinone], were isolated. All these compounds have been isolated from different plants before with the exception of 1,7-dihydroxy-2-methoxy-5-(methoxymethyl)anthraquinone and 1,6-dihydroxy-2-methoxy-5-(methoxymethyl)anthraquinone which were tentatively assigned the structures due to insufficient data. To the best of our knowledge, this is the first report on the identification of all of the mentioned compounds, with the exception of ß-sitosterol and stigmasterol, from M. lucida. Molecular docking was performed on one of the isolated anthraquinones (5,15-O- dimethylmorindol) to check if it can bind to cytochrome bci, a known target for atovaquone. This compound interacted with the same amino acids that atovaquone, a well known antimalarial agent, interacted with on cytochrome bc1 indicating a possible similar mode of action.
- Full Text:
- Date Issued: 2017
- Authors: Chithambo, Bertha
- Date: 2017
- Subjects: Botanical chemistry , Anthraquinones , Antimalarials , Rubiaceae -- Therapeutic use , Malaria -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/45730 , vital:25535
- Description: Antimalarial activities of secondary metabolites from Morinda lucida (Rubiaceae), were investigated. Even though M. lucida is traditionally used to treat malaria, diabetes, jaundice, hypertension, dysentery and many other diseases, the compounds in this plant have not yet been fully investigated and characterised. Most of the studies that have been done on this plant focused on the medicinal properties of the crude extracts but have not gone further to isolate and characterise the compounds. In this study, the methanol - dichloromethane crude extract from the bark of M. lucida was fractionated into fractions 1-8. Fractions 2-5 were purified in order to isolate active secondary metabolites. The isolated pure compounds were characterised and identified. An in vitro antimalarial assay was carried out on the crude extract, fractions, pure compounds and solutions made from different combinations of pure compounds using the parasite lactate dehydrogenase (pLDH) assay. An IC50 done on the methanolic crude extract gave a value of 25 µg/mL. The % cell viability for the crude extract in cell toxicity assay remained at 100%. Each of the pure compounds tested had very little activity. Their activities were increased when samples from the different compounds were mixed. One of these mixtures reduced malaria viability to about 22 % at 20 µM and gave an IC50 value of 17 µM. Antibacterial assays were also carried out on the crude extract and fractions. Fractions 2 and 3 were relatively active (MIC values ranging between 125-1000 µg/mL) against M. cattarhalis and E. faecalis. Fraction 2 was also the most active on S. typhimurium and S. aureus (MIC value of 1000 µg/mL) compared with the other fractions. This same fraction also showed some activity against M. tuberculosis with MIC90 and MIC99 values of 40.9 and 46.3 µg/mL respectively in an anti-tuberculosis assay.The following compounds, comprising of iridoids (asperuloside and asperulosidic acid), terpenoids (stigmasterol, P-sitosterol, campesterol, lanosterol and cycloartenol) and anthraquinones [5,15-O-dimethylmorindol, 1,7-dihydroxy-2-methoxy-5-(methoxymethyl) anthraquinone and 1,6-dihydroxy-2-methoxy-5-(methoxymethyl)anthraquinone], were isolated. All these compounds have been isolated from different plants before with the exception of 1,7-dihydroxy-2-methoxy-5-(methoxymethyl)anthraquinone and 1,6-dihydroxy-2-methoxy-5-(methoxymethyl)anthraquinone which were tentatively assigned the structures due to insufficient data. To the best of our knowledge, this is the first report on the identification of all of the mentioned compounds, with the exception of ß-sitosterol and stigmasterol, from M. lucida. Molecular docking was performed on one of the isolated anthraquinones (5,15-O- dimethylmorindol) to check if it can bind to cytochrome bci, a known target for atovaquone. This compound interacted with the same amino acids that atovaquone, a well known antimalarial agent, interacted with on cytochrome bc1 indicating a possible similar mode of action.
- Full Text:
- Date Issued: 2017
Application of computer-aided drug design for identification of P. falciparum inhibitors
- Authors: Diallo, Bakary N’tji
- Date: 2021-10-29
- Subjects: Plasmodium falciparum , Malaria -- Chemotherapy , Molecular dynamics , Antimalarials , Cheminformatics , Drug development , Ligand binding (Biochemistry) , Plasmodium falciparum1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) , South African Natural Compounds Database
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/192798 , vital:45265 , 10.21504/10962/192798
- Description: Malaria is a millennia-old disease with the first recorded cases dating back to 2700 BC found in Chinese medical records, and later in other civilizations. It has claimed human lives to such an extent that there are a notable associated socio-economic consequences. Currently, according to the World Health Organization (WHO), Africa holds the highest disease burden with 94% of deaths and 82% of cases with P. falciparum having ~100% prevalence. Chemotherapy, such as artemisinin combination therapy, has been and continues to be the work horse in the fight against the disease, together with seasonal malaria chemoprevention and the use of insecticides. Natural products such as quinine and artemisinin are particularly important in terms of their antimalarial activity. The emphasis in current chemotherapy research is the need for time and cost-effective workflows focussed on new mechanisms of action (MoAs) covering the target candidate profiles (TCPs). Despite a decline in cases over the past decades with, countries increasingly becoming certified malaria free, a stalling trend has been observed in the past five years resulting in missing the 2020 Global Technical Strategy (GTS) milestones. With no effective vaccine, a reduction in funding, slower drug approval than resistance emergence from resistant and invasive vectors, and threats in diagnosis with the pfhrp2/3 gene deletion, malaria remains a major health concern. Motivated by these reasons, the primary aim of this work was a contribution to the antimalarial pipeline through in silico approaches focusing on P. falciparum. We first intended an exploration of malarial targets through a proteome scale screening on 36 targets using multiple metrics to account for the multi-objective nature of drug discovery. The continuous growth of structural data offers the ideal scenario for mining new MoAs covering antimalarials TCPs. This was combined with a repurposing strategy using a set of orally available FDA approved drugs. Further, use was made of time- and cost-effective strategies combining QVina-W efficiency metrics that integrate molecular properties, GRIM rescoring for molecular interactions and a hydrogen mass repartitioning (HMR) molecular dynamics (MD) scheme for accelerated development of antimalarials in the context of resistance. This pipeline further integrates a complex ranking for better drug-target selectivity, and normalization strategies to overcome docking scoring function bias. The different metrics, ranking, normalization strategies and their combinations were first assessed using their mean ranking error (MRE). A version combining all metrics was used to select 36 unique protein-ligand complexes, assessed in MD, with the final retention of 25. From the 16 in vitro tested hits of the 25, fingolimod, abiraterone, prazosin, and terazosin showed antiplasmodial activity with IC50 2.21, 3.37, 16.67 and 34.72 μM respectively and of these, only fingolimod was found to be not safe with respect to human cell viability. These compounds were predicted active on different molecular targets, abiraterone was predicted to interact with a putative liver-stage essential target, hence promising as a transmission-blocking agent. The pipeline had a promising 25% hit rate considering the proteome-scale and use of cost-effective approaches. Secondly, we focused on Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) using a more extensive screening pipeline to overcome some of the current in silico screening limitations. Starting from the ZINC lead-like library of ~3M, hierarchical ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS) approaches with molecular docking and re-scoring using eleven scoring functions (SFs) were used. Later ranking with an exponential consensus strategy was included. Selected hits were further assessed through Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA), advanced MD sampling in a ligand pulling simulations and (Weighted Histogram Analysis Method) WHAM analysis for umbrella sampling (US) to derive binding free energies. Four leads had better predicted affinities in US than LC5, a 280 nM potent PfDXR inhibitor with ZINC000050633276 showing a promising binding of -20.43 kcal/mol. As shown with fosmidomycin, DXR inhibition offers fast acting compounds fulfilling antimalarials TCP1. Yet, fosmidomycin has a high polarity causing its short half-life and hampering its clinical use. These leads scaffolds are different from fosmidomycin and hence may offer better pharmacokinetic and pharmacodynamic properties and may also be promising for lead optimization. A combined analysis of residues’ contributions to the free energy of binding in MM-PBSA and to steered molecular dynamics (SMD) Fmax indicated GLU233, CYS268, SER270, TRP296, and HIS341 as exploitable for compound optimization. Finally, we updated the SANCDB library with new NPs and their commercially available analogs as a solution to NP availability. The library is extended to 1005 compounds from its initial 600 compounds and the database is integrated to Mcule and Molport APIs for analogs automatic update. The new set may contribute to virtual screening and to antimalarials as the most effective ones have NP origin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Diallo, Bakary N’tji
- Date: 2021-10-29
- Subjects: Plasmodium falciparum , Malaria -- Chemotherapy , Molecular dynamics , Antimalarials , Cheminformatics , Drug development , Ligand binding (Biochemistry) , Plasmodium falciparum1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) , South African Natural Compounds Database
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/192798 , vital:45265 , 10.21504/10962/192798
