In silico substrate binding profiling for SARS-COV-2 main protease (mpro) using hexapeptide substrates
- Authors: Zabo, Sophakama
- Date: 2022-10-14
- Subjects: COVID-19 (Disease) , Peptides , Chymotrypsin like , Chymotrypsin , Proteases , Proteolytic enzymes
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365566 , vital:65760
- Description: COVID-19, as a disease resulting from SARS-CoV-2 infection, and a pandemic has had a devastating effect on the world. There are limited effective measures that control the spread and treatment of COVID-19 illness. The homodimeric cysteine main protease (Mpro) is crucial to the life cycle of the virus, as it cleaves the large polyproteins 1a and 1ab into matured, functional non-structural proteins. The Mpro exhibits high degrees of conservation in sequence, structure and specificity across coronavirus species, making it an ideal drug target. The Mpro substrate-binding profiles remain, despite the resolution of its recognition sequence and cleavage points (Leu-Gln↓(Ser/Ala/Gly)). In this study, a series of hexapeptide sequences containing the appropriate recognition sequence and cleavage points were generated and screened against the Mpro to study these binding profiles, and to further be the basis for efficiency-driven drug design. A multi-conformer hexapeptide substrate library comprising optimised 81000 models of 810 unique sequences was generated using RDKit within the context of python. Terminal capping with ACE and NMe was effected using SMILES and SMARTS matching. Multiple hexapeptides were complexed with chain B of crystallographic Mpro (PDS ID: 6XHM), following the validation of chain B for this purpose using AutoDock Vina at high levels of exhaustiveness (480). The resulting Vina scores ranged between -8.7 and -7.0 kcal.mol-1, and the reproducibility of best poses was validated through redocking. Ligand efficiency indices were calculated to identify substrate residues with high binding efficiency at their respective positions, revealing Val (P3), Ala (P1′); and Gly and Ala (P2′ and P3′) as leading efficient binders. Binding efficiencies were lowered by molecular weight. Substrate recognition was assessed by mapping of binding subsites, and Mpro specificity was evaluated through the resolution of intermolecular interaction at the binding interface. Molecular dynamics simulations for 20 ns were performed to assess the stability and behaviour of 132 Mpro systems complexed with KLQ*** substrates. Principal component analysis (PCA), was performed to assess II protein motions and conformational changes during the simulations. A strategy was formulated to classify and evaluate relations in the Mpro PCA motions, revealing four main clades of similarity. Similarity within a clade (Group 2) and dissimilarity between clades were confirmed. Trajectory visualisation revealed complex stability, substrate unbinding and dimer dissociation for various Mpro systems. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Zabo, Sophakama
- Date: 2022-10-14
- Subjects: COVID-19 (Disease) , Peptides , Chymotrypsin like , Chymotrypsin , Proteases , Proteolytic enzymes
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365566 , vital:65760
- Description: COVID-19, as a disease resulting from SARS-CoV-2 infection, and a pandemic has had a devastating effect on the world. There are limited effective measures that control the spread and treatment of COVID-19 illness. The homodimeric cysteine main protease (Mpro) is crucial to the life cycle of the virus, as it cleaves the large polyproteins 1a and 1ab into matured, functional non-structural proteins. The Mpro exhibits high degrees of conservation in sequence, structure and specificity across coronavirus species, making it an ideal drug target. The Mpro substrate-binding profiles remain, despite the resolution of its recognition sequence and cleavage points (Leu-Gln↓(Ser/Ala/Gly)). In this study, a series of hexapeptide sequences containing the appropriate recognition sequence and cleavage points were generated and screened against the Mpro to study these binding profiles, and to further be the basis for efficiency-driven drug design. A multi-conformer hexapeptide substrate library comprising optimised 81000 models of 810 unique sequences was generated using RDKit within the context of python. Terminal capping with ACE and NMe was effected using SMILES and SMARTS matching. Multiple hexapeptides were complexed with chain B of crystallographic Mpro (PDS ID: 6XHM), following the validation of chain B for this purpose using AutoDock Vina at high levels of exhaustiveness (480). The resulting Vina scores ranged between -8.7 and -7.0 kcal.mol-1, and the reproducibility of best poses was validated through redocking. Ligand efficiency indices were calculated to identify substrate residues with high binding efficiency at their respective positions, revealing Val (P3), Ala (P1′); and Gly and Ala (P2′ and P3′) as leading efficient binders. Binding efficiencies were lowered by molecular weight. Substrate recognition was assessed by mapping of binding subsites, and Mpro specificity was evaluated through the resolution of intermolecular interaction at the binding interface. Molecular dynamics simulations for 20 ns were performed to assess the stability and behaviour of 132 Mpro systems complexed with KLQ*** substrates. Principal component analysis (PCA), was performed to assess II protein motions and conformational changes during the simulations. A strategy was formulated to classify and evaluate relations in the Mpro PCA motions, revealing four main clades of similarity. Similarity within a clade (Group 2) and dissimilarity between clades were confirmed. Trajectory visualisation revealed complex stability, substrate unbinding and dimer dissociation for various Mpro systems. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Molecular characterization of microbial communities in the Sundays and Swartkops estuaries impacted by anthropogenic activities
- Kgomokhumo, Tlhoafalang Evah
- Authors: Kgomokhumo, Tlhoafalang Evah
- Date: 2022-04-06
- Subjects: Microbial ecology South Africa Sundays Estuary (Eastern Cape) , Microbial ecology South Africa Swartkops River Estuary , Estuarine health Effect of human beings on South Africa Sundays Estuary (Eastern Cape) , Estuarine health Effect of human beings on South Africa Swartkops River Estuary , Microorganisms South Africa Sundays Estuary (Eastern Cape) Molecular aspects , Microorganisms South Africa Swartkops River Estuary Molecular aspects , Eutrophication South Africa Sundays Estuary (Eastern Cape) , Eutrophication South Africa Swartkops River Estuary , Algal blooms South Africa Sundays Estuary (Eastern Cape) , Algal blooms South Africa Swartkops River Estuary
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/290994 , vital:56806
- Description: Anthropogenic activities are of concern in estuarine systems as they are the main source of water degradation. Water pollution in estuaries is indicated by eutrophication and the presence of pathogens and bacterial indicators which affect biodiversity and energy flow. This study focused on two geographically linked estuaries, namely the Sundays and Swartkops Estuaries. The Sundays Estuary is primarily impacted by agricultural activities in the river catchment with increased nutrients levels, particularly of total oxidised nitrogen (TOxN), likely derived from these farming activities. In contrast, the Swartkops Estuary, which is heavily influenced by urban/industrial activities, reflected increased levels of phosphates likely from wastewater and sewage water contamination from residential areas, leaking pipes, and poorly managed sewage treatment plants. The central objective of this study was to assess microbial population profiles and diversity impacted by agricultural activities in Sundays Estuary and industrial/urban-influenced Swartkops Estuary using 16S and 18S rRNA gene metabarcoding. A distinct difference in eukaryotic composition and diversity was evident between the two sampling exercises in 2018 and 2019 in Sundays Estuary. The eutrophication of both the Sundays and Swartkops estuaries was evident in the repeated occurrences of bloom events. In the Sundays Estuary, a bloom of Heterosigma akashiwo was observed in 2018 whilst Cyclotella dominated the estuary in 2019. The Swartkops Estuary exhibited seasonal variation in phytoplankton composition with Bacillariophyceae blooms in the upper reaches of the estuary in summer and increased prevalence of Dinophyceae in spring. Bacterial taxonomic variation was also noted between the two contrasting estuaries. Although members of the Proteobacteria dominated both estuaries, Gammaproteobacteria were in increased abundance in Sundays Estuary while members of Alphaproteobacteria were in high relative abundance in the marine dominated Swartkops Estuary. Members of the Bacteroidetes were the second most abundant bacterial phylum in both estuaries. Bacterial indicators of agricultural anthropogenic impacts identified in Sundays Estuary included members of Sporichthyaceae, Erysipelotrichaceae, Nostocaceae, and NS11-12_marine_group while some taxa such as the Flavobacteriaceae, Cryomorphaceae, and Halieaceae reflected their capability in degrading the phytoplankton bloom biomass present in the estuary. The urban impacts on the Swartkops Estuary was reflected by the contamination of the estuary with potential pathogens including Aeromonas caviae, Vibrio fluvialis, Mycobacterium intracellulare, Vibrio cholerae, and Bacillus cereus. Bacterial community profiles of the major water inflow points into the Swartkops Estuary included members of the Burkholderiaceae, Rhodocyclaceae, Aeromonadaceae, and Arcobacteriaceae which are typically indicative of raw sewage contamination. The Motherwell canal, which runs through informal settlements, was the most polluted input source with high levels of anthropogenic nutrients and pathogenic bacteria. The Chatty river, which also runs through townships, recorded increased nutrient concentrations and low bacterial richness and diversity which was likely due to an Arthrospira bloom at the time of sampling. The overall results of this study identified sources of pollution in Sundays and Swartkops Estuaries and highlighted the impacts of anthropogenic inputs on microbial population profiles and diversity. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
- Authors: Kgomokhumo, Tlhoafalang Evah
- Date: 2022-04-06
- Subjects: Microbial ecology South Africa Sundays Estuary (Eastern Cape) , Microbial ecology South Africa Swartkops River Estuary , Estuarine health Effect of human beings on South Africa Sundays Estuary (Eastern Cape) , Estuarine health Effect of human beings on South Africa Swartkops River Estuary , Microorganisms South Africa Sundays Estuary (Eastern Cape) Molecular aspects , Microorganisms South Africa Swartkops River Estuary Molecular aspects , Eutrophication South Africa Sundays Estuary (Eastern Cape) , Eutrophication South Africa Swartkops River Estuary , Algal blooms South Africa Sundays Estuary (Eastern Cape) , Algal blooms South Africa Swartkops River Estuary
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/290994 , vital:56806
- Description: Anthropogenic activities are of concern in estuarine systems as they are the main source of water degradation. Water pollution in estuaries is indicated by eutrophication and the presence of pathogens and bacterial indicators which affect biodiversity and energy flow. This study focused on two geographically linked estuaries, namely the Sundays and Swartkops Estuaries. The Sundays Estuary is primarily impacted by agricultural activities in the river catchment with increased nutrients levels, particularly of total oxidised nitrogen (TOxN), likely derived from these farming activities. In contrast, the Swartkops Estuary, which is heavily influenced by urban/industrial activities, reflected increased levels of phosphates likely from wastewater and sewage water contamination from residential areas, leaking pipes, and poorly managed sewage treatment plants. The central objective of this study was to assess microbial population profiles and diversity impacted by agricultural activities in Sundays Estuary and industrial/urban-influenced Swartkops Estuary using 16S and 18S rRNA gene metabarcoding. A distinct difference in eukaryotic composition and diversity was evident between the two sampling exercises in 2018 and 2019 in Sundays Estuary. The eutrophication of both the Sundays and Swartkops estuaries was evident in the repeated occurrences of bloom events. In the Sundays Estuary, a bloom of Heterosigma akashiwo was observed in 2018 whilst Cyclotella dominated the estuary in 2019. The Swartkops Estuary exhibited seasonal variation in phytoplankton composition with Bacillariophyceae blooms in the upper reaches of the estuary in summer and increased prevalence of Dinophyceae in spring. Bacterial taxonomic variation was also noted between the two contrasting estuaries. Although members of the Proteobacteria dominated both estuaries, Gammaproteobacteria were in increased abundance in Sundays Estuary while members of Alphaproteobacteria were in high relative abundance in the marine dominated Swartkops Estuary. Members of the Bacteroidetes were the second most abundant bacterial phylum in both estuaries. Bacterial indicators of agricultural anthropogenic impacts identified in Sundays Estuary included members of Sporichthyaceae, Erysipelotrichaceae, Nostocaceae, and NS11-12_marine_group while some taxa such as the Flavobacteriaceae, Cryomorphaceae, and Halieaceae reflected their capability in degrading the phytoplankton bloom biomass present in the estuary. The urban impacts on the Swartkops Estuary was reflected by the contamination of the estuary with potential pathogens including Aeromonas caviae, Vibrio fluvialis, Mycobacterium intracellulare, Vibrio cholerae, and Bacillus cereus. Bacterial community profiles of the major water inflow points into the Swartkops Estuary included members of the Burkholderiaceae, Rhodocyclaceae, Aeromonadaceae, and Arcobacteriaceae which are typically indicative of raw sewage contamination. The Motherwell canal, which runs through informal settlements, was the most polluted input source with high levels of anthropogenic nutrients and pathogenic bacteria. The Chatty river, which also runs through townships, recorded increased nutrient concentrations and low bacterial richness and diversity which was likely due to an Arthrospira bloom at the time of sampling. The overall results of this study identified sources of pollution in Sundays and Swartkops Estuaries and highlighted the impacts of anthropogenic inputs on microbial population profiles and diversity. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
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