In silico identification of natural inhibitory compounds against the Mycobacterium tuberculosis Enzyme Pyrazinamidase using high-throughput virtual screening techniques
- Authors: Kenyon, Thomas
- Date: 2021-10-29
- Subjects: Mycobacterium tuberculosis , Pyrazinamide , Molecular dynamics , High throughput screening (Drug development) , Mutagenesis , South African Natural Compounds database (SANCDB)
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192074 , vital:45193
- Description: Tuberculosis (TB) is most commonly a pulmonary infection caused by the bacterium Mycobacterium tuberculosis. With the exception of the COVID-19 pandemic, TB was the most common cause of death due to an infectious disease for a number of years up until 2020. In 2019, 10 million people fell ill with TB worldwide and 1.4 million people died (WHO, 2020a). Additionally, multidrug-resistant TB (MDR-TB) remains a public health crisis and a health security threat. A global total of 206 030 people with multidrug- or rifampicin-resistant TB (MDR/RR-TB) were reported in 2019, a 10% increase from 186 883 in 2018. South Africa is ranked among the 48 high TB burden countries, with an estimated 360 000 people falling ill in 2019, resulting in 58 000 deaths, the majority of which being among people living with HIV. Unlike HIV, however, TB is a curable disease when managed correctly with long durations of antitubercular chemotherapy. Pyrazinamide (PZA) is an important first-line tuberculosis drug unique for its activity against latent TB. PZA is a prodrug, being converted into its active form, pyrazinoic acid (POA) by the Mtb gene pncA, coding for the pyrazinamidase enzyme (PZase). TB resistance to first-line drugs such as PZA is commonly associated with mutations in the pncA/PZase enzyme. This study aimed to identify potential novel inhibitors that bind to the active site of PZase. By making use of molecular docking studies and molecular dynamics (MD) simulations, high throughput virtual screening was performed on 623 compounds from the South African Natural Compounds database (SANCDB; https://sancdb.rubi.ru.ac.za). Ligands that selectively bound to the PZase active site were identified using docking studies, followed by MD simulations to assess ligand-PZase complex stability, Finally, hit compounds identified from the first round of MD simulations were screened again against PZase structures with high confidence point mutations known to infer PZA resistance in order to identify any novel compounds which had inhibitory potential against both WT and mutant forms of the PZase enzyme. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Kenyon, Thomas
- Date: 2021-10-29
- Subjects: Mycobacterium tuberculosis , Pyrazinamide , Molecular dynamics , High throughput screening (Drug development) , Mutagenesis , South African Natural Compounds database (SANCDB)
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192074 , vital:45193
- Description: Tuberculosis (TB) is most commonly a pulmonary infection caused by the bacterium Mycobacterium tuberculosis. With the exception of the COVID-19 pandemic, TB was the most common cause of death due to an infectious disease for a number of years up until 2020. In 2019, 10 million people fell ill with TB worldwide and 1.4 million people died (WHO, 2020a). Additionally, multidrug-resistant TB (MDR-TB) remains a public health crisis and a health security threat. A global total of 206 030 people with multidrug- or rifampicin-resistant TB (MDR/RR-TB) were reported in 2019, a 10% increase from 186 883 in 2018. South Africa is ranked among the 48 high TB burden countries, with an estimated 360 000 people falling ill in 2019, resulting in 58 000 deaths, the majority of which being among people living with HIV. Unlike HIV, however, TB is a curable disease when managed correctly with long durations of antitubercular chemotherapy. Pyrazinamide (PZA) is an important first-line tuberculosis drug unique for its activity against latent TB. PZA is a prodrug, being converted into its active form, pyrazinoic acid (POA) by the Mtb gene pncA, coding for the pyrazinamidase enzyme (PZase). TB resistance to first-line drugs such as PZA is commonly associated with mutations in the pncA/PZase enzyme. This study aimed to identify potential novel inhibitors that bind to the active site of PZase. By making use of molecular docking studies and molecular dynamics (MD) simulations, high throughput virtual screening was performed on 623 compounds from the South African Natural Compounds database (SANCDB; https://sancdb.rubi.ru.ac.za). Ligands that selectively bound to the PZase active site were identified using docking studies, followed by MD simulations to assess ligand-PZase complex stability, Finally, hit compounds identified from the first round of MD simulations were screened again against PZase structures with high confidence point mutations known to infer PZA resistance in order to identify any novel compounds which had inhibitory potential against both WT and mutant forms of the PZase enzyme. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
Infant health: a community-based assessment and educational intervention in two rural communities in the Eastern Cape.
