Shonhai, A.Ncube, Hlomphani Rachel2024-10-192024-10-192024-09-06Ncube, H.R. 2024. Development of high-throughput screening assays to identify small molecule inhibitors of Plasmodium falciparum Hsp70-1. . .https://univendspace.univen.ac.za/handle/11602/2747M.Sc. (Biochemistry)Department of Biochemistry and MicrobiologyPlasmodium falciparum causes the most lethal form of malaria infection. Identification and development of novel antimalarial drugs is limited by the shortage of simple, cost-effective screening assays. Heat shock proteins (Hsps) are a special class of molecular chaperones essential for parasite survival and development within the human host. Hsps are also implicated in drug resistance. Hsp70 forms an integral part of the molecular chaperone system, participating in essential processes such as protein refolding, preventing aggregation and aiding degradation of misfolded proteins. The molecular chaperone PfHsp70-1 is essential for cell survival during stress and it is expressed throughout all the blood stages of the parasite. Thus, Hsp70(s) are essential for functionality in the cell and their function has been demonstrated using the complementation assay. The E. coli dnaK756 mutant strain can grow between 30- 37 ℃ but its viability diminishes at temperatures >40 ℃. In addition, the strain produces non-functional Hsp70. As such, this strain is suitable for studying Hsp70 function using the complementation assay. A high-throughput complementation assay was optimized and developed to screen potential inhibitors of Hsp70. Cells heterogously expressing DnaK were the positive control, while those expressing KPf-V436F were the negative control and the cells heterologously expressing KPf were the test cells. KPf contains a substrate binding domain (SBD) from PfHsp70-1. Protein expression was confirmed by sodium dodecyl sulfate-polyacyrlyamide and Western blot analysis. Cells grown with DMSO managed to grow normally under stress, showing that DnaK and KPf conferred cytoprotection to E. coli dnaK756 cells. Those heterologously expressing the negative control could not withstand the thermal stress. However, treatment with inhibitors colistin sulfate and pifithrin μ hindered the growth of cells heterologously expressing DnaK and KPf under non-permissive temperatures by 50-fold. Interestingly, the inhibitors seemed not to have any effect on the cells heterologously expressing the mutant protein KPf-V436F. The cells grown with DMSO seemed to die at the same rate as those treated with inhibitors. The colistin sulfate IC50 values were 0.22 μM for DnaK and 0.07 μM for KPf, whilst pifithrin μ IC50 values were found to be 14.48 μM and 10.41 μM for DnaK and KPf respectively. Statical analysis using Two-way ANOVA utilizing multiple comparison analysis proved the validity of this assay. Solubility studies were conducted to check the solubility status of E. coli dnaK756 proteome in the presence of the inhibitors. Findings from this study show that the inhibition interrupted the interaction of DnaK/KPf with some of its client proteins consequently leading to aggregation. This was a confirmation of the compromised function of DnaK/KPf due to the presence of the inhibitor. Tryptophan fluorescence studies were done to validate the findings of the complementation assay, the results show that pifithrin μ and colistin sulfate perturb the tertiary conformation of DnaK, KPf and PfHsp70-1. Since a protein’s tertiary conformation is crucial for its function, perturbations in its conformation might have altered the protein functional activity. These findings further validated the complementation assay results which show cell growth defects in the presence of inhibitors. Hence, the current study demonstrated the potential of using the complementation assay as a screening assay to identify potential inhibitors of P. falciparum Hsp70-1.1 online resource (xii, 87 leaves) : color illustrationsenUniversity of VendaPlasmodium falciparumUCTDPfHsp70-1KPfDnaKhigh-throughputinhibitorHeat shock proteinsADPATPDissertationNcube HR. Development of high-throughput screening assays to identify small molecule inhibitors of Plasmodium falciparum Hsp70-1. []. , 2024 [cited yyyy month dd]. Available from:Ncube, H. R. (2024). <i>Development of high-throughput screening assays to identify small molecule inhibitors of Plasmodium falciparum Hsp70-1</i>. (). . Retrieved fromNcube, Hlomphani Rachel. <i>"Development of high-throughput screening assays to identify small molecule inhibitors of Plasmodium falciparum Hsp70-1."