- Description: Malaria is a millennia-old disease with the first recorded cases dating back to 2700 BC found in Chinese medical records, and later in other civilizations. It has claimed human lives to such an extent that there are a notable associated socio-economic consequences. Currently, according to the World Health Organization (WHO), Africa holds the highest disease burden with 94% of deaths and 82% of cases with P. falciparum having ~100% prevalence. Chemotherapy, such as artemisinin combination therapy, has been and continues to be the work horse in the fight against the disease, together with seasonal malaria chemoprevention and the use of insecticides. Natural products such as quinine and artemisinin are particularly important in terms of their antimalarial activity. The emphasis in current chemotherapy research is the need for time and cost-effective workflows focussed on new mechanisms of action (MoAs) covering the target candidate profiles (TCPs). Despite a decline in cases over the past decades with, countries increasingly becoming certified malaria free, a stalling trend has been observed in the past five years resulting in missing the 2020 Global Technical Strategy (GTS) milestones. With no effective vaccine, a reduction in funding, slower drug approval than resistance emergence from resistant and invasive vectors, and threats in diagnosis with the pfhrp2/3 gene deletion, malaria remains a major health concern. Motivated by these reasons, the primary aim of this work was a contribution to the antimalarial pipeline through in silico approaches focusing on P. falciparum. We first intended an exploration of malarial targets through a proteome scale screening on 36 targets using multiple metrics to account for the multi-objective nature of drug discovery. The continuous growth of structural data offers the ideal scenario for mining new MoAs covering antimalarials TCPs. This was combined with a repurposing strategy using a set of orally available FDA approved drugs. Further, use was made of time- and cost-effective strategies combining QVina-W efficiency metrics that integrate molecular properties, GRIM rescoring for molecular interactions and a hydrogen mass repartitioning (HMR) molecular dynamics (MD) scheme for accelerated development of antimalarials in the context of resistance. This pipeline further integrates a complex ranking for better drug-target selectivity, and normalization strategies to overcome docking scoring function bias. The different metrics, ranking, normalization strategies and their combinations were first assessed using their mean ranking error (MRE). A version combining all metrics was used to select 36 unique protein-ligand complexes, assessed in MD, with the final retention of 25. From the 16 in vitro tested hits of the 25, fingolimod, abiraterone, prazosin, and terazosin showed antiplasmodial activity with IC50 2.21, 3.37, 16.67 and 34.72 μM respectively and of these, only fingolimod was found to be not safe with respect to human cell viability. These compounds were predicted active on different molecular targets, abiraterone was predicted to interact with a putative liver-stage essential target, hence promising as a transmission-blocking agent. The pipeline had a promising 25% hit rate considering the proteome-scale and use of cost-effective approaches. Secondly, we focused on Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) using a more extensive screening pipeline to overcome some of the current in silico screening limitations. Starting from the ZINC lead-like library of ~3M, hierarchical ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS) approaches with molecular docking and re-scoring using eleven scoring functions (SFs) were used. Later ranking with an exponential consensus strategy was included. Selected hits were further assessed through Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA), advanced MD sampling in a ligand pulling simulations and (Weighted Histogram Analysis Method) WHAM analysis for umbrella sampling (US) to derive binding free energies. Four leads had better predicted affinities in US than LC5, a 280 nM potent PfDXR inhibitor with ZINC000050633276 showing a promising binding of -20.43 kcal/mol. As shown with fosmidomycin, DXR inhibition offers fast acting compounds fulfilling antimalarials TCP1. Yet, fosmidomycin has a high polarity causing its short half-life and hampering its clinical use. These leads scaffolds are different from fosmidomycin and hence may offer better pharmacokinetic and pharmacodynamic properties and may also be promising for lead optimization. A combined analysis of residues’ contributions to the free energy of binding in MM-PBSA and to steered molecular dynamics (SMD) Fmax indicated GLU233, CYS268, SER270, TRP296, and HIS341 as exploitable for compound optimization. Finally, we updated the SANCDB library with new NPs and their commercially available analogs as a solution to NP availability. The library is extended to 1005 compounds from its initial 600 compounds and the database is integrated to Mcule and Molport APIs for analogs automatic update. The new set may contribute to virtual screening and to antimalarials as the most effective ones have NP origin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
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
Exploring para-thiophenols to expand the SAR of antimalarial 3-indolylethanones
- Authors: Chisango, Ruramai Lissa
- Date: 2018
- Subjects: Antimalarials , Malaria Chemotherapy , Thiols , Plasmodium falciparum , Blood-brain barrier
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63515 , vital:28428
- Description: According to the WHO, malaria is responsible for over half a million deaths annually especially in populations from disadvantaged settings. Although there has been a documented improvement in the mortality rates, malaria has proved to be a global emergency. Mostly affecting the poor population, this disease is perpetuating a vicious cycle of poverty in the developing world as current preventive measures are not adequate unless adopted in addition to effective treatment. However, there has been a worldwide increase in resistance to available treatment which presents a need for novel, affordable treatment. A study conducted in our laboratory identified two hit thiophenol containing compounds 2.24 and 2.25. These molecules provided initial insight into the SAR and potential pharmacophore of this class of compounds. We decided to further explore these compounds by making bioisosteric replacements to optimize the structure as we monitor the effect of these modifications on the anti-plasmodial activity. The synthetic pathway to form the target compounds of our study comprised of three steps which were initiated by the Friedel-Crafts acetylation of the indoles resulting in compounds 3.5 - 3.7. A bromination step followed which yielded the -bromo ketones (3.8 - 3.11). Some of the thiophenols (3.14 and 3.16) were not readily available in our laboratory and so were synthesized for the final synthetic step. This step involved the nucleophilic displacement of the -bromine to generate the -aryl substituted 3-indolylethanones (3.17 - 3.27). The thioethers displayed improved antimalarial activity from 2.24 and 2.25 against the chloroquine sensitive 3D7 Plasmodium falciparum strain. In addition, these compounds were non-toxic against HeLa cells which indicated this potential novel class of antimalarials is selective for the malaria parasite as hypothesized in the previous study conducted in our laboratory. In an attempt to predict the bioavailability of some of our compounds, in silico studies were conducted revealing that these compounds could be passively absorbed by the gastrointestinal tract, a positive result for bioavailability purposes. However, results from these studies indicate that modifications of these compounds would be necessary to allow for permeation through the blood brain barrier (BBB) for instances when the patient has cerebral malaria. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Date Issued: 2018
- Authors: Chisango, Ruramai Lissa
- Date: 2018
- Subjects: Antimalarials , Malaria Chemotherapy , Thiols , Plasmodium falciparum , Blood-brain barrier
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/63515 , vital:28428
- Description: According to the WHO, malaria is responsible for over half a million deaths annually especially in populations from disadvantaged settings. Although there has been a documented improvement in the mortality rates, malaria has proved to be a global emergency. Mostly affecting the poor population, this disease is perpetuating a vicious cycle of poverty in the developing world as current preventive measures are not adequate unless adopted in addition to effective treatment. However, there has been a worldwide increase in resistance to available treatment which presents a need for novel, affordable treatment. A study conducted in our laboratory identified two hit thiophenol containing compounds 2.24 and 2.25. These molecules provided initial insight into the SAR and potential pharmacophore of this class of compounds. We decided to further explore these compounds by making bioisosteric replacements to optimize the structure as we monitor the effect of these modifications on the anti-plasmodial activity. The synthetic pathway to form the target compounds of our study comprised of three steps which were initiated by the Friedel-Crafts acetylation of the indoles resulting in compounds 3.5 - 3.7. A bromination step followed which yielded the -bromo ketones (3.8 - 3.11). Some of the thiophenols (3.14 and 3.16) were not readily available in our laboratory and so were synthesized for the final synthetic step. This step involved the nucleophilic displacement of the -bromine to generate the -aryl substituted 3-indolylethanones (3.17 - 3.27). The thioethers displayed improved antimalarial activity from 2.24 and 2.25 against the chloroquine sensitive 3D7 Plasmodium falciparum strain. In addition, these compounds were non-toxic against HeLa cells which indicated this potential novel class of antimalarials is selective for the malaria parasite as hypothesized in the previous study conducted in our laboratory. In an attempt to predict the bioavailability of some of our compounds, in silico studies were conducted revealing that these compounds could be passively absorbed by the gastrointestinal tract, a positive result for bioavailability purposes. However, results from these studies indicate that modifications of these compounds would be necessary to allow for permeation through the blood brain barrier (BBB) for instances when the patient has cerebral malaria. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Date Issued: 2018
Identification of novel compounds against Plasmodium falciparum Cytochrome bc1 Complex inhibiting the trans-membrane electron transfer pathway: an In Silico study
- Authors: Chebon, Lorna Jemosop
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium falciparum , Molecular dynamics , Antimalarials , Molecules Models , Docking , Cytochromes , Drug resistance , Computer simulation , Drugs Computer-aided design , System analysis
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365666 , vital:65774 , DOI https://doi.org/10.21504/10962/365666
- Description: Malaria continues to be a burden globally with a myriad of challenges deterring eradication efforts. With most antimalarials facing drug resistance, such as atovaquone (ATQ), alternative compounds that can withstand resistance are warranted. The Plasmodium falciparum cytochrome b (PfCytb), a subunit of P. falciparum cytochrome bc1 complex, is a validated drug target. Structurally, cytochrome b, cytochrome c1, and iron sulphur protein (ISP) subunits form the catalytic domain of the protein complex having heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors. These cofactos have redox centres to aid in the electron transfer (ET) process. These subunits promote ET mainly through the enzyme’s ubiquinol oxidation (Qo) and ubiquinone reduction (Qi) processes in the catalytic domain. ATQ drug has been used in the prevention and treatment of uncomplicated malaria by targeting PfCytb protein. Once the mitochondrial transmembrane ET pathway is inhibited, it causes a collapse in its membrane potential. Previously reported ATQ drug resistance has been associated with the point mutations Y268C, Y268N and Y268S. Thus, in finding alternatives to the ATQ drug, this research aimed to: i) employ in silico approaches incorporating protein into phospholipid bilayer for the first time to understand the parasites’ resistance mechanism; ii) determine any sequence and structural differences that could be explored in drug design studies; and iii) screen for PfCytb-iron sulphur protein (Cytb-ISP) hit compounds from South African natural compound database (SANCDB) and Medicines for Malaria Venture (MMV) that can withstand the identified mutations. Using computational tools, comparative sequence and structural analyses were performed on the cytochrome b protein, where the ultimate focus was on