- Authors: Kuzeeko, Faith
- Date: 2021
- Subjects: Angus Gillis Foundation (South Africa) , Infants -- Mortality -- South Africa , Infants -- Health and hygiene -- South Africa , Breastfeeding -- South Africa -- Eastern Cape , Infants -- Care -- Equipment and supplies , Infants -- Care -- South Africa -- Eastern Cape -- Case studies , Children -- Mortality -- South Africa , High throughput screening (Drug development)
- Language: English
- Type: text , Thesis , Masters , MPharm
- Identifier: http://hdl.handle.net/10962/170837 , vital:41964
- Description: South Africa is on track to attaining the 2030 Agenda on reducing under-five deaths to 25 deaths per 1000 live births at its current momentum, however an unacceptable amount of infants are still at risk from preventable illnesses. Malnutrition is the major underlined cause of infant mortality rates in South Africa. Exclusive breastfeeding rates are low nationwide due to low exposure to breastfeeding information, some mothers having other commitments and others having breastfeeding difficulties. Implementation and expansion of simple, cost-effective interventions, such as exclusive breastfeeding for six months to reduce and/or prevent infant mortality rates, remains low in South Africa. The aim of the study was to determine one infant health issue of major concern to participants in two rural villages in the Eastern Cape, namely Glenmore and Ndwayana. The identified infant health issue was used to design an educational intervention in the villages. This was the second phase of this study. A community-based participatory approach was utilized in which the Angus Gillis Foundation, a non-profit organization that works in these communities, was one of the stakeholders. Stock status of WHO priority medicines for infants, semi-structured interviews and focus group discussions were carried out during the baseline study. The intervention phase contained pre-2 intervention semi-structured interviews with ten pregnant women followed by an educational intervention with nine out of the ten; and finally, a post-intervention with seven out of the ten women. A questionnaire was completed by members of the Angus Gillis Foundation to provide feedback on the sustainability of the intervention. Semi-structured interviews revealed that medicines stocked at the clinic parallel those indicated in the WHO priority medicines list for infants. The results from the focus group discussions indicated that mothers do not exclusively breastfeed their infants during the first six months. Pre- and post-intervention results on exclusive breastfeeding illustrated a positive change in participants’ knowledge and intent to breastfeed exclusively for six months. They showed a better understanding of the importance of exclusive breastfeeding and indicated a more focussed intention and confidence to carry out optimal breastfeeding practices. In the questionnaire the members of the Angus Gillis Foundation stated that the intervention is sustainable as it was linked with the existing networks. These include educational programs carried out in the villages by the foundation together with positive health champions, community health workers and women self-help groups; which will be able to build on the present knowledge base. Finally, the study also included the design of a booklet on the identified infant health issue. In conclusion, participants highlighted lack of understanding regarding breastfeeding as an issue of concern during the baseline phase of the study. This community-based educational intervention improved the understanding of breastfeeding among the participants, resulting in a positive change in perception with regards to exclusive breastfeeding practices.
- Full Text:
- Date Issued: 2021
- Authors: Kuzeeko, Faith
- Date: 2021
- Subjects: Angus Gillis Foundation (South Africa) , Infants -- Mortality -- South Africa , Infants -- Health and hygiene -- South Africa , Breastfeeding -- South Africa -- Eastern Cape , Infants -- Care -- Equipment and supplies , Infants -- Care -- South Africa -- Eastern Cape -- Case studies , Children -- Mortality -- South Africa , High throughput screening (Drug development)
- Language: English
- Type: text , Thesis , Masters , MPharm
- Identifier: http://hdl.handle.net/10962/170837 , vital:41964
- Description: South Africa is on track to attaining the 2030 Agenda on reducing under-five deaths to 25 deaths per 1000 live births at its current momentum, however an unacceptable amount of infants are still at risk from preventable illnesses. Malnutrition is the major underlined cause of infant mortality rates in South Africa. Exclusive breastfeeding rates are low nationwide due to low exposure to breastfeeding information, some mothers having other commitments and others having breastfeeding difficulties. Implementation and expansion of simple, cost-effective interventions, such as exclusive breastfeeding for six months to reduce and/or prevent infant mortality rates, remains low in South Africa. The aim of the study was to determine one infant health issue of major concern to participants in two rural villages in the Eastern Cape, namely Glenmore and Ndwayana. The identified infant health issue was used to design an educational intervention in the villages. This was the second phase of this study. A community-based participatory approach was utilized in which the Angus Gillis Foundation, a non-profit organization that works in these communities, was one of the stakeholders. Stock status of WHO priority medicines for infants, semi-structured interviews and focus group discussions were carried out during the baseline study. The intervention phase contained pre-2 intervention semi-structured interviews with ten pregnant women followed by an educational intervention with nine out of the ten; and finally, a post-intervention with seven out of the ten women. A questionnaire was completed by members of the Angus Gillis Foundation to provide feedback on the sustainability of the intervention. Semi-structured interviews revealed that medicines stocked at the clinic parallel those indicated in the WHO priority medicines list for infants. The results from the focus group discussions indicated that mothers do not exclusively breastfeed their infants during the first six months. Pre- and post-intervention results on exclusive breastfeeding illustrated a positive change in participants’ knowledge and intent to breastfeed exclusively for six months. They showed a better understanding of the importance of exclusive breastfeeding and indicated a more focussed intention and confidence to carry out optimal breastfeeding practices. In the questionnaire the members of the Angus Gillis Foundation stated that the intervention is sustainable as it was linked with the existing networks. These include educational programs carried out in the villages by the foundation together with positive health champions, community health workers and women self-help groups; which will be able to build on the present knowledge base. Finally, the study also included the design of a booklet on the identified infant health issue. In conclusion, participants highlighted lack of understanding regarding breastfeeding as an issue of concern during the baseline phase of the study. This community-based educational intervention improved the understanding of breastfeeding among the participants, resulting in a positive change in perception with regards to exclusive breastfeeding practices.
- Full Text:
- Date Issued: 2021
Application of quality by design to the manufacture of a multiparticulate prednisone dosage form
- Authors: Manda, Arthur
- Date: 2020-04
- Subjects: Drugs -- Quality control , Drugs -- Design -- Quality control , Drugs -- Dosage forms , Drug development -- Quality control , Pharmaceutical industry -- Quality control , Prednisone , High throughput screening (Drug development)
- Language: English
- Type: text , Thesis , Masters , MSc (Pharmacy)
- Identifier: http://hdl.handle.net/10962/117986 , vital:34583
