Screening drug candidates for sars-cov-2 spike protein variants within the SANCDB (South African Natural Compounds Database) and drug bank
- Authors: van der Merwe, Vicky
- Date: 2024-12
- Subjects: COVID-19 (Disease) , Vaccines -- Development , Viral vaccines
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/69499 , vital:77263
- Description: The WHO declared the Coronavirus Infectious Disease 2019 (COVID-19) pandemic as over on the 5th of May 2023. However, the Severe Acute Respiratory Syndrome Coronavirus 2 Virus (SARS-CoV-2) is still prevalently spread within populations, causing hospitalization and death in serious infections. Most anti-COVID-19 agents are weakly or conditionally or even strongly recommended against in non-severe cases with low to medium risk of hospitalisation and further highlight the significance of effective treatments for SARS-CoV-2 infected patients. The host’s Angiotensin-Converting Enzyme 2 (ACE2) interaction with the viral spike protein’s Receptor-Binding Domain (RBD) of SARS-CoV-2 leads to the subsequent infection, viral replication, and further propagation of the virus. This establishes the spike protein’s RBD as a vital target for drug design. Through HADDOCK and AlphaFold driven structural refinement, the then novel SARS-CoV-2 delta [3.34 Å (PDB ID: 7WBQ)] and omicron [3.00 Å (PDB ID: 7WBP)] crystalline structures of the variants’ RBD, in complex with its host receptor human ACE2, were modelled to improve the low-resolution starting structures. Models predicted by HADDOCK independently yielded refined structures with improved resolutions of 2.49 Å (delta) and 2.41 Å (omicron). SARS-CoV-2 spike protein RBD of delta and omicron variants were screened for less frequently considered allosteric binding sites that could block ACE2-RBD engagement. Eight potential allosteric binding sites were identified through CavityPlus for both variants, with a single best druggable potential allosteric site identified for each. The search for potential inhibiting hit compounds involved using the SANCDB and DrugBank databanks and screening both the allosteric and orthosteric binding sites through molecular docking. Despite differences in mutated interacting residues and bond interactions, a lead compound for orthosteric binding sites (SANC00290) and potential allosteric modulators of both delta and omicron (SANC00746 and DB01029) were found. These compounds show promise in blocking ACE2-RBD engagement to potentially reduce viral interaction and infection of, and potentially withstanding further mutational residues. This study contributes to further investigation of affordable SA natural compound COVID-19 treatment and a starting point for in vitro analysis studies. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-12
- Authors: van der Merwe, Vicky
- Date: 2024-12
- Subjects: COVID-19 (Disease) , Vaccines -- Development , Viral vaccines
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/69499 , vital:77263
- Description: The WHO declared the Coronavirus Infectious Disease 2019 (COVID-19) pandemic as over on the 5th of May 2023. However, the Severe Acute Respiratory Syndrome Coronavirus 2 Virus (SARS-CoV-2) is still prevalently spread within populations, causing hospitalization and death in serious infections. Most anti-COVID-19 agents are weakly or conditionally or even strongly recommended against in non-severe cases with low to medium risk of hospitalisation and further highlight the significance of effective treatments for SARS-CoV-2 infected patients. The host’s Angiotensin-Converting Enzyme 2 (ACE2) interaction with the viral spike protein’s Receptor-Binding Domain (RBD) of SARS-CoV-2 leads to the subsequent infection, viral replication, and further propagation of the virus. This establishes the spike protein’s RBD as a vital target for drug design. Through HADDOCK and AlphaFold driven structural refinement, the then novel SARS-CoV-2 delta [3.34 Å (PDB ID: 7WBQ)] and omicron [3.00 Å (PDB ID: 7WBP)] crystalline structures of the variants’ RBD, in complex with its host receptor human ACE2, were modelled to improve the low-resolution starting structures. Models predicted by HADDOCK independently yielded refined structures with improved resolutions of 2.49 Å (delta) and 2.41 Å (omicron). SARS-CoV-2 spike protein RBD of delta and omicron variants were screened for less frequently considered allosteric binding sites that could block ACE2-RBD engagement. Eight potential allosteric binding sites were identified through CavityPlus for both variants, with a single best druggable potential allosteric site identified for each. The search for potential inhibiting hit compounds involved using the SANCDB and DrugBank databanks and screening both the allosteric and orthosteric binding sites through molecular docking. Despite differences in mutated interacting residues and bond interactions, a lead compound for orthosteric binding sites (SANC00290) and potential allosteric modulators of both delta and omicron (SANC00746 and DB01029) were found. These compounds show promise in blocking ACE2-RBD engagement to potentially reduce viral interaction and infection of, and potentially withstanding further mutational residues. This study contributes to further investigation of affordable SA natural compound COVID-19 treatment and a starting point for in vitro analysis studies. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-12
