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
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
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