- Description: For many years, quality by testing was the only approach to guarantee quality of drug products before the Food and Drug Administration launched the concept of current Good Manufacturing Practice. In order to gain more knowledge of the manufacturing process, a new system known as Quality by Design was introduced into the pharmaceutical industry. Quality by Design is based on thorough understanding of how materials, process parameters and interaction thereof impact final product quality. Quality by Design is a systematic approach to product development which ensures that quality is built into a product during product development and not just tested into it. The aim of Quality by Design is to achieve optimum product quality with consistent dosage form performance and minimal risk of failure in patients. The objective of these studies was to implement a Quality by Design approach to establish a design space for the development and manufacture of a safe, effective and stable multi-partite solid oral dosage form for prednisone as an alternative to currently marketed prednisone formulations. Multi-particulate dosage forms offer significant advantages over conventional technologies. In addition to lowering the incidence of gastrointestinal irritation they exhibit a reduced risk of dose dumping and a large surface area which favours dissolution. Furthermore, their free flowing nature facilitates reproducible capsule filling and consequently uniformity of dosing. Different multi-particulate dosage forms exist however a multiple-unit pellet system was investigated during these studies. Quality by Design principles were used to develop and establish a reversed-phase high performance liquid chromatographic method for quantifying prednisone from solid oral dosage forms. A Central Composite Design was used to generate multivariate experiments and to investigate the impact of input variables on the quality and performance of the analytical method. The optimized method was validated according to International Council for Harmonization guidelines and was found to be linear, precise, accurate and specific for the quantitation of prednisone. Pre-formulation studies were conducted and included the assessment of particle size, particle shape, powder flow properties and compatibility studies. Carr’s index, Hausner ratio and the Angle of Repose were used to evaluate powder flow properties and results generated from all studies suggest the need for adding a glidant and lubricant to improve pellet flow. The images generated from Scanning Electron Microscopy were used to analyze particle shape and size. Differential Scanning Calorimetry and Fourier Transform Infrared Spectroscopy were used to evaluate API-excipient compatibility. All excipients investigated were found to be compatible with prednisone and suitable for formulation development studies. Extrusion-spheronization was used to manufacture prednisone pellets. Extrusion-spheronization is a multi-step process involving many factors. Quality risk management tools particularly an Ishikawa Fishbone (cause and effect) diagram and failure mode and effects analysis were used to narrow down potentially significant factors to a reasonable number that could be investigated experimentally. Risk priority numbers were used to quantify risk and factors above a set threshold value were considered to be of high risk. A total of eleven risk factors were identified as high. A Plackett-Burman study was conducted to narrow down the eleven high risk factors to identify the most impactful factors viz., microcrystalline cellulose content, sodium starch glycolate content, extrusion speed and spheronization time. Evaluation of four factors was carried over to optimization studies using a Box-Behnken Design and following identifaction of the optimum process settings and excipient content a design space for the manufacture of a multi-partite dosage form containing prednisone was established.
- Full Text:
- Date Issued: 2020-04
- Authors: Manda, Arthur
- Date: 2020-04
- Subjects: Drugs -- Quality control , Drugs -- Design -- Quality control , Drugs -- Dosage forms , Drug development -- Quality control , Pharmaceutical industry -- Quality control , Prednisone , High throughput screening (Drug development)
- Language: English
- Type: text , Thesis , Masters , MSc (Pharmacy)
- Identifier: http://hdl.handle.net/10962/117986 , vital:34583
- Description: For many years, quality by testing was the only approach to guarantee quality of drug products before the Food and Drug Administration launched the concept of current Good Manufacturing Practice. In order to gain more knowledge of the manufacturing process, a new system known as Quality by Design was introduced into the pharmaceutical industry. Quality by Design is based on thorough understanding of how materials, process parameters and interaction thereof impact final product quality. Quality by Design is a systematic approach to product development which ensures that quality is built into a product during product development and not just tested into it. The aim of Quality by Design is to achieve optimum product quality with consistent dosage form performance and minimal risk of failure in patients. The objective of these studies was to implement a Quality by Design approach to establish a design space for the development and manufacture of a safe, effective and stable multi-partite solid oral dosage form for prednisone as an alternative to currently marketed prednisone