Synthesis, characterisation and evaluation of ferrocene-containing Novobiocin analogues for anticancer and antiplasmodial activity through inhibition of Hsp90
- Authors: Mbaba, Mziyanda
- Date: 2017
- Subjects: Antibiotics Synthesis , Ferrocene , Heat shock proteins , Antimalarials , Cancer Chemotherapy
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65111 , vital:28690
- Description: Novobiocin (Nb) is a coumarin type antibiotic isolated from the bacterium species of Streptomyces and possesses modest anticancer and antimalarial activities. Nb and analogues have been extensively explored as potential anticancer agents through inhibition of the C- terminal domain of heat shock protein 90 (Hsp90), which plays a pivotal role in the proteinfolding machinery of cells. There has been little effort in the exploration of Nb and derivatives for antimalarial activity. Incorporation of organometallic units, such as ferrocene (Fc), into bioactive chemical scaffolds remains an attractive approach for developing new therapeutic agents for treatment of several ailments. The current study sought to investigate the anticancer and antiplasmodial effects of incorporating ferrocene (Fc) into Nb scaffold presumably through inhibition of Hsp90. The ferrocenyl Nb analogues containing simplified structural motifs such as phenyl, benzyl, and piperidine were synthesized in six to nine steps employing conventional synthetic organic protocols adapted from literature, and the compounds were accessed in reasonable yields. For comparison purposes, a selection of organic Nb analogues were also included in the study. The target compounds were characterized by spectroscopic techniques including 1-dimensional nuclear magnetic resonance (1D NMR) and high-resolution mass spectroscopy. The synthesized compounds were evaluated in vitro for potential anticancer and antiplasmodial activities using the breast cancer cell line (HCC38) and chloroquine-sensitive strain (3D7) of the malaria parasite, Plasmodium falciparum. The presence of the Fc unit was found to enhance both anticancer and antiplasmodial activities of the resultant ferrocenyl Nb compounds with IC50 values in the low to mid micromolar range. Hsp90 inhibitory studies of the ferrocenyl Nb analogues possessing superior activities (2.13a and 2.20c) were also conducted using different yeast strains expressing both human and malarial Hsp90 isoforms: hHsp90a/p and PfHsp90, respectively. The results of Hsp90 inhibitory studies suggested no direct correlation between the observed activities of the analogues and Hsp90 inhibition. However, since the conditions of the assay were not optimised due to time constrains of the project, these observed data remained to be confirmed. , Thesis (MSc) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Date Issued: 2017
- Authors: Mbaba, Mziyanda
- Date: 2017
- Subjects: Antibiotics Synthesis , Ferrocene , Heat shock proteins , Antimalarials , Cancer Chemotherapy
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65111 , vital:28690
- Description: Novobiocin (Nb) is a coumarin type antibiotic isolated from the bacterium species of Streptomyces and possesses modest anticancer and antimalarial activities. Nb and analogues have been extensively explored as potential anticancer agents through inhibition of the C- terminal domain of heat shock protein 90 (Hsp90), which plays a pivotal role in the proteinfolding machinery of cells. There has been little effort in the exploration of Nb and derivatives for antimalarial activity. Incorporation of organometallic units, such as ferrocene (Fc), into bioactive chemical scaffolds remains an attractive approach for developing new therapeutic agents for treatment of several ailments. The current study sought to investigate the anticancer and antiplasmodial effects of incorporating ferrocene (Fc) into Nb scaffold presumably through inhibition of Hsp90. The ferrocenyl Nb analogues containing simplified structural motifs such as phenyl, benzyl, and piperidine were synthesized in six to nine steps employing conventional synthetic organic protocols adapted from literature, and the compounds were accessed in reasonable yields. For comparison purposes, a selection of organic Nb analogues were also included in the study. The target compounds were characterized by spectroscopic techniques including 1-dimensional nuclear magnetic resonance (1D NMR) and high-resolution mass spectroscopy. The synthesized compounds were evaluated in vitro for potential anticancer and antiplasmodial activities using the breast cancer cell line (HCC38) and chloroquine-sensitive strain (3D7) of the malaria parasite, Plasmodium falciparum. The presence of the Fc unit was found to enhance both anticancer and antiplasmodial activities of the resultant ferrocenyl Nb compounds with IC50 values in the low to mid micromolar range. Hsp90 inhibitory studies of the ferrocenyl Nb analogues possessing superior activities (2.13a and 2.20c) were also conducted using different yeast strains expressing both human and malarial Hsp90 isoforms: hHsp90a/p and PfHsp90, respectively. The results of Hsp90 inhibitory studies suggested no direct correlation between the observed activities of the analogues and Hsp90 inhibition. However, since the conditions of the assay were not optimised due to time constrains of the project, these observed data remained to be confirmed. , Thesis (MSc) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Date Issued: 2017
The Role of HOP in Emerin-Mediated Nuclear Structure
- Authors: Kituyi, Sarah Naulikha
- Date: 2017
- Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
- Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
- Full Text:
- Date Issued: 2017
- Authors: Kituyi, Sarah Naulikha
- Date: 2017
- Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
- Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
- Full Text:
- Date Issued: 2017
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