P. falciparum cytochrome b and its human homolog. Through multiple sequence alignment, motif discovery and phylogeny, differences between P. falciparum and H. sapiens cytochrome b were identified. Protein modelling of both P. falciparum and H. sapiens cytochrome b - iron sulphur protein (PfCytb-ISP and HsCytb-ISP) was performed. Results showed that at the sequence level, there were few amino acid residue differences because the protein is highly conserved. Important to note is the four-residue deletion in Plasmodium spp. absent in the human homolog. Motif analysis discovered five unique motifs in P. falciparum cytochrome b protein which were mapped onto the predicted protein model. These motifs were not in regions of functional importance; hence their function is still unknown. At a structural level, the four-residue deletion was observed to alter the Qo substrate binding pocket as reported in previous studies and confirmed in this study. This deletion resulted in a 0.83 Å structural displacement. Also, there are currently no in silico studies that have performed experiments with P. falciparum cytochrome b protein incorporated into a phospholipid bilayer. Using 350 ns molecular dynamics (MD) simulations of the holo and ATQ-bound systems, the study highlighted the resistance mechanism of the parasite protein where the loss of active site residue-residue interactions was identified, all linked to the three mutations. The identified compromised interactions are likely to destabilise the protein’s function, specifically in the Qo substrate binding site. This showed the possible effect of mutations on ATQ drug activity, where all three mutations were reported to share a similar resistance mechanism. Thereafter, this research work utilised in silico approaches where both Qo active site and interface pocket were targeted by screening the South African natural compounds database (SANCDB) and Medicines for Malaria Venture (MMV) compounds to identify novel selective hits. SANCDB compounds are known for their structural complexity that preserves the potency of the drug molecule. Both SANCDB and MMV compounds have not been explored as inhibitors against the PfCytb drug target. Molecular docking, molecular dynamics (MD) simulations, principal component, and dynamic residue network (DRN; global and local) analyses were utilised to identify and confirm the potential selective inhibitors. Docking results identified compounds that bound selectively onto PfCytb-ISP with a binding energy ≤ -8.7 kcal/mol-1. Further, this work validated a total of eight potential selective compounds to inhibit PfCytb-ISP protein (Qo active site) not only in the wild-type but also in the presence of the point mutations Y268C, Y268N and Y268S. The selective binding of these hit compounds could be linked to the differences reported at sequence/residue level in chapter 3. DRN and residue contact map analyses of the eight compounds in holo and ligand-bound systems revealed reduced residue interactions and decreased protein communication. This suggests that the eight compounds show the possibility of inhibiting the parasite and disrupting important residue-residue interactions. Additionally, 13 selective compounds were identified to bind at the protein’s heterodimer interface, where global and local analysis confirmed their effect on active site residues (distal location) as well as on the communication network. Based on the sequence differences between PfCytb and the human homolog, these findings suggest these selective compounds as potential allosteric modulators of the parasite enzyme, which may serve as possible replacements of the already resistant ATQ drug. Therefore, these findings pave the way for further in vitro studies to establish their anti-plasmodial inhibition levels. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Chebon, Lorna Jemosop
- Date: 2022-10-14
- Subjects: Malaria , Plasmodium falciparum , Molecular dynamics , Antimalarials , Molecules Models , Docking , Cytochromes , Drug resistance , Computer simulation , Drugs Computer-aided design , System analysis
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365666 , vital:65774 , DOI https://doi.org/10.21504/10962/365666
- Description: Malaria continues to be a burden globally with a myriad of challenges deterring eradication efforts. With most antimalarials facing drug resistance, such as atovaquone (ATQ), alternative compounds that can withstand resistance are warranted. The Plasmodium falciparum cytochrome b (PfCytb), a subunit of P. falciparum cytochrome bc1 complex, is a validated drug target. Structurally, cytochrome b, cytochrome c1, and iron sulphur protein (ISP) subunits form the catalytic domain of the protein complex having heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors. These cofactos have redox centres to aid in the electron transfer (ET) process. These subunits promote ET mainly through the enzyme’s ubiquinol oxidation (Qo) and ubiquinone reduction (Qi) processes in the catalytic domain. ATQ drug has been used in the prevention and treatment of uncomplicated malaria by targeting PfCytb protein. Once the mitochondrial transmembrane ET pathway is inhibited, it causes a collapse in its membrane potential. Previously reported ATQ drug resistance has been associated with the point mutations Y268C, Y268N and Y268S. Thus, in finding alternatives to the ATQ drug, this research aimed to: i) employ in silico approaches incorporating protein into phospholipid bilayer for the first time to understand the parasites’ resistance mechanism; ii) determine any sequence and structural differences that could be explored in drug design studies; and iii) screen for PfCytb-iron sulphur protein (Cytb-ISP) hit compounds from South African natural compound database (SANCDB) and Medicines for Malaria Venture (MMV) that can withstand the identified mutations. Using computational tools, comparative sequence and structural analyses were performed on the cytochrome b protein, where the ultimate focus was on P. falciparum cytochrome b and its human homolog. Through multiple sequence alignment, motif discovery and phylogeny, differences between P. falciparum and H. sapiens cytochrome b were identified. Protein modelling of both P. falciparum and H. sapiens cytochrome b - iron sulphur protein (PfCytb-ISP and HsCytb-ISP) was performed. Results showed that at the sequence level, there were few amino acid residue differences because the protein is highly conserved. Important to note is the four-residue deletion in Plasmodium spp. absent in the human homolog. Motif analysis discovered five unique motifs in P. falciparum cytochrome b protein which were mapped onto the predicted protein model. These motifs were not in regions of functional importance; hence their function is still unknown. At a structural level, the four-residue deletion was observed to alter the Qo substrate binding pocket as reported in previous studies and confirmed in this study. This deletion resulted in a 0.83 Å structural displacement. Also, there are currently no in silico studies that have performed experiments with P. falciparum cytochrome b protein incorporated into a phospholipid bilayer. Using 350 ns molecular dynamics (MD) simulations of the holo and ATQ-bound systems, the study highlighted the resistance mechanism of the parasite protein where the loss of active site residue-residue interactions was identified, all linked to the three mutations. The identified compromised interactions are likely to destabilise the protein’s function, specifically in the Qo substrate binding site. This showed the possible effect of mutations on ATQ drug activity, where all three mutations were reported to share a similar resistance mechanism. Thereafter, this research work utilised in silico approaches where both Qo active site and interface pocket were targeted by screening the South African natural compounds database (SANCDB) and Medicines for Malaria Venture (MMV) compounds to identify novel selective hits. SANCDB compounds are known for their structural complexity that preserves the potency of the drug molecule. Both SANCDB and MMV compounds have not been explored as inhibitors against the PfCytb drug target. Molecular docking, molecular dynamics (MD) simulations, principal component, and dynamic residue network (DRN; global and local) analyses were utilised to identify and confirm the potential selective inhibitors. Docking results identified compounds that bound selectively onto PfCytb-ISP with a binding energy ≤ -8.7 kcal/mol-1. Further, this work validated a total of eight potential selective compounds to inhibit PfCytb-ISP protein (Qo active site) not only in the wild-type but also in the presence of the point mutations Y268C, Y268N and Y268S. The selective binding of these hit compounds could be linked to the differences reported at sequence/residue level in chapter 3. DRN and residue contact map analyses of the eight compounds in holo and ligand-bound systems revealed reduced residue interactions and decreased protein communication. This suggests that the eight compounds show the possibility of inhibiting the parasite and disrupting important residue-residue interactions. Additionally, 13 selective compounds were identified to bind at the protein’s heterodimer interface, where global and local analysis confirmed their effect on active site residues (distal location) as well as on the communication network. Based on the sequence differences between PfCytb and the human homolog, these findings suggest these selective compounds as potential allosteric modulators of the parasite enzyme, which may serve as possible replacements of the already resistant ATQ drug. Therefore, these findings pave the way for further in vitro studies to establish their anti-plasmodial inhibition levels. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
In silico study of Plasmodium 1-deoxy-dxylulose 5-phosphate reductoisomerase (DXR) for identification of novel inhibitors from SANCDB
- Authors: Diallo, Bakary N'tji
- Date: 2018
- Subjects: Plasmodium 1-deoxy-dxylulose 5-phosphate reductoisomerase , Isoprenoids , Plasmodium , Antimalarials , Malaria -- Chemotherapy , Molecules -- Models , Molecular dynamics , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64012 , vital:28523
- Description: Malaria remains a major health concern with a complex parasite constantly developing resistance to the different drugs introduced to treat it, threatening the efficacy of the current ACT treatment recommended by WHO (World Health Organization). Different antimalarial compounds with different mechanisms of action are ideal as this decreases chances of resistance occurring. Inhibiting DXR and consequently the MEP pathway is a good strategy to find a new antimalarial with a novel mode of action. From literature, all the enzymes of the MEP pathway have also been shown to be indispensable for the synthesis of isoprenoids. They have been validated as drug targets and the X-ray structure of each of the enzymes has been solved. DXR is a protein which catalyses the second step of the MEP pathway. There are currently 255 DXR inhibitors in the Binding Database (accessed November 2017) generally based on the fosmidomycin structural scaffold and thus often showing poor drug likeness properties. This study aims to research new DXR inhibitors using in silico techniques. We analysed the protein sequence and built 3D models in close and open conformations for the different Plasmodium sequences. Then SANCDB compounds were screened to identify new potential DXR inhibitors with new chemical scaffolds. Finally, the identified hits were submitted to molecular dynamics studies, preceded by a parameterization of the manganese atom in the protein active site.
