Computational studies in human African trypanosomiasis
- Authors: Muronzi, Tendai
- Date: 2023-10-13
- Subjects: African trypanosomiasis , Apolipoprotein L1 , Docking , Protein-protein interactions , Homology modeling , Tetrahydrofolate dehydrogenase , Pteridine reductase
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/431883 , vital:72812 , DOI 10.21504/10962/431885
- Description: Human African trypanosomiasis (HAT) is a neglected tropical disease (NTD) caused by two subspecies of the parasite, namely Trypanosoma brucei (Tb) gambiense (g-HAT) and rhodesiense (r-HAT). HAT is endemic in sub-Saharan countries, where the parasite transmission vectors, tsetse flies, breed. An estimated 70 million people remain at risk of contracting the disease, where the infection is classified as acute or chronic for g-HAT and r-HAT, respectively, with both forms ending in fatal meningoencephalitis when left untreated. Both g-HAT and r-HAT are responsible for widespread fatal epidemics throughout sub-Saharan African history, resulting from the complex molecular interplay between trypanosomes and humans through unique, innate immunity evasion mechanisms. Of interest, the Tbr subspecies expresses a serum resistance-associated protein (SRA), which binds to human serum lytic factor, apolipoprotein L1 (ApoL1), nullifying any trypanocidal activity. In response, ApoL1 (G1 and G2) variants found in humans of sub-Saharan African lineage have been cited for conferring resistance to the r-HAT infection in an interaction that is not fully elucidated In the event of successful infection, current HAT chemotherapeutics are plagued with complexity of administration, poor efficacy, toxicity, and potential drug resistance, highlighting a need for improved approaches. The parasite folate pathway provides a strategic target for alternative anti-trypanosomal drug development as trypanosomatids are folate auxotrophs, requiring host folate for growth and survival. Validated drug targets pteridine reductase (TbPTR1) and dihydrofolate reductase (TbDHFR) are essential for salvaging cofactors folate and folate biopterin crucial to parasite survival, making them viable targets for anti-folate investigation. The overall aims of this thesis were to a) provide insights into the molecular and dynamic basis of the SRA and ApoL1 interplay in HAT infection and b) identify safer and more efficient anti-folate anti-trypanosomal drug alternatives through in silico approaches. To achieve our first aim, in silico structure prediction was applied to generate 3D models of ApoL1 C-terminal variants G0, G1, G1G/M, G2 and G1G2, and four SRA variants retrieved from the NCBI database. The SRA and ApoL1 structures were inspected dynamically to identify the effect of the variants through molecular dynamics (MD) simulations. Dynamic residue network (DRN) analysis of MD trajectories was fundamental in identifying residues playing a vital role in the intramolecular