</i> ., , 2024.TY - Dissertation AU - Ncube, Hlomphani Rachel AB - Plasmodium falciparum causes the most lethal form of malaria infection. Identification and development of novel antimalarial drugs is limited by the shortage of simple, cost-effective screening assays. Heat shock proteins (Hsps) are a special class of molecular chaperones essential for parasite survival and development within the human host. Hsps are also implicated in drug resistance. Hsp70 forms an integral part of the molecular chaperone system, participating in essential processes such as protein refolding, preventing aggregation and aiding degradation of misfolded proteins. The molecular chaperone PfHsp70-1 is essential for cell survival during stress and it is expressed throughout all the blood stages of the parasite. Thus, Hsp70(s) are essential for functionality in the cell and their function has been demonstrated using the complementation assay. The E. coli dnaK756 mutant strain can grow between 30- 37 ℃ but its viability diminishes at temperatures >40 ℃. In addition, the strain produces non-functional Hsp70. As such, this strain is suitable for studying Hsp70 function using the complementation assay. A high-throughput complementation assay was optimized and developed to screen potential inhibitors of Hsp70. Cells heterogously expressing DnaK were the positive control, while those expressing KPf-V436F were the negative control and the cells heterologously expressing KPf were the test cells. KPf contains a substrate binding domain (SBD) from PfHsp70-1. Protein expression was confirmed by sodium dodecyl sulfate-polyacyrlyamide and Western blot analysis. Cells grown with DMSO managed to grow normally under stress, showing that DnaK and KPf conferred cytoprotection to E. coli dnaK756 cells. Those heterologously expressing the negative control could not withstand the thermal stress. However, treatment with inhibitors colistin sulfate and pifithrin μ hindered the growth of cells heterologously expressing DnaK and KPf under non-permissive temperatures by 50-fold. Interestingly, the inhibitors seemed not to have any effect on the cells heterologously expressing the mutant protein KPf-V436F. The cells grown with DMSO seemed to die at the same rate as those treated with inhibitors. The colistin sulfate IC50 values were 0.22 μM for DnaK and 0.07 μM for KPf, whilst pifithrin μ IC50 values were found to be 14.48 μM and 10.41 μM for DnaK and KPf respectively. Statical analysis using Two-way ANOVA utilizing multiple comparison analysis proved the validity of this assay. Solubility studies were conducted to check the solubility status of E. coli dnaK756 proteome in the presence of the inhibitors. Findings from this study show that the inhibition interrupted the interaction of DnaK/KPf with some of its client proteins consequently leading to aggregation. This was a confirmation of the compromised function of DnaK/KPf due to the presence of the inhibitor. Tryptophan fluorescence studies were done to validate the findings of the complementation assay, the results show that pifithrin μ and colistin sulfate perturb the tertiary conformation of DnaK, KPf and PfHsp70-1. Since a protein’s tertiary conformation is crucial for its function, perturbations in its conformation might have altered the protein functional activity. These findings further validated the complementation assay results which show cell growth defects in the presence of inhibitors. Hence, the current study demonstrated the potential of using the complementation assay as a screening assay to identify potential inhibitors of P. falciparum Hsp70-1. DA - 2024-09-06 DB - ResearchSpace DP - Univen KW - Plasmodium falciparum KW - PfHsp70-1 KW - KPf KW - DnaK KW - high-throughput KW - inhibitor KW - Heat shock proteins KW - ADP KW - ATP LK - https://univendspace.univen.ac.za PY - 2024 T1 - Development of high-throughput screening assays to identify small molecule inhibitors of Plasmodium falciparum Hsp70-1 TI - Development of high-throughput screening assays to identify small molecule inhibitors of Plasmodium falciparum Hsp70-1 UR - ER -