- Full Text:
- Date Issued: 2018
- Authors: Diallo, Bakary N'tji
- Date: 2018
- Subjects: Plasmodium 1-deoxy-dxylulose 5-phosphate reductoisomerase , Isoprenoids , Plasmodium , Antimalarials , Malaria -- Chemotherapy , Molecules -- Models , Molecular dynamics , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64012 , vital:28523
- Description: Malaria remains a major health concern with a complex parasite constantly developing resistance to the different drugs introduced to treat it, threatening the efficacy of the current ACT treatment recommended by WHO (World Health Organization). Different antimalarial compounds with different mechanisms of action are ideal as this decreases chances of resistance occurring. Inhibiting DXR and consequently the MEP pathway is a good strategy to find a new antimalarial with a novel mode of action. From literature, all the enzymes of the MEP pathway have also been shown to be indispensable for the synthesis of isoprenoids. They have been validated as drug targets and the X-ray structure of each of the enzymes has been solved. DXR is a protein which catalyses the second step of the MEP pathway. There are currently 255 DXR inhibitors in the Binding Database (accessed November 2017) generally based on the fosmidomycin structural scaffold and thus often showing poor drug likeness properties. This study aims to research new DXR inhibitors using in silico techniques. We analysed the protein sequence and built 3D models in close and open conformations for the different Plasmodium sequences. Then SANCDB compounds were screened to identify new potential DXR inhibitors with new chemical scaffolds. Finally, the identified hits were submitted to molecular dynamics studies, preceded by a parameterization of the manganese atom in the protein active site.
- Full Text:
- Date Issued: 2018
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
Malarial drug targets cysteine proteases as hemoglobinases
- Authors: Mokoena, Fortunate
- Date: 2012
- Subjects: Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4005 , http://hdl.handle.net/10962/d1004065 , Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Description: Malaria has consistently been rated as the worst parasitic disease in the world. This disease affects an estimated 5 billion households annually. Malaria has a high mortality rate leading to distorted socio-economic development of the world at large. The major challenge pertaining to malaria is its continuous and rapid spread together with the emergence of drug resistance in Plasmodium species (vector agent of the disease). For this reason, researchers throughout the world are following new leads for possible drug targets and therefore, investigating ways of curbing the spread of the disease. Cysteine proteases have emerged as potential antimalarial chemotherapeutic targets. These particular proteases are found in all living organisms, Plasmodium cysteine proteases are known to degrade host hemoglobin during the life cycle of the parasite within the human host. The main objective of this study was to use various in silico methods to analyze the hemoglobinase function of cysteine proteases in P. falciparum and P. vivax. Falcipain-2 (FP2) of P. falciparum is the best characterized of these enzymes, it is a validated drug target. Both the three-dimensional structures of FP2 and its close homologue falcipain-3 (FP3) have been solved by the experimental technique X-ray crystallography. However, the homologue falcipain-2 (FP2’)’ and orthologues from P.vivax vivapain-2 (VP2) and vivapain-3 (VP3) have yet to be elucidated by experimental techniques. In an effort to achieve the principal goal of the study, homology models of the protein structures not already elucidated by experimental methods (FP2’, VP2 and VP3) were calculated using the well known spatial restraint program MODELLER. The derived models, FP2 and FP3 were docked to hemoglobin (their natural substrate). The protein-protein docking was done using the unbound docking program ZDOCK. The substrate-enzyme interactions were analyzed and amino acids involved in binding were observed. It is anticipated that the results obtained from the study will help focus inhibitor design for potential drugs against malaria. The residues found in both the P. falciparum and P. vivax cysteine proteases involved in hemoglobin binding have been identified and some of these are proposed to be the main focus for the design of a peptidomimetric inhibitor.
- Full Text:
- Date Issued: 2012
- Authors: Mokoena, Fortunate
- Date: 2012
- Subjects: Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4005 , http://hdl.handle.net/10962/d1004065 , Malaria -- Chemotherapy , Antimalarials , Hemoglobin , Proteolytic enzymes , Cysteine proteinases , Plasmodium falciparum , Plasmodium vivax , Papain
- Description: Malaria has consistently been rated as the worst parasitic disease in the world. This disease affects an estimated 5 billion households annually. Malaria has a high mortality rate leading to distorted socio-economic development of the world at large. The major challenge pertaining to malaria is its continuous and rapid spread together with the emergence of drug resistance in Plasmodium species (vector agent of the disease). For this reason, researchers throughout the world are following new leads for possible drug targets and therefore, investigating ways of curbing the spread of the disease. Cysteine proteases have emerged as potential antimalarial chemotherapeutic targets. These particular proteases are found in all living organisms, Plasmodium cysteine proteases are known to degrade host hemoglobin during the life cycle of the parasite within the human host. The main objective of this study was to use various in silico methods to analyze the hemoglobinase function of cysteine proteases in P. falciparum and P. vivax. Falcipain-2 (FP2) of P. falciparum is the best characterized of these enzymes, it is a validated drug target. Both the three-dimensional structures of FP2 and its close homologue falcipain-3 (FP3) have been solved by the experimental technique X-ray crystallography. However, the homologue falcipain-2 (FP2’)’ and orthologues from P.vivax vivapain-2 (VP2) and vivapain-3 (VP3) have yet to be elucidated by experimental techniques. In an effort to achieve the principal goal of the study, homology models of the protein structures not already elucidated by experimental methods (FP2’, VP2 and VP3) were calculated using the well known spatial restraint program MODELLER. The derived models, FP2 and FP3 were docked to hemoglobin (their natural substrate). The protein-protein docking was done using the unbound docking program ZDOCK. The substrate-enzyme interactions were analyzed and amino acids involved in binding were observed. It is anticipated that the results obtained from the study will help focus inhibitor design for potential drugs against malaria. The residues found in both the P. falciparum and P. vivax cysteine proteases involved in hemoglobin binding have been identified and some of these are proposed to be the main focus for the design of a peptidomimetric inhibitor.
- Full Text:
- Date Issued: 2012
Marine anti-malarial isonitriles : a synthetic and computational study
- Authors: Adendorff, Matthew Ralph
- Date: 2011 , 2010-05-17
- Subjects: Isocyanides , Isocyanates , Marine pharmacology , Antimalarials , Antimalarials -- Development , Drug development
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4398 , http://hdl.handle.net/10962/d1006674 , Isocyanides , Isocyanates , Marine pharmacology , Antimalarials , Antimalarials -- Development , Drug development
- Description: The development of Plasmodium falciparum malarial resistance to the current armoury of anti-malarial drugs requires the development of new treatments to help combat this disease. The marine environment is a well established source of potential pharmaceuticals. Of interest to us are isonitrile, isocyanate and isothiocyanate compounds isolated from marine sponges and molluscs which have exhibited nano-molar anti-plasmodial activities. Through quantitative structure-activity relation studies (QSAR), a literature precedent exists for a pseudoreceptor model from which a pharmacophore for the design of novel anti-malarial agents was proposed. The current theory suggests that these marine compounds exert their inhibitory action through interfering with the heme detoxification pathway in P. falciparum. We propose that the computational methods used to draw detailed conclusions about the mode of action of these marine compounds were inadequate. This thesis addresses this problem using contemporary computational methodologies and seeks to propose a more robust method for the rational design of new anti-malarial drug compounds that inhibit heme polymerization to hemozoin. In order to investigate the interactions of the marine compounds with their heme targets, a series of modern computational procedures were formulated, validated and then applied to theoretical systems. The validations of these algorithms, before their application to the marine compound-heme systems, were achieved through two case studies. The first was used to investigate the applicability of the statistical docking algorithm AutoDock to be used for the exploration of conformational space around the heme target. A theoretical P. falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) enzyme model, constructed by the Biochemistry Department at Rhodes University, provided the ideal model to validate the AutoDock program. The protein model was accordingly subjected to rigorous docking simulations with over 30 different ligand molecules using the AutoDock algorithm which allowed for the docking algorithm’s limitations to be ascertained and improved upon. This investigation facilitated the successful validation of the protein model, which can now be used for the rational design of new PfDXR-inhibiting anti-plasmodial compounds, as well as enabling us to propose an improvement of the docking algorithm for application to the heme systems. The second case study was used to investigate the applicability of an ab initio molecular dynamics algorithm for simulation of bond breaking/forming events between the marine compounds and their heme target. This validation involved the exploration of intermolecular interactions in a naturally occurring nonoligomeric zipper using the Car-Parrinello Molecular Dynamics (CPMD) method. This study allowed us to propose a model for the intermolecular forces responsible for zipper self-assembly and showcased the CPMD method’s abilities to simulate and predict bond forming/breaking events. Data from the computational analyses suggested that the interactions between marine isonitriles, isocyanates and isothiocyanates occur through bond-less electrostatic attractions rather than through formal intermolecular bonds as had been previously suggested. Accordingly, a simple bicyclic tertiary isonitrile (5.14) was synthesized using Kitano et al’s relatively underutilized isonitrile synthetic method for the conversion of tertiary alcohols to their corresponding isonitriles. This compound’s potential for heme detoxification inhibition was then explored in vitro via the pyridine-hemochrome assay. The assay data suggested that the synthesized isonitrile was capable of inhibiting heme polymerization in a similar fashion to the known inhibitor chloroquine. Attempts to synthesize tricyclic analogues of 5.14 were unsuccessful and highlighted the limitation of Kitano et al’s isonitrile synthetic methodology.
- Full Text:
- Date Issued: 2011
- Authors: Adendorff, Matthew Ralph
- Date: 2011 , 2010-05-17
- Subjects: Isocyanides , Isocyanates , Marine pharmacology , Antimalarials , Antimalarials -- Development , Drug development
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4398 , http://hdl.handle.net/10962/d1006674 , Isocyanides , Isocyanates , Marine pharmacology , Antimalarials , Antimalarials -- Development , Drug development
- Description: The development of Plasmodium falciparum malarial resistance to the current armoury of anti-malarial drugs requires the development of new treatments to help combat this disease. The marine environment is a well established source of potential pharmaceuticals. Of interest to us are isonitrile, isocyanate and isothiocyanate compounds isolated from marine sponges and molluscs which have exhibited nano-molar anti-plasmodial activities. Through quantitative structure-activity relation studies (QSAR), a literature precedent exists for a pseudoreceptor model from which a pharmacophore for the design of novel anti-malarial agents was proposed. The current theory suggests that these marine compounds exert their inhibitory action through interfering with the heme detoxification pathway in P. falciparum. We propose that the computational methods used to draw detailed conclusions about the mode of action of these marine compounds were inadequate. This thesis addresses this problem using contemporary computational methodologies and seeks to propose a more robust method for the rational design of new anti-malarial drug compounds that inhibit heme polymerization to hemozoin. In order to investigate the interactions of the marine compounds with their heme targets, a series of modern computational procedures were formulated, validated and then applied to theoretical systems. The validations of these algorithms, before their application to the marine compound-heme systems, were achieved through two case studies. The first was used to investigate the applicability of the statistical docking algorithm AutoDock to be used for the exploration of conformational space around the heme target. A theoretical P. falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) enzyme model, constructed by the Biochemistry Department at Rhodes University, provided the ideal model to validate the AutoDock program. The protein model was accordingly subjected to rigorous docking simulations with over 30 different ligand molecules using the AutoDock algorithm which allowed for the docking algorithm’s limitations to be ascertained and improved upon. This investigation facilitated the successful validation of the protein model, which can now be used for the rational design of new PfDXR-inhibiting anti-plasmodial compounds, as well as enabling us to propose an improvement of the docking algorithm for application to the heme systems. The second case study was used to investigate the applicability of an ab initio molecular dynamics algorithm for simulation of bond breaking/forming events between the marine compounds and their heme target. This validation involved the exploration of intermolecular interactions in a naturally occurring nonoligomeric zipper using the Car-Parrinello Molecular Dynamics (CPMD) method. This study allowed us to propose a model for the intermolecular forces responsible for zipper self-assembly and showcased the CPMD method’s abilities to simulate and predict bond forming/breaking events. Data from the computational analyses suggested that the interactions between marine isonitriles, isocyanates and isothiocyanates occur through bond-less electrostatic attractions rather than through formal intermolecular bonds as had been previously suggested. Accordingly, a simple bicyclic tertiary isonitrile (5.14) was synthesized using Kitano et al’s relatively underutilized isonitrile synthetic method for the conversion of tertiary alcohols to their corresponding isonitriles. This compound’s potential for heme detoxification inhibition was then explored in vitro via the pyridine-hemochrome assay. The assay data suggested that the synthesized isonitrile was capable of inhibiting heme polymerization in a similar fashion to the known inhibitor chloroquine. Attempts to synthesize tricyclic analogues of 5.14 were unsuccessful and highlighted the limitation of Kitano et al’s isonitrile synthetic methodology.
- Full Text:
- Date Issued: 2011
Structural analysis of prodomain inhibition of cysteine proteases in plasmodium species
- Authors: Njuguna, Joyce Njoki
- Date: 2012
- Subjects: Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4021 , http://hdl.handle.net/10962/d1004081 , Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Description: Plasmodium is a genus of parasites causing malaria, a virulent protozoan infection in humans resulting in over a million deaths annually. Treatment of malaria is increasingly limited by parasite resistance to available drugs. Hence, there is a need to identify new drug targets and authenticate antimalarial compounds that act on these targets. A relatively new therapeutic approach targets proteolytic enzymes responsible for parasite‟s invasion, rupture and hemoglobin degradation at the erythrocytic stage of infection. Cysteine proteases (CPs) are essential for these crucial roles in the intraerythrocytic parasite. CPs are a diverse group of enzymes subdivided into clans and further subdivided into families. Our interest is in Clan CA, papain family C1 proteases, whose members play numerous roles in human and parasitic metabolism. These proteases are produced as zymogens having an N-terminal extension known as the prodomain which regulates the protease activity by selectively inhibiting its active site, preventing substrate access. A Clan CA protease Falcipain-2 (FP-2) of Plasmodium falciparum is a validated drug target but little is known of its orthologs in other malarial Plasmodium species. This study uses various structural bioinformatics approaches to characterise the prodomain‟s regulatory effect in FP-2 and its orthologs in Plasmodium species (P. vivax, P. berghei, P. knowlesi, P. ovale, P. chabaudi and P. yoelii). This was in an effort to discover short peptides with essential residues to mimic the prodomain‟s inhibition of these proteases, as potential peptidomimetic therapeutic agents. Residues in the prodomain region that spans over the active site are most likely to interact with the subsite residues inhibiting the protease. Sequence analysis revealed conservation of residues in this region of Plasmodium proteases that differed significantly in human proteases. Further prediction of the 3D structure of these proteases by homology modelling allowed visualisation of these interactions revealing differences between parasite and human proteases which will lead to significant contribution in structure based malarial inhibitor design.
- Full Text:
- Date Issued: 2012
- Authors: Njuguna, Joyce Njoki
- Date: 2012
- Subjects: Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4021 , http://hdl.handle.net/10962/d1004081 , Plasmodium , Cysteine proteinases , Proteolytic enzymes , Malaria -- Chemotherapy , Antimalarials , Plasmodium falciparum
- Description: Plasmodium is a genus of parasites causing malaria, a virulent protozoan infection in humans resulting in over a million deaths annually. Treatment of malaria is increasingly limited by parasite resistance to available drugs. Hence, there is a need to identify new drug targets and authenticate antimalarial compounds that act on these targets. A relatively new therapeutic approach targets proteolytic enzymes responsible for parasite‟s invasion, rupture and hemoglobin degradation at the erythrocytic stage of infection. Cysteine proteases (CPs) are essential for these crucial roles in the intraerythrocytic parasite. CPs are a diverse group of enzymes subdivided into clans and further subdivided into families. Our interest is in Clan CA, papain family C1 proteases, whose members play numerous roles in human and parasitic metabolism. These proteases are produced as zymogens having an N-terminal extension known as the prodomain which regulates the protease activity by selectively inhibiting its active site, preventing substrate access. A Clan CA protease Falcipain-2 (FP-2) of Plasmodium falciparum is a validated drug target but little is known of its orthologs in other malarial Plasmodium species. This study uses various structural bioinformatics approaches to characterise the prodomain‟s regulatory effect in FP-2 and its orthologs in Plasmodium species (P. vivax, P. berghei, P. knowlesi, P. ovale, P. chabaudi and P. yoelii). This was in an effort to discover short peptides with essential residues to mimic the prodomain‟s inhibition of these proteases, as potential peptidomimetic therapeutic agents. Residues in the prodomain region that spans over the active site are most likely to interact with the subsite residues inhibiting the protease. Sequence analysis revealed conservation of residues in this region of Plasmodium proteases that differed significantly in human proteases. Further prediction of the 3D structure of these proteases by homology modelling allowed visualisation of these interactions revealing differences between parasite and human proteases which will lead to significant contribution in structure based malarial inhibitor design.
- Full Text:
- Date Issued: 2012
Studies towards the development of novel antimalarial agents
- Authors: Adeyemi, Christiana Modupe
- Date: 2015
- Subjects: Antimalarials , Malaria , Drug resistance , Drug development , Enzyme inhibitors , Plasmodium
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54645 , vital:26596
- Description: Considerable efforts have been made in the modification of existing antimalarial drugs, and the support of incentive programmes have led to a drastic decrease in malaria cases reported by WHO during the past 6 years. However, the development of drug resistance threatens the eradication of this deadly disease and has prompted research on the synthesis of novel antimalarial drugs. Our research has involved the design and synthesis of novel benzylated phosphonate esters as potential 1-deoxy-D-xylose-5-phosphate reductoisomerase (DXR) inhibitors. A series of amidoalkylphosphonate esters were obtained by reacting various 3-subsituted anilines and heterocyclic amines with chloroalkanoyl chlorides and reacting the resulting chloroalkanamides with triethyl phosphite using Michaelis-Arbuzov methodology. Benzylation of the phosphonate esters afforded a series of novel N-benzylated derivatives in good yields and these compounds were fully characterised by NMR and HRMS methods. Several approaches to the introduction of a benzyl group at the C-2 position of the phosphonate ester derivatives have been explored, leading unexpectedly to the isolation of unprecedented tetrahydrofuranyl derivatives. Studies towards the preparation of potential bi-functional PfDXR / HIV-1 RT inhibitors have also been initiated. Preliminary in silico docking studies of selected non-benzylated and benzylated phosphonated derivatives into the Pf-DXR active-site has provided useful insight into the binding potential of these ligands. Bioassays have revealed a very low toxicity for all the synthesised phosphonated compounds and a number of these ligands also exhibit a promising inhibitory activity against the Plasmodium parasite.
- Full Text:
- Date Issued: 2015
- Authors: Adeyemi, Christiana Modupe
- Date: 2015
- Subjects: Antimalarials , Malaria , Drug resistance , Drug development , Enzyme inhibitors , Plasmodium
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54645 , vital:26596
- Description: Considerable efforts have been made in the modification of existing antimalarial drugs, and the support of incentive programmes have led to a drastic decrease in malaria cases reported by WHO during the past 6 years. However, the development of drug resistance threatens the eradication of this deadly disease and has prompted research on the synthesis of novel antimalarial drugs. Our research has involved the design and synthesis of novel benzylated phosphonate esters as potential 1-deoxy-D-xylose-5-phosphate reductoisomerase (DXR) inhibitors. A series of amidoalkylphosphonate esters were obtained by reacting various 3-subsituted anilines and heterocyclic amines with chloroalkanoyl chlorides and reacting the resulting chloroalkanamides with triethyl phosphite using Michaelis-Arbuzov methodology. Benzylation of the phosphonate esters afforded a series of novel N-benzylated derivatives in good yields and these compounds were fully characterised by NMR and HRMS methods. Several approaches to the introduction of a benzyl group at the C-2 position of the phosphonate ester derivatives have been explored, leading unexpectedly to the isolation of unprecedented tetrahydrofuranyl derivatives. Studies towards the preparation of potential bi-functional PfDXR / HIV-1 RT inhibitors have also been initiated. Preliminary in silico docking studies of selected non-benzylated and benzylated phosphonated derivatives into the Pf-DXR active-site has provided useful insight into the binding potential of these ligands. Bioassays have revealed a very low toxicity for all the synthesised phosphonated compounds and a number of these ligands also exhibit a promising inhibitory activity against the Plasmodium parasite.
- Full Text:
- Date Issued: 2015
Synthesis and biolgical screening of potential plasmodium falciparum DXR inhibitors
- Authors: Adeyemi, Christiana Modupe
- Date: 2017-04
- Subjects: Plasmodium falciparum , Enzyme inhibitors , Malaria , Antimalarials , Drug development , Malaria -- Chemotherapy , Isopentenoids -- Synthesis , Fosmidomycin , 1-Deoxy-D-xylulose 5-phosphate
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61790 , vital:28060
- Description: The non-mevalonate isoprenoid pathway, also known as the 1-deoxy-D-xylulose-5- phosphate DXP pathway, is absent in humans, but present in the anopheles mosquito responsible for the transmission of malaria. DXP reductoisomerase - a key enzyme in the DXP pathway in Plasmodium falciparum (PfDXR) has been identified as a target for the design of novel anti-malarial drugs. Fosmidomycin and its acetyl analogue (FR900098) are known to be inhibitors of PfDXR and, in this study, synthetic variations of the fosmidomycin scaffold have led to four series of novel analogues. Particular attention has been centred on the introduction of various substituted benzyl groups in each of these series in order to occupy a recently discovered vacant pocket in the PfDXR active-site and thus enhance ligand-enzyme binding. In the process 160 ligands and precursors have been prepared, no less than 119 of them novel. Fistly, a series of C-benzylated phosphonate esters and phosphonic acids were synthesised, in which the fosmidomycin hydroxamate Mg2+- coordinating moiety was replaced by an amide funtionality and the number of methylene groups in the “hydrophobic patch” between the phosphonate and the hydroxamate moiety was decreased from two to one. Several approaches were explored for this series, the most successful involving reaction of 3- substituted anilines with a-bromo propanoic acid in the presence of the coupling agent 1,1'- carbonyldiimidazole (CDI), followed by Michaelis-Arbuzov phosphonation using triethyl phosphite. Reaction of the resulting chiral phosphonate esters with bromotrimethylsilane gave the corresponding phosphonic acids in good yields. In order to obviate chirality issues, a second series of potential “reverse” fosmidomycin analogues was synthesised by replacing the methylene group adjacent to the the phosphonate moiety with a nitrogen atom. Deprotonation, alkylation and phosphorylation of various amines gave diethyl #-benzylphosphoramidate ester intermediate. Aza-Michael addition of these intermediates, followed by hydrolysis gave the corresponding carboxylic acids which could be reacted with different hydroxylamine hydrochloride derivatives to afford the novel hydroxamic acid derivatives in good yields. Thirdly, a series of a novel #-benzylated phosphoramidate derivatives were prepared as aza- FR900098 analogues. Alkylation of different amines using bromoacetalde-hyde diethylacetal gave a series of N-benzyl-2,2-diethoxyethylamine compounds, which were then elaborated via a futher six steps to afford novel #-benzylated phosphoramidate derivatives. Finally, in order to ensure syn-orientation of the donor atoms in the Mg - coordinating group and, at the same time, introduce conformational constraints in the ligand, the hydrophobic patch and the hydroxamate moiety were replaced by cyclic systems. Several approaches towards the synthesis of such conformationally constrained phosphoramidate analogues from maleic anhydride led to the unexpected isolation of an unprecedented acyclic furfuryl compound, and 1H NMR and DFT-level theoretical studies have been initiated to explore the reaction sequence. A series of #-benzylated phosphoramidate derivatives containing dihydroxy aromatic rings (as the conformationally constrained groups) to replace the hydroxamate moiety, were successfully obtained in six steps from the starting material, 3,4-dihydroxylbenzaldehyde. While in vitro assays have been conducted on all of the synthesised compounds, and some of the ligands show promising anti-malarial inhibitory activity - most especially the conformationally constrained cyclic #-benzylated phosphoramidate series. Interestingly, a number of these compounds has also shown activity against T.brucei - the causative agent of sleeping sickness. In silico docking studies of selected compounds has revealed the capacity of some of the ligands to bind effectively in the PfDXR active-site with the newly introduced benzyl group occupying the adjacent vacant pocket. The physico-chemical properties of these ligands were also explored in order to predict the oral-bioavailability. Most of the ligands obeyed the Lipinski rule of 5, while QSAR methods have been used in an attempt to correlate structural variations and calculated molecular properties with the bioassay data. , Thesis (PhD) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Date Issued: 2017-04
- Authors: Adeyemi, Christiana Modupe
- Date: 2017-04
- Subjects: Plasmodium falciparum , Enzyme inhibitors , Malaria , Antimalarials , Drug development , Malaria -- Chemotherapy , Isopentenoids -- Synthesis , Fosmidomycin , 1-Deoxy-D-xylulose 5-phosphate
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61790 , vital:28060
- Description: The non-mevalonate isoprenoid pathway, also known as the 1-deoxy-D-xylulose-5- phosphate DXP pathway, is absent in humans, but present in the anopheles mosquito responsible for the transmission of malaria. DXP reductoisomerase - a key enzyme in the DXP pathway in Plasmodium falciparum (PfDXR) has been identified as a target for the design of novel anti-malarial drugs. Fosmidomycin and its acetyl analogue (FR900098) are known to be inhibitors of PfDXR and, in this study, synthetic variations of the fosmidomycin scaffold have led to four series of novel analogues. Particular attention has been centred on the introduction of various substituted benzyl groups in each of these series in order to occupy a recently discovered vacant pocket in the PfDXR active-site and thus enhance ligand-enzyme binding. In the process 160 ligands and precursors have been prepared, no less than 119 of them novel. Fistly, a series of C-benzylated phosphonate esters and phosphonic acids were synthesised, in which the fosmidomycin hydroxamate Mg2+- coordinating moiety was replaced by an amide funtionality and the number of methylene groups in the “hydrophobic patch” between the phosphonate and the hydroxamate moiety was decreased from two to one. Several approaches were explored for this series, the most successful involving reaction of 3- substituted anilines with a-bromo propanoic acid in the presence of the coupling agent 1,1'- carbonyldiimidazole (CDI), followed by Michaelis-Arbuzov phosphonation using triethyl phosphite. Reaction of the resulting chiral phosphonate esters with bromotrimethylsilane gave the corresponding phosphonic acids in good yields. In order to obviate chirality issues, a second series of potential “reverse” fosmidomycin analogues was synthesised by replacing the methylene group adjacent to the the phosphonate moiety with a nitrogen atom. Deprotonation, alkylation and phosphorylation of various amines gave diethyl #-benzylphosphoramidate ester intermediate. Aza-Michael addition of these intermediates, followed by hydrolysis gave the corresponding carboxylic acids which could be reacted with different hydroxylamine hydrochloride derivatives to afford the novel hydroxamic acid derivatives in good yields. Thirdly, a series of a novel #-benzylated phosphoramidate derivatives were prepared as aza- FR900098 analogues. Alkylation of different amines using bromoacetalde-hyde diethylacetal gave a series of N-benzyl-2,2-diethoxyethylamine compounds, which were then elaborated via a futher six steps to afford novel #-benzylated phosphoramidate derivatives. Finally, in order to ensure syn-orientation of the donor atoms in the Mg - coordinating group and, at the same time, introduce conformational constraints in the ligand, the hydrophobic patch and the hydroxamate moiety were replaced by cyclic systems. Several approaches towards the synthesis of such conformationally constrained phosphoramidate analogues from maleic anhydride led to the unexpected isolation of an unprecedented acyclic furfuryl compound, and 1H NMR and DFT-level theoretical studies have been initiated to explore the reaction sequence. A series of #-benzylated phosphoramidate derivatives containing dihydroxy aromatic rings (as the conformationally constrained groups) to replace the hydroxamate moiety, were successfully obtained in six steps from the starting material, 3,4-dihydroxylbenzaldehyde. While in vitro assays have been conducted on all of the synthesised compounds, and some of the ligands show promising anti-malarial inhibitory activity - most especially the conformationally constrained cyclic #-benzylated phosphoramidate series. Interestingly, a number of these compounds has also shown activity against T.brucei - the causative agent of sleeping sickness. In silico docking studies of selected compounds has revealed the capacity of some of the ligands to bind effectively in the PfDXR active-site with the newly introduced benzyl group occupying the adjacent vacant pocket. The physico-chemical properties of these ligands were also explored in order to predict the oral-bioavailability. Most of the ligands obeyed the Lipinski rule of 5, while QSAR methods have been used in an attempt to correlate structural variations and calculated molecular properties with the bioassay data. , Thesis (PhD) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Date Issued: 2017-04
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:
- Date Issued: 2014
- 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:
- Date Issued: 2014
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:
- Date Issued: 2015
- 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:
- Date Issued: 2015
Synthesis, characterisation and biological activity of 2-(methylthiomethyl)anilines, 2-(methylthio)anilines, their Schiff-base derivatives and metal(II) (Co, Ni, Cu) complexes
- Olalekan, Temitope Elizabeth
- Authors: Olalekan, Temitope Elizabeth
- Date: 2013
- Subjects: Aniline , Schiff bases , Ligands , Nuclear magnetic resonance spectroscopy , Chelates , X-ray crystallography , Antimalarials
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4562 , http://hdl.handle.net/10962/d1020868
- Description: A series of 31 sulfur-nitrogen donor ligands and 64 metal(II) complexes have been investigated. The thiomethylated aniline ligands 2–(methylthiomethyl)aniline 2MT and 2–(methylthio)aniline 2MA were synthesized with their substituted derivatives (-Me, -MeO, -Cl, -Br, -NO2) to serve as chelating agents. These ligands behave as bidentate ligands with SN donor group with Co(II), Ni(II) and Cu(II). The Co(II) and Ni(II) complexes have the ML2Cl2 molecular formula while the Cu(II) complexes formed with MLCl2 stoichiometry where L is the bidentate ligand. The ligands and their metal(II) complexes have been characterized by elemental analysis and with spectroscopic techniques. The trend observed in the NMR spectra and IR frequencies of the thiomethylated compounds shows there is a significant difference between the 2MT and 2MA series as a result of sulfur lone pairs extending the conjugation of the aromatic ring in the case of the latter. The effect of the position and electronic nature of ring substituent on the NMR shifts of the amine protons is discussed. The 6- and 5-membered chelate complexes formed by the 2MT and 2MA ligands respectively do not show significant diversity in their spectroscopic properties. From the elemental analysis for the Co(II) and Ni(II) complexes, their compositions reveal 1:2 M:L stoichiometry with 2 chlorine atoms from the respective metal salts. In addition, the spectroscopic data are largely indicative of tetragonally distorted structures for these solid complexes. The X-ray crystallography data reveal the Cu(II) complexes exist as square pyramidal dimers and with long Cu–Cl equitorial bonds fit into the tetragonally distorted octahedral structure. The electrolytic nature of Co(II) and Cu(II) complexes in DMF were found to be similar, they behave as non electrolytes in contrast to Ni(II) complexes which are 1:1 electrolytes. The electronic spectra of these metal(II) complexes were found to be different for both their solid forms and in solutions of DMF and DMSO and this has been discussed. The thiomethylated aniline ligands possess the amine and thioether groups which are present in many known biologically active compounds, hence the biological activity of the ligands and their metal complexes were tested against three strains of bacteria and one fungus. The methoxy-substituted derivatives were found to possess better inhibitory activity and this was similarly reflected in the metal(II) complexes. The activity of the complexes can be said to be in the order, Cu(II) > Co(II) > Ni(II). The Schiff-base derivatives were prepared from the ligands and para-methoxysalicylaldehyde and their Cu(II) complexes were synthesized in order to determine their biological activity. The Schiff-base ligands were found to be less active than their parent ligands. The Cu(II) complexes are not soluble in water, DMSO or DMF, as a result and could not be evaluated for their biological activity. Based on the good results from the antimicrobial evaluation, the antiplasmodial activity of some of the Co(II), Ni(II) and Cu(II) complexes of the thiomethylated ligands against Plasmodium falciparum (FCR-3) was determined. At 50 μM concentration level, the Cu(II) complexes show activity equal or better than the prophylactic chloroquine. The Cu(II) complexes with the methoxy-substituted demonstrated exceptional activity but their Co(II) and Ni(II) analogues did not show any activity. The cytotoxicity of the active Cu(II) complexes at 50 μM concentration was determined against the breast cancer cell line (MDA-MB-231). The compounds destroyed the cancer cell in the range of 28–40%, thus showing their preferred activity against the parasitic cell instead of the cancer cell. The selectivity demonstrated by these compounds have shown them to be potential antimalarial agents and this could be further investigated.
- Full Text:
- Date Issued: 2013
- Authors: Olalekan, Temitope Elizabeth
- Date: 2013
- Subjects: Aniline , Schiff bases , Ligands , Nuclear magnetic resonance spectroscopy , Chelates , X-ray crystallography , Antimalarials
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4562 , http://hdl.handle.net/10962/d1020868
- Description: A series of 31 sulfur-nitrogen donor ligands and 64 metal(II) complexes have been investigated. The thiomethylated aniline ligands 2–(methylthiomethyl)aniline 2MT and 2–(methylthio)aniline 2MA were synthesized with their substituted derivatives (-Me, -MeO, -Cl, -Br, -NO2) to serve as chelating agents. These ligands behave as bidentate ligands with SN donor group with Co(II), Ni(II) and Cu(II). The Co(II) and Ni(II) complexes have the ML2Cl2 molecular formula while the Cu(II) complexes formed with MLCl2 stoichiometry where L is the bidentate ligand. The ligands and their metal(II) complexes have been characterized by elemental analysis and with spectroscopic techniques. The trend observed in the NMR spectra and IR frequencies of the thiomethylated compounds shows there is a significant difference between the 2MT and 2MA series as a result of sulfur lone pairs extending the conjugation of the aromatic ring in the case of the latter. The effect of the position and electronic nature of ring substituent on the NMR shifts of the amine protons is discussed. The 6- and 5-membered chelate complexes formed by the 2MT and 2MA ligands respectively do not show significant diversity in their spectroscopic properties. From the elemental analysis for the Co(II) and Ni(II) complexes, their compositions reveal 1:2 M:L stoichiometry with 2 chlorine atoms from the respective metal salts. In addition, the spectroscopic data are largely indicative of tetragonally distorted structures for these solid complexes. The X-ray crystallography data reveal the Cu(II) complexes exist as square pyramidal dimers and with long Cu–Cl equitorial bonds fit into the tetragonally distorted octahedral structure. The electrolytic nature of Co(II) and Cu(II) complexes in DMF were found to be similar, they behave as non electrolytes in contrast to Ni(II) complexes which are 1:1 electrolytes. The electronic spectra of these metal(II) complexes were found to be different for both their solid forms and in solutions of DMF and DMSO and this has been discussed. The thiomethylated aniline ligands possess the amine and thioether groups which are present in many known biologically active compounds, hence the biological activity of the ligands and their metal complexes were tested against three strains of bacteria and one fungus. The methoxy-substituted derivatives were found to possess better inhibitory activity and this was similarly reflected in the metal(II) complexes. The activity of the complexes can be said to be in the order, Cu(II) > Co(II) > Ni(II). The Schiff-base derivatives were prepared from the ligands and para-methoxysalicylaldehyde and their Cu(II) complexes were synthesized in order to determine their biological activity. The Schiff-base ligands were found to be less active than their parent ligands. The Cu(II) complexes are not soluble in water, DMSO or DMF, as a result and could not be evaluated for their biological activity. Based on the good results from the antimicrobial evaluation, the antiplasmodial activity of some of the Co(II), Ni(II) and Cu(II) complexes of the thiomethylated ligands against Plasmodium falciparum (FCR-3) was determined. At 50 μM concentration level, the Cu(II) complexes show activity equal or better than the prophylactic chloroquine. The Cu(II) complexes with the methoxy-substituted demonstrated exceptional activity but their Co(II) and Ni(II) analogues did not show any activity. The cytotoxicity of the active Cu(II) complexes at 50 μM concentration was determined against the breast cancer cell line (MDA-MB-231). The compounds destroyed the cancer cell in the range of 28–40%, thus showing their preferred activity against the parasitic cell instead of the cancer cell. The selectivity demonstrated by these compounds have shown them to be potential antimalarial agents and this could be further investigated.
- Full Text:
- Date Issued: 2013
Synthesis, characterisation and evaluation of benzoxaborole-based hybrids as antiplasmodial agents
- Authors: Gumbo, Maureen
- Date: 2017
- Subjects: Malaria Chemotherapy , Antimalarials , Boron compounds , Drug resistance , Plasmodium falciparum , Drug development
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59193 , vital:27456
- Description: Malaria is a mosquito-borne disease, which continues to pose a threat to the entire humanity. About 40% of the world population is estimated to be at risk of infections by malaria. Despite efforts undertaken by scientific community, government entities and international organizations, malaria is still rampant. The major problem is drug resistance, where the Plasmodium spp have over the past decades developed drug resistance against available drugs. In order to counter this problem, novel antimalarial drugs that are efficacious and with novel mode of action are of great necessity. Benzoxaborole derivatives have been shown to exhibit promising antimalarial activity against Plasmodium falciparum strains. Previous studies reported on the compounds such as 6-(2- (alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaboroles, which showed good antimalarial activity against both W7 and 3D7 strains without significant toxicity. On the other hand, chloroquine (CQ) and cinnamic acids have a wide variety of biological activity including antimalarial activity. Herein, a hybridisation strategy was employed to synthesise new CQ-benzoxaborole and cinnamoyl-benzoxaborole hybrids. CQ-Benzoxaborole 2.12a-c and cinnamoylbenzoxaborole 2.11a-g hydrid molecules were synthesised in low to good yields. Their structural identities were confirmed using conventional spectroscopic techniques (1H and 13C NMR, and mass spectrometry). CQ-benzoxaborole compounds, however, showed instability, and only 2.12b was used for in vitro biological assay and showed activity comparable to CQ. Furthermore, in vitro biological assay revealed that compounds 2.11a-g poorly inhibited the growth of P. falciparum parasites. Interestingly, these compounds, however, exhibited satisfactory activity against Trypanosoma brucei with IC50 = 0.052 μM for compound 2.11g. The cell cytotoxicity assay of all final compounds confirmed that all CQ-benzoxaborole 2.12b and cinnamoyl-benzoxaborole 2.11a-g hybrids were non-toxic against HeLa cell lines. However, efforts to further expand the structure-activity relationship (SAR) of CQbenzoxaborole by increasing the length of the linker with one extra carbon (Scheme 2.10) were not possible as an important precursor 6-formylbenzoxaborole 2.29 could not be synthesized in sufficient yields. , Thesis (MSc) -- Faculty of Faculty of Science, Chemistry, 2017
- Full Text:
- Date Issued: 2017
- Authors: Gumbo, Maureen
- Date: 2017
- Subjects: Malaria Chemotherapy , Antimalarials , Boron compounds , Drug resistance , Plasmodium falciparum , Drug development
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59193 , vital:27456
- Description: Malaria is a mosquito-borne disease, which continues to pose a threat to the entire humanity. About 40% of the world population is estimated to be at risk of infections by malaria. Despite efforts undertaken by scientific community, government entities and international organizations, malaria is still rampant. The major problem is drug resistance, where the Plasmodium spp have over the past decades developed drug resistance against available drugs. In order to counter this problem, novel antimalarial drugs that are efficacious and with novel mode of action are of great necessity. Benzoxaborole derivatives have been shown to exhibit promising antimalarial activity against Plasmodium falciparum strains. Previous studies reported on the compounds such as 6-(2- (alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaboroles, which showed good antimalarial activity against both W7 and 3D7 strains without significant toxicity. On the other hand, chloroquine (CQ) and cinnamic acids have a wide variety of biological activity including antimalarial activity. Herein, a hybridisation strategy was employed to synthesise new CQ-benzoxaborole and cinnamoyl-benzoxaborole hybrids. CQ-Benzoxaborole 2.12a-c and cinnamoylbenzoxaborole 2.11a-g hydrid molecules were synthesised in low to good yields. Their structural identities were confirmed using conventional spectroscopic techniques (1H and 13C NMR, and mass spectrometry). CQ-benzoxaborole compounds, however, showed instability, and only 2.12b was used for in vitro biological assay and showed activity comparable to CQ. Furthermore, in vitro biological assay revealed that compounds 2.11a-g poorly inhibited the growth of P. falciparum parasites. Interestingly, these compounds, however, exhibited satisfactory activity against Trypanosoma brucei with IC50 = 0.052 μM for compound 2.11g. The cell cytotoxicity assay of all final compounds confirmed that all CQ-benzoxaborole 2.12b and cinnamoyl-benzoxaborole 2.11a-g hybrids were non-toxic against HeLa cell lines. However, efforts to further expand the structure-activity relationship (SAR) of CQbenzoxaborole by increasing the length of the linker with one extra carbon (Scheme 2.10) were not possible as an important precursor 6-formylbenzoxaborole 2.29 could not be synthesized in sufficient yields. , Thesis (MSc) -- Faculty of Faculty of Science, Chemistry, 2017
- Full Text:
- Date Issued: 2017