The development of techniques for the identification of novel viruses associated with acute infantile gastroenteritis in South Africa
- Authors: Jaquet, Brittany J
- Date: 2017
- Subjects: Gastroenteritis in children -- Treatment , Gastroenteritis in children -- Treatment -- South Africa , Antiviral agents , Viral vaccines , Rotaviruses , Virus diseases in children
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/38013 , vital:24725
- Description: Gastroenteritis is a serious disease affecting both children and adults globally, but is more predominant in children with over half a million deaths reported each year. The leading cause of this disease is rotavirus, which accounts for 38% of all hospitalised cases. There has, however, been a significant decrease in the number of deaths associated with rotavirus worldwide since the introduction of the two vaccines, Rotarix® and RotaTeq®. A large number of cases are therefore either associated with other viruses, such as norovirus, Aichi virus (AiV) or Saffold virus (SAFV), or are of unknown aetiology. This study thus aims to develop techniques for the identification of viruses associated with gastroenteritis. Theiler’s murine encephalomyelitis virus (TMEV) was used to develop the sample preparation, transmission electron microscopy and RT-PCR techniques used in this study. This virus was chosen as a replication system using baby hamster kidney cells can be used to create high concentrations of viral particles from which RNA can be extracted preventing the waste of the limited samples. The virus particles are also similar in size and morphology to the viruses to be identified in this study, as it belongs to the same family. After sample preparation, TEM analysis showed the presence of small, round, non-enveloped virus particles in the TMEV sample. Due to the low concentration of virus particles, PEG precipitation was performed using both 0.15 M and 0.25 M NaCl and 8% (w/v) PEG 6000. TEM analysis then showed an increase in viral particle concentration, with the highest concentration observed at 0.25 M NaCl and 8% PEG 6000. RNA was successfully extracted and RT-PCR assays were performed for both the VP1 and 2B coding regions of TMEV. A method for creating a positive control for the RT-PCR assay was developed by the in vitro transcription of RNA from pTMEV, which contains the cDNA of TMEV. The RNA was then used as the template for the 2B two-step RT-PCR assay. A product of 412 bp was successfully amplified from the in vitro transcribed RNA and the sensitivity of the RT-PCR assay was determined. Using a Norovirus GII positive stool sample provided by Maureen Taylor, a nested RT-PCR assay was developed for the NoV GII N/S domain using a previously-published primer set and cycling parameters. A 342 bp product was successfully amplified from the RNA extracted from the stool sample and cloned into pGEM®-T Easy to produce pNoVGII. Using the plasmid containing the AiV 5’UTR and the PCR amplicon for AiV 3CD, RT-PCR assays were developed for AiV 5’UTR and partial 3CD. The RT-PCR assays produced a 1008 bp product for AiV 5’UTR and 266 bp for AiV 3CD, which were cloned into pGEM®-T Easy to produce pAiV5’UTR and pAiV3CD, respectively. Using in vitro transcribed RNA from pNoVGII, pAiV5’UTR, pAiV3CD and pSAFV, which contains the SAFV cDNA, positive controls were developed for the RT-PCR assays for NoV GII, AiV 5’UTR, AiV 3CD and SAFV 2C. The sensitivity of these assays was determined. The samples chosen for this study include wastewater collected from the Belmont Valley water treatment plant, oysters suspected to be infected with viruses collected from Port Elizabeth, South Africa and 30 stool samples from symptomatic patients. With the methods developed using TMEV, the wastewater, oysters and 30 stool samples were filter-sterilised, concentrated and screened by TEM. All samples showed the presence of virus particles. RNA was successfully extracted and the wastewater, oyster and 30 stool samples were screened for NoV GII using the NoV GII RT-PCR assay. The wastewater, oysters and 11 of the stool samples produced the 342 bp NoV GII PCR product and BLAST analysis determined the nucleotide sequences to be NoV GII.4. This shows that this study was able to develop sample preparation techniques and TEM analysis for selected samples and RT-PCR assays for NoV GII, AiV and SAFV. The NoV GII RT-PCR assay was successfully used for the screening of the wastewater, oysters and 30 stool samples for NoV GII. Due to the high number of gastroenteritis cases with unknown aetiology in South Africa, the development of techniques for the identification of NoV, AiV, SAFV and other viruses is very important. The identification of these viruses will allow for better surveillance, treatment and prevention of gastroenteritis in South Africa.
- Full Text:
- Date Issued: 2017
- Authors: Jaquet, Brittany J
- Date: 2017
- Subjects: Gastroenteritis in children -- Treatment , Gastroenteritis in children -- Treatment -- South Africa , Antiviral agents , Viral vaccines , Rotaviruses , Virus diseases in children
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/38013 , vital:24725
- Description: Gastroenteritis is a serious disease affecting both children and adults globally, but is more predominant in children with over half a million deaths reported each year. The leading cause of this disease is rotavirus, which accounts for 38% of all hospitalised cases. There has, however, been a significant decrease in the number of deaths associated with rotavirus worldwide since the introduction of the two vaccines, Rotarix® and RotaTeq®. A large number of cases are therefore either associated with other viruses, such as norovirus, Aichi virus (AiV) or Saffold virus (SAFV), or are of unknown aetiology. This study thus aims to develop techniques for the identification of viruses associated with gastroenteritis. Theiler’s murine encephalomyelitis virus (TMEV) was used to develop the sample preparation, transmission electron microscopy and RT-PCR techniques used in this study. This virus was chosen as a replication system using baby hamster kidney cells can be used to create high concentrations of viral particles from which RNA can be extracted preventing the waste of the limited samples. The virus particles are also similar in size and morphology to the viruses to be identified in this study, as it belongs to the same family. After sample preparation, TEM analysis showed the presence of small, round, non-enveloped virus particles in the TMEV sample. Due to the low concentration of virus particles, PEG precipitation was performed using both 0.15 M and 0.25 M NaCl and 8% (w/v) PEG 6000. TEM analysis then showed an increase in viral particle concentration, with the highest concentration observed at 0.25 M NaCl and 8% PEG 6000. RNA was successfully extracted and RT-PCR assays were performed for both the VP1 and 2B coding regions of TMEV. A method for creating a positive control for the RT-PCR assay was developed by the in vitro transcription of RNA from pTMEV, which contains the cDNA of TMEV. The RNA was then used as the template for the 2B two-step RT-PCR assay. A product of 412 bp was successfully amplified from the in vitro transcribed RNA and the sensitivity of the RT-PCR assay was determined. Using a Norovirus GII positive stool sample provided by Maureen Taylor, a nested RT-PCR assay was developed for the NoV GII N/S domain using a previously-published primer set and cycling parameters. A 342 bp product was successfully amplified from the RNA extracted from the stool sample and cloned into pGEM®-T Easy to produce pNoVGII. Using the plasmid containing the AiV 5’UTR and the PCR amplicon for AiV 3CD, RT-PCR assays were developed for AiV 5’UTR and partial 3CD. The RT-PCR assays produced a 1008 bp product for AiV 5’UTR and 266 bp for AiV 3CD, which were cloned into pGEM®-T Easy to produce pAiV5’UTR and pAiV3CD, respectively. Using in vitro transcribed RNA from pNoVGII, pAiV5’UTR, pAiV3CD and pSAFV, which contains the SAFV cDNA, positive controls were developed for the RT-PCR assays for NoV GII, AiV 5’UTR, AiV 3CD and SAFV 2C. The sensitivity of these assays was determined. The samples chosen for this study include wastewater collected from the Belmont Valley water treatment plant, oysters suspected to be infected with viruses collected from Port Elizabeth, South Africa and 30 stool samples from symptomatic patients. With the methods developed using TMEV, the wastewater, oysters and 30 stool samples were filter-sterilised, concentrated and screened by TEM. All samples showed the presence of virus particles. RNA was successfully extracted and the wastewater, oyster and 30 stool samples were screened for NoV GII using the NoV GII RT-PCR assay. The wastewater, oysters and 11 of the stool samples produced the 342 bp NoV GII PCR product and BLAST analysis determined the nucleotide sequences to be NoV GII.4. This shows that this study was able to develop sample preparation techniques and TEM analysis for selected samples and RT-PCR assays for NoV GII, AiV and SAFV. The NoV GII RT-PCR assay was successfully used for the screening of the wastewater, oysters and 30 stool samples for NoV GII. Due to the high number of gastroenteritis cases with unknown aetiology in South Africa, the development of techniques for the identification of NoV, AiV, SAFV and other viruses is very important. The identification of these viruses will allow for better surveillance, treatment and prevention of gastroenteritis in South Africa.
- Full Text:
- Date Issued: 2017
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