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
- Full Text:
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
- Full Text:
Falcipain 2 and 3 as malarial drug targets: deciphering the effects of missense mutations and identification of allosteric modulators via computational approaches
- Authors: Okeke, Chiamaka Jessica
- Date: 2023-10-13
- Subjects: Antimalarials , Cysteine proteinases , Missense mutation , Allostery , Cysteine proteinase falcipain 2a , Cysteine proteinase falcipain 3
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/432170 , vital:72848 , DOI 10.21504/10962/432170
- Description: Malaria, caused by an obligate unicellular protozoan parasite of the genus Plasmodium, is a disease of global health importance that remains a major cause of morbidity and mortality in developing countries. The World Health Organization (WHO) reported nearly 247 million malaria cases in 2021, causing 619,000 deaths, the vast majority ascribed to pregnant women and young children in sub-Saharan Africa. A critical component of malaria mitigation and elimination efforts worldwide is antimalarial drugs. However, resistance to available antimalarial drugs jeopardizes the treatment, prevention, and eradication of the disease. The recent emergence and spread of resistance to artemisinin (ART), the currently recommended first-line antimalarial drug, emphasizes the need to understand the resistance mechanism and apply this knowledge in developing new drugs that are effective against malaria. An insight into ART's mechanism of action indicates that ferrous iron (Fe2+) or heme, released when hemoglobin is degraded, cleaves the endoperoxide bridge. As a result, free radicals are formed, which alkylate many intracellular targets and result in plasmodial proteopathy. Aside from the existing evidence that mutations in the Kelch 13 protein propeller domain affect ART sensitivity and clearance rate by Plasmodium falciparum (Pf) parasites, recent investigations raise the possibility that additional target loci may be involved, and these include a nonsense (S69stop) and four missense variants (K255R, N257E, T343P, and D345G) in falcipain 2 (FP-2) protein. FP-2 and falcipain 3 (FP-3) are cysteine proteases responsible for hydrolyzing hemoglobin in the host erythrocytic cycle, a key virulence factor for malaria parasite growth and metabolism. Due to the obligatory nature of the hemoglobin degradation process, both proteases have become potential antimalarial drug targets attracting attention in recent years for the development of blood-stage antimalarial drugs. The alteration of the expression profile of FP-2 and FP-3 through gene manipulation approaches (knockout) or compound inhibition assays, respectively, induced parasites with swollen food vacuoles due to the accumulation of undegraded hemoglobin. Furthermore, missense mutations in FP-2 confer parasites with decreased ART sensitivity, probably due to altered enzyme efficiency and momentary decreased hemoglobin degradation. Hence, understanding how these mutations affect FP-2 (including those implicated in ART resistance) and FP-3 is imperative to finding potentially effective inhibitors. The first aim of this thesis is to characterize the effects of missense mutations on the partial zymogen complex and the catalytic domain of FP-2 and FP-3 using a range of computational approaches and tools such as homology modeling, molecular dynamics (MD) simulations, comparative essential dynamics, dynamic residue network (DRN) analysis, weighted residue contact map analysis, amongst others. The Pf genomic resource database (PlasmoDB) identified 41 missense mutations located in the partial zymogen and catalytic domains of FP-2 and FP-3. Using structure-based tools, six putative allosteric pockets were identified in FP-2 and FP-3. The effect of mutations on the whole protein, the central core, binding pocket residues and allosteric pockets was evaluated. The accurate 3D homology models of the WT and mutants were calculated. MD simulations were performed on the various systems as a quick starting point. MD simulations have provided a cornerstone for establishing numerous computational tools for describing changes arising from mutations, ligand binding, and environmental changes such as pH and temperature. Post-MD analysis was performed in two stages viz global and local analysis. Global analysis via radius of gyration (Rg) and comparative essential dynamic analysis revealed the conformational variability associated with all mutations. In the catalytic domain of FP-2, the presence of M245I mutation triggered the formation of a cryptic pocket via an exclusive mechanism involving the fusion of pockets 2 and 6. This striking observation was also detected in the partial zymogen complex of FP-2 and induced by A159V, M245I and E249A mutations. A similar observation was uncovered in the presence of A422T mutation in the catalytic domain of FP-3. Local DRN and contact map analyses identified conserved inter-residue interaction changes on important communication networks. This study brings a novel understanding of the effects of missense mutations in FP-2 and FP-3 and provides important insight which may help discover new anti-hemoglobinase drugs. The second aim is the identification of potential allosteric ligands against the WT and mutant systems of FP-2 and FP-3 using various computational tools. Of the six potential allosteric pockets identified in FP-2 and FP-3, pocket 1 was evaluated by SiteMap as the most druggable in both proteins. This pipeline was implemented to screen pocket 1 of FP-2 and FP-3 against 2089 repositionable compounds obtained from the DrugBank database. In order to ensure selectivity and specificity to the Plasmodium protein, the human homologs (Cat K and Cat L) were screened, and compounds binding to these proteins were exempted from further analysis. Subsequently, eight compounds (DB00128, DB00312, DB00766, DB00951, DB02893, DB03754, DB13972, and DB14159) were identified as potential allosteric hits for FP-2 and five (DB00853, DB00951, DB01613, DB04173 and DB09419) for FP-3. These compounds were subjected to MD simulation and post-MD trajectory analysis to ascertain their stability in their respective protein structures. The effects of the stable compounds on the WT and mutant systems of FP-2 and FP-3 were then evaluated using DRN analysis. Attention has recently been drawn towards identifying novel allosteric compounds targeting FP-2 and FP-3; hence this study explores the potential allosteric inhibitory mechanisms in the presence and absence of mutations in FP-2 and FP-3. Overall, the results presented in this thesis provide (i) an understanding of the role mutations in the partial zymogen complex play in the activation of the active enzyme, (ii) an insight into the possible allosteric mechanisms induced by mutations on the active enzymes, and (iii) a computational pipeline for the development of novel allosteric modulators for malaria inhibition studies. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text: