An investigation on the effects of Afrocentric missense variations on the structure and function of CYP2A6 protein
- Authors: Makombe, Chipo Perpetual
- Date: 2025-04-02
- Subjects: Missense mutation , Structural dynamics , Enzyme activity , Drugs Metabolism , CYP2A6
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/479119 , vital:78262
- Description: Pharmacogenomics, the foundation of personalized medicine distinguishes patients into different categories based on their response to the risk of a disease. Cytochrome P450 (CYPs) proteins are a family of enzymes critical in the metabolism of drugs and other substances. Genetic polymorphisms in CYPs can result in different enzymatic activity in individuals influencing the efficacy and toxicity of drugs. One of the CYPs which primarily metabolizes nicotine and other pharmaceutical drugs such as Artemisinin and Artesunate, Pilocarpine, Valproic Acid and Letrozole is CYP2A6. The gene encoding the protein is highly polymorphic and this can affect the rate of metabolism of drugs in individuals. Previously most studies unveiled connections between CYP2A6 variants and nicotine. Implications concerning the effects of specific missense variations in CYP2A6 drug metabolism have deficiencies. This study aimed to critically examine the structural and functional implications of 13 CYP2A6 allele variations on CYP2A6 protein using Bioinformatics techniques. Methods used were template selection, mutagenesis, parameter assignment and protonation. Molecular Dynamics to get insights regarding protein behavior at an atomic level, clustering to identify conformations during a simulation and DSSP for secondary structure analysis to monitor how secondary structures evolve. Berendsen and Parinello-Rahman barostats at production run were used for comparison. A global analysis was conducted to identify structural transitions (RMSD, RMSF, and Rg), clustering, and secondary structure prediction. Results from Berendsen barostat were inconsistent compared to Parrinello-Rahman barostat implying that CYP2A6 is sensitive to the pressure coupling parameter for precise and accurate results. Our clustering results showed each system in one conformation, fluctuations and shifts on the C-D, H-I loops and F, G, and L helices on variants I149M, F118l, K476R, and E390K_N418D_E419D. This indicated a potential loss of function limiting the protein’s ability to conformational flexibility for catalysis and substrate recognition. Certain regions of CYP2A6 became more rigid due to variations, which could have a negative impact on the catalytic activity, regulatory interactions, and general function of the enzyme in metabolism. Globally the variations did not cause large changes to the protein, there is need for a local analysis using Dynamic Residue Networks to study how residue interactions affect the function of CYP2A6. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Bioinformatics, 2025
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The effect of Afrocentric missense variations on the structural dynamics of CYP2B6
- Authors: Govender, Shaylyn Ashley
- Date: 2025-04-02
- Subjects: CYP2B6 , Structural dynamics , Metabolism , Missense mutation , Molecular dynamics
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/479108 , vital:78261
- Description: Cytochrome P450s are a superfamily of enzymes with over 50 members involved in metabolizing 90% of xenobiotics. Among the these, families 1, 2, and 3 are responsible for approximately 80% of clinical drug metabolism. This study investigates the effect of Afrocentric missense variants on the structural dynamics of CYP2B6. Molecular dynamic simulations reveal that specific variants affect the enzyme’s flexibility and stability, potentially altering catalytic activity and drug binding properties. These findings highlight the importance of considering genetic variants in personalized medicine and drug development. By investigating CYP2B6’s function and structural changes induced by missense variants, this research advances our understanding of the enzyme’s role in drug metabolism. The study utilized computational tools such as GROMACS and AMBER for pre- and post-simulation analysis, with clustering and DSSP used to assess protein structures. Variants I328T, K282R, P428T and R140Q exhibited significant deviations in enzyme dynamics, while other variants caused minor shifts. Overall, the findings provide insight into the relationship between genetic variants and enzyme function, contributing to bioinformatics and molecular modelling approaches in drug discovery. Future studies could explore the structural and fuctional impacts of CYP2B6 bound to substrates such as antimalarials, expanding the investigation to a broader range of missense variants. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology and Bioinformatics, 2025
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In-silico investigation of the effects of genetic mutations on the structural dynamics of thiopurine s-methyltransferase and their implications on the metabolism of 6-mercaptopurine
- Authors: Mwaniki, Rehema Mukami
- Date: 2023-10-13
- Subjects: Mutation , Thiopurine S-methyltransferase , Mercaptopurine , Molecular dynamics , Protein structure , Structural dynamics
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/432553 , vital:72880
- Description: Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds such as 6-mercaptopurine (6MP), 6-thioguanine (6TG) and azathioprine (AZA) which is first converted to 6MP through reduction by glutathione S- transferases (GST). The compounds, generally referred to as thiopurines, are immunosuppressants used to treat childhood acute lymphoblastic leukemia (ALL), autoimmune disorders and transplant rejection. Thiopurines are prodrugs which require metabolic activation to give thioguanine nucleotides that exert their cytotoxic effects by incorporation into DNA or inhibiting purine synthesis. The methylation reaction by TPMT utilizing S-adenosylmethionine (SAM) as the methyl donor prevents their conversion to these toxic compounds. The catalytic activity of TPMT in metabolising these compounds has been associated with occurrence of genetic variations. The variations that result to missense mutations cause amino-acid changes and in turn alter the polypeptide sequence of the protein. This could alter functionality and structural dynamics of the enzyme. This study sought to understand the underlying mechanism by which 7 specially selected mutations impede metabolic activity of the enzyme on 6-MP using in silico techniques. VAPOR and PredictSNP were used to predict the effects of single nucleotide polymorphisms (SNPs) on the stability and function of the enzyme. Of the 7 mutations, only H227Q was predicted to be functionally benign while the rest (L49S, L69V, A80P, R163H, R163C and R163P) were predicted to be deleterious or associated with disease. All the SNPs were predicted to destabilize the enzyme. Molecular dynamics (MD) simulations were preformed to mimic the behaviour of the apo, holo and drug-bound WT and mutant enzymes in vivo. This was followed by post-MD analysis to identify changes in the local and global motions of the protein in the presence of mutations and changes in intra-protein communication networks through contact map and centrality metrics calculations. RMSD and Rg analyses were performed to assess changes in global motions and compactness of the enzyme in the apo, holo and drug-bound states and in the presence of mutations. These revealed that binding of the ligand had a stabilizing effect on the WT enzyme evident from more steady trends from the analyses across trajectories in the holo and drug-bound enzymes compared to the apoenzyme. The occurrence of mutations had an effect on the global motions and compactness of the enzyme across the trajectories. Most mutations resulted in destabilized systems and less compact structures shown by unsteady RMSD and Rg across trajectories respectively. The drugbound systems appeared to be more stable in most of the systems meaning that the binding of 6MP stabilized the enzyme regardless of the presence of a mutation. RMSF analysis recorded local changes in residue flexibility due to the presence of mutations in all the systems. All the drug-bound mutant systems lost flexibility on the αAhelix which caps the active site. This could have an effect on drug binding and result to defective drug metabolism. The A80P mutation resulted to a more rigid structure from both global and local motions compared to the WT enzyme which could be associated with its nearly loss of function in vivo and in vitro. Dynamic cross correlation calculations were performed to assess how the atoms moved together. Correlated, anti-correlated and areas of no correlations were recorded in all the systems and in similar places when compared to each other. This meant that occurrence of mutations had no effect on how the atoms moved together. Contact map analysis showed that occurrence of mutations caused changes in interactions around the positions where the mutations occurred, which could have an effect on protein structural dynamics. The A80P substitution which occurred on the surface away from the binding site was identified as an allosteric mutation that resulted to changes in the catalytic site. Contact maps for the drug-cofactor complex in the mutant systems in comparison with the WT protein revealed changes that could suggest reorientation of the drug at the catalytic site. This could be an implication to altered drug metabolism. Eigenvector centrality (EC) and betweenness centrality (BC) for the most equilibrated portions of the trajectories were calculated for all the studied systems to identify residues connected to the most important residues and those that were spanned the most in shortest paths connecting other residues. Areas that scored highest in these metrics where mostly found in regions surrounding the catalytic site. Top 5% centrality hubs calculations showed loss of major hubs due to mutations with gaining of new ones. This means that mutations affected communication networks within the protein. The gained hubs were in areas close-by the lost ones which could have been an attempt of the protein to accommodate the mutations. Persistent top 5% BC hubs were identified at positions 90 and 151 while one persistent top 5% EC hub was identified at position 70. This positions play important roles in shaping the catalytic site and are in direct contact with the ligands. It was concluded that in silico techniques and analysis applied in this study revealed possible mechanisms in which genetic variations affected the structural dynamics of TMPT enzyme an affecte 6MP metabolism. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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Identification of selective novel hits against Mycobacterium tuberculosis KasA potential allosteric sites using bioinformatics approaches
- Authors: Hare, Fadzayi Faith
- Date: 2022-10-14
- Subjects: Tuberculosis , Docking , Molecules Models , Virtual screening , Multidrug-resistant tuberculosis , Fatty acids Synthesis , Drugs Design
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362842 , vital:65367
- Description: Tuberculosis (TB) is a global health threat that has led to approximately 1.5 million deaths annually. According to the World Health Organization (WHO), TB is among the top ten deadly diseases and is the leading cause of death due to a single infectious agent. The main challenge in the effective treatment and control of TB is the ongoing emergence of resistant strains of Mycobacterium tuberculosis (Mtb) which lead to multi-drug resistant (MDR) and extensive-drug resistant (XDR) TB. Hence, the identification and characterization of novel drug targets and drugs that modulate the activity of the pathogen are an urgent priority. The current situation even necessitates the reengineering or repurposing of drugs in order to achieve effective control. The β-ketoacyl-acyl carrier protein synthase I (KasA) of Mycobacterium tuberculosis is an essential enzyme in the mycobacterial fatty acid synthesis (FAS-II) pathway and is believed to be a promising target for drug discovery in TB. It is one of the five main proteins of the FAS-II pathway and catalyzes a key condensation reaction in the synthesis of meromycolate chains, the precursors of mycolic acids involved in cell wall formation. Although this protein has been extensively studied, little research has been devoted to the allosteric inhibition of potential drug compounds. The main aim of this research was to identify the allosteric sites on the protein that could be involved in the inhibition of substrate binding activities and novel drug compounds that bind to these sites by use of in-silico approaches. The bioinformatics approaches used in this study were divided into four main objectives namely identification of KasA homolog sequences, sequence analysis and protein characterization, allosteric site search and lastly virtual screening of DrugBank compounds via molecular docking. Fifteen homolog sequences were identified from the BLASTP analysis and were derived from bacteria, fungi and mammals. In order to discover important residues and regions within the KasA proteins, sequence alignment, motif analysis and phylogenetic studies were performed using Mtb KasA as a reference. Sequence alignment revealed conserved residues in all KasA proteins that have functional importance such as the catalytic triad residues (Cys171, His311 and His345). Motif analysis identified 18 highly conserved motifs within the KasA proteins with structural and functional roles. In addition, motifs unique to the Mtb KasA protein were also identified and explored for inhibitor drug design purposes. Phylogenetic analysis of the homolog sequences showed a distinct clustering of prokaryotes and eukaryotes. A distinctive clustering was also observed for species belonging to the same genus. Since the mechanism of action of most drugs involves the active site, allosteric site search was conducted on Mtb KasA and the human homolog protein using a combination of pocket detection algorithms with the aim of identifying sites that could be utilized in allosteric modulator drug discovery. This was followed by the virtual screening of 2089 FDA approved DrugBank compounds against the entire protein surfaces of Mtb KasA and Hsmt KasA, performed via molecular docking using AutoDock Vina. Screening of the compounds was based on the binding energies, with more focus on identifying ligands that bound exclusively to the acyl-binding tunnel of Mtb KasA. This reduced the data set to 27 promising drug compounds with a relatively high binding affinity for Mtb KasA, however, further experiments need to be performed to validate this result. Among these compounds were DB08889, DB06755, DB09270, DB11226, DB00392, DB12278, DB08936, DB00781, DB13720 and DB00392, which displayed relatively low binding energies for Mtb KasA when compared to the human homolog protein. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
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