formulations. Multi-particulate dosage forms offer significant advantages over conventional technologies. In addition to lowering the incidence of gastrointestinal irritation they exhibit a reduced risk of dose dumping and a large surface area which favours dissolution. Furthermore, their free flowing nature facilitates reproducible capsule filling and consequently uniformity of dosing. Different multi-particulate dosage forms exist however a multiple-unit pellet system was investigated during these studies. Quality by Design principles were used to develop and establish a reversed-phase high performance liquid chromatographic method for quantifying prednisone from solid oral dosage forms. A Central Composite Design was used to generate multivariate experiments and to investigate the impact of input variables on the quality and performance of the analytical method. The optimized method was validated according to International Council for Harmonization guidelines and was found to be linear, precise, accurate and specific for the quantitation of prednisone. Pre-formulation studies were conducted and included the assessment of particle size, particle shape, powder flow properties and compatibility studies. Carr’s index, Hausner ratio and the Angle of Repose were used to evaluate powder flow properties and results generated from all studies suggest the need for adding a glidant and lubricant to improve pellet flow. The images generated from Scanning Electron Microscopy were used to analyze particle shape and size. Differential Scanning Calorimetry and Fourier Transform Infrared Spectroscopy were used to evaluate API-excipient compatibility. All excipients investigated were found to be compatible with prednisone and suitable for formulation development studies. Extrusion-spheronization was used to manufacture prednisone pellets. Extrusion-spheronization is a multi-step process involving many factors. Quality risk management tools particularly an Ishikawa Fishbone (cause and effect) diagram and failure mode and effects analysis were used to narrow down potentially significant factors to a reasonable number that could be investigated experimentally. Risk priority numbers were used to quantify risk and factors above a set threshold value were considered to be of high risk. A total of eleven risk factors were identified as high. A Plackett-Burman study was conducted to narrow down the eleven high risk factors to identify the most impactful factors viz., microcrystalline cellulose content, sodium starch glycolate content, extrusion speed and spheronization time. Evaluation of four factors was carried over to optimization studies using a Box-Behnken Design and following identifaction of the optimum process settings and excipient content a design space for the manufacture of a multi-partite dosage form containing prednisone was established.
- Full Text:
- Date Issued: 2020-04
Cyclooxygenase-1 as an anti-stroke target: potential inhibitor identification and non-synonymous single nucleotide polymorphism analysis
- Authors: Muronzi, Tendai
- Date: 2020
- Subjects: Cerebrovascular disease , Cerebrovascular disease -- Treatment , Cerebrovascular disease -- Chemotherapy , Cyclooxygenases , High throughput screening (Drug development) , Drug development , Molecular dynamics , South African Natural Compounds Database , ZINC database
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/143404 , vital:38243
- Description: Stroke is the third leading cause of death worldwide, with 87% of cases being ischemic stroke. The two primary therapeutic strategies to reduce post-ischemic brain damage are cellular and vascular approaches. The vascular strategy aims to rapidly re-open obstructed blood vessels, while the cellular approach aims to interfere with the signalling pathways that facilitate neuron damage and death. Unfortunately, popular vascular treatments have adverse side effects, necessitating the need for alternative chemotherapeutics. In this study, cyclooxygenase-1 (COX-1), which plays a significant role in the post- ischemic neuroinflammation and neuronal death, was targeted for identification of novel drug compounds and to assess the effect of nsSNPs on its structure and function. In a drug discovery part, ligands from the South African Natural Compounds Database (SANCDB-https://sancdb.rubi.ru.ac.za/) and ZINC database (http://zinc15.docking.org/) were used for high-throughput virtual screening (HVTS) against COX-1. Additionally, five nsSNPs were being investigated to assess their impact on protein structure and function. Three of these SNPs were in the COX-1 dimer interface. Molecular docking and molecular dynamics simulations revealed asymmetric nature of the protein. Several ligands, peculiar to each monomer, exhibited favourable binding energies in the respective active sites. SNP analysis indicated effects on inter-monomer interactions and protein stability.
- Full Text:
- Date Issued: 2020
- Authors: Muronzi, Tendai
- Date: 2020
- Subjects: Cerebrovascular disease , Cerebrovascular disease -- Treatment , Cerebrovascular disease -- Chemotherapy , Cyclooxygenases , High throughput screening (Drug development) , Drug development , Molecular dynamics , South African Natural Compounds Database , ZINC database
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/143404 , vital:38243
- Description: Stroke is the third leading cause of death worldwide, with 87% of cases being ischemic stroke. The two primary therapeutic strategies to reduce post-ischemic brain damage are cellular and vascular approaches. The vascular strategy aims to rapidly re-open obstructed blood vessels, while the cellular approach aims to interfere with the signalling pathways that facilitate neuron damage and death. Unfortunately, popular vascular treatments have adverse side effects, necessitating the need for alternative chemotherapeutics. In this study, cyclooxygenase-1 (COX-1), which plays a significant role in the post- ischemic neuroinflammation and neuronal death, was targeted for identification of novel drug compounds and to assess the effect of nsSNPs on its structure and function. In a drug discovery part, ligands from the South African Natural Compounds Database (SANCDB-https://sancdb.rubi.ru.ac.za/) and ZINC database (http://zinc15.docking.org/) were used for high-throughput virtual screening (HVTS) against COX-1. Additionally, five nsSNPs were being investigated to assess their impact on protein structure and function. Three of these SNPs were in the COX-1 dimer interface. Molecular docking and molecular dynamics simulations revealed asymmetric nature of the protein. Several ligands, peculiar to each monomer, exhibited favourable binding energies in the respective active sites. SNP analysis indicated effects on inter-monomer interactions and protein stability.
- Full Text:
- Date Issued: 2020
A dynamics based analysis of allosteric modulation in heat shock proteins
- Authors: Penkler, David Lawrence
- Date: 2019
- Subjects: Heat shock proteins , Molecular chaperones , Allosteric regulation , Homeostasis , Protein kinases , Transcription factors , Adenosine triphosphatase , Cancer -- Chemotherapy , Molecular dynamics , High throughput screening (Drug development)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115948 , vital:34273
- Description: The 70 kDa and 90 kDa heat shock proteins (Hsp70 and Hsp90) are molecular chaperones that play central roles in maintaining cellular homeostasis in all organisms of life with the exception of archaea. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the regulation and activity of some 200 known natively folded protein clients which include protein kinases, transcription factors and receptors, many of which are implicated as key regulators of essential signal transduction pathways. Both chaperones are considered to be large multi-domain proteins that rely on ATPase activity and co-chaperone interactions to regulate their conformational cycles for peptide binding and release. The unique positioning of Hsp90 at the crossroads of several fundamental cellular pathways coupled with its known association with diverse oncogenic peptide clients has brought the molecular chaperone under increasing interest as a potential anti-cancer target that is crucially implicated with all eight hallmarks of the disease. Current orthosteric drug discovery efforts aimed at the inhibition of the ATPase domain of Hsp90 have been limited due to high levels of associated toxicity. In an effort to circumnavigate this, the combined focus of research efforts is shifting toward alternative approaches such as interference with co-chaperone binding and the allosteric inhibition/activation of the molecular chaperone. The overriding aim of this thesis was to demonstrate how the computational technique of Perturbation response scanning (PRS) coupled with all-atom molecular dynamics simulations (MD) and dynamic residue interaction network (DRN) analysis can be used as a viable strategy to efficiently scan and accurately identify allosteric control element capable of modulating the functional dynamics of a protein. In pursuit of this goal, this thesis also contributes to the current understanding of the nucleotide dependent allosteric mechanisms at play in cellular functionality of both Hsp70 and Hsp90. All-atom MD simulations of E. coli DnaK provided evidence of nucleotide driven modulation of conformational dynamics in both the catalytically active and inactive states. PRS analysis employed on these trajectories demonstrated sensitivity toward bound nucleotide and peptide substrate, and provided evidence of a putative allosterically active intermediate state between the ATPase active and inactive conformational states. Simultaneous binding of ATP and peptide substrate was found to allosterically prime the chaperone for interstate conversion regardless of the transition direction. Detailed analysis of these allosterically primed states revealed select residue sites capable of selecting a coordinate shift towards the opposite conformational state. In an effort to validate these results, the predicted allosteric hot spot sites were cross-validated with known experimental works and found to overlap with functional sites implicated in allosteric signal propagation and ATPase activation in Hsp70. This study presented for the first time, the application of PRS as a suitable diagnostic tool for the elucidation and quantification of the allosteric potential of select residues to effect functionally relevant global conformational rearrangements. The PRS methodology described in this study was packaged within the Python programming environment in the MD-TASK software suite for command-line ease of use and made freely available. Homology modelling techniques were used to address the lack of experimental structural data for the human cytosolic isoform of Hsp90 and for the first time provided accurate full-length structural models of human Hsp90α in fully-closed and partially-open conformations. Long-range all-atom MD simulations of these structures revealed nucleotide driven modulation of conformational dynamics in Hsp90. Subsequent DRN and PRS analysis of these MD trajectories allowed for the quantification and elucidation of nucleotide driven allosteric modulation in the molecular chaperone. A detailed PRS analysis revealed allosteric inter-domain coupling between the extreme terminals of the chaperone in response to external force perturbations at either domain. Furthermore PRS also identified several individual residue sites that are capable of selecting conformational rearrangements towards functionally relevant states which may be considered to be putative allosteric target sites for future drug discovery efforts Molecular docking techniques were employed to investigate the modulation of conformational dynamics of human Hsp90α in response to ligand binding interactions at two identified allosteric sites at the C-terminal. High throughput screening of a small library of natural compounds indigenous to South Africa revealed three hit compounds at these sites: Cephalostatin 17, 20(29)-Lupene-3β isoferulate and 3'-Bromorubrolide F. All-atom MD simulations on these protein-ligand complexes coupled with DRN analysis and several advanced trajectory based analysis techniques provided evidence of selective allosteric modulation of Hsp90α conformational dynamics in response to the identity and location of the bound ligands. Ligands bound at the four-helix bundle presented as putative allosteric inhibitors of Hsp90α, driving conformational dynamics in favour of dimer opening and possibly dimer separation. Meanwhile, ligand interactions at an adjacent sub-pocket located near the interface between the middle and C-terminal domains demonstrated allosteric activation of the chaperone, modulating conformational dynamics in favour of the fully-closed catalytically active conformational state. Taken together, the data presented in this thesis contributes to the understanding of allosteric modulation of conformational dynamics in Hsp70 and Hsp90, and provides a suitable platform for future biochemical and drug discovery studies. Furthermore, the molecular docking and computational identification of allosteric compounds with suitable binding affinity for allosteric sites at the CTD of human Hsp90α provide for the first time “proof-of-principle” for the use of PRS in conjunction with MD simulations and DRN analysis as a suitable method for the rapid identification of allosteric sites in proteins that can be probed by small molecule interaction. The data presented in this section could pave the way for future allosteric drug discovery studies for the treatment of Hsp90 associated pathologies.
- Full Text:
- Date Issued: 2019
- Authors: Penkler, David Lawrence
- Date: 2019
- Subjects: Heat shock proteins , Molecular chaperones , Allosteric regulation , Homeostasis , Protein kinases , Transcription factors , Adenosine triphosphatase , Cancer -- Chemotherapy , Molecular dynamics , High throughput screening (Drug development)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115948 , vital:34273
- Description: The 70 kDa and 90 kDa heat shock proteins (Hsp70 and Hsp90) are molecular chaperones that play central roles in maintaining cellular homeostasis in all organisms of life with the exception of archaea. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the regulation and activity of some 200 known natively folded protein clients which include protein kinases, transcription factors and receptors, many of which are implicated as key regulators of essential signal transduction pathways. Both chaperones are considered to be large multi-domain proteins that rely on ATPase activity and co-chaperone interactions to regulate their conformational cycles for peptide binding and release. The unique positioning of Hsp90 at the crossroads of several fundamental cellular pathways coupled with its known association with diverse oncogenic peptide clients has brought the molecular chaperone under increasing interest as a potential anti-cancer target that is crucially implicated with all eight hallmarks of the disease. Current orthosteric drug discovery efforts aimed at the inhibition of the ATPase domain of Hsp90 have been limited due to high levels of associated toxicity. In an effort to circumnavigate this, the combined focus of research efforts is shifting toward alternative approaches such as interference with co-chaperone binding and the allosteric inhibition/activation of the molecular chaperone. The overriding aim of this thesis was to demonstrate how the computational technique of Perturbation response scanning (PRS) coupled with all-atom molecular dynamics simulations (MD) and dynamic residue interaction network (DRN) analysis can be used as a viable strategy to efficiently scan and accurately identify allosteric control element capable of modulating the functional dynamics of a protein. In pursuit of this goal, this thesis also contributes to the current understanding of the nucleotide dependent allosteric mechanisms at play in cellular functionality of both Hsp70 and Hsp90. All-atom MD simulations of E. coli DnaK provided evidence of nucleotide driven modulation of conformational dynamics in both the catalytically active and inactive states. PRS analysis employed on these trajectories demonstrated sensitivity toward bound nucleotide and peptide substrate, and provided evidence of a putative allosterically active intermediate state between the ATPase active and inactive conformational states. Simultaneous binding of ATP and peptide substrate was found to allosterically prime the chaperone for interstate conversion regardless of the transition direction. Detailed analysis of these allosterically primed states revealed select residue sites capable of selecting a coordinate shift towards the opposite conformational state. In an effort to validate these results, the predicted allosteric hot spot sites were cross-validated with known experimental works and found to overlap with functional sites implicated in allosteric signal propagation and ATPase activation in Hsp70. This study presented for the first time, the application of PRS as a suitable diagnostic tool for the elucidation and quantification of the allosteric potential of select residues to effect functionally relevant global conformational rearrangements. The PRS methodology described in this study was packaged within the Python programming environment in the MD-TASK software suite for command-line ease of use and made freely available. Homology modelling techniques were used to address the lack of experimental structural data for the human cytosolic isoform of Hsp90 and for the first time provided accurate full-length structural models of human Hsp90α in fully-closed and partially-open conformations. Long-range all-atom MD simulations of these structures revealed nucleotide driven modulation of conformational dynamics in Hsp90. Subsequent DRN and PRS analysis of these MD trajectories allowed for the quantification and elucidation of nucleotide driven allosteric modulation in the molecular chaperone. A detailed PRS analysis revealed allosteric inter-domain coupling between the extreme terminals of the chaperone in response to external force perturbations at either domain. Furthermore PRS also identified several individual residue sites that are capable of selecting conformational rearrangements towards functionally relevant states which may be considered to be putative allosteric target sites for future drug discovery efforts Molecular docking techniques were employed to investigate the modulation of conformational dynamics of human Hsp90α in response to ligand binding interactions at two identified allosteric sites at the C-terminal. High throughput screening of a small library of natural compounds indigenous to South Africa revealed three hit compounds at these sites: Cephalostatin 17, 20(29)-Lupene-3β isoferulate and 3'-Bromorubrolide F. All-atom MD simulations on these protein-ligand complexes coupled with DRN analysis and several advanced trajectory based analysis techniques provided evidence of selective allosteric modulation of Hsp90α conformational dynamics in response to the identity and location of the bound ligands. Ligands bound at the four-helix bundle presented as putative allosteric inhibitors of Hsp90α, driving conformational dynamics in favour of dimer opening and possibly dimer separation. Meanwhile, ligand interactions at an adjacent sub-pocket located near the interface between the middle and C-terminal domains demonstrated allosteric activation of the chaperone, modulating conformational dynamics in favour of the fully-closed catalytically active conformational state. Taken together, the data presented in this thesis contributes to the understanding of allosteric modulation of conformational dynamics in Hsp70 and Hsp90, and provides a suitable platform for future biochemical and drug discovery studies. Furthermore, the molecular docking and computational identification of allosteric compounds with suitable binding affinity for allosteric sites at the CTD of human Hsp90α provide for the first time “proof-of-principle” for the use of PRS in conjunction with MD simulations and DRN analysis as a suitable method for the rapid identification of allosteric sites in proteins that can be probed by small molecule interaction. The data presented in this section could pave the way for future allosteric drug discovery studies for the treatment of Hsp90 associated pathologies.
- Full Text:
- Date Issued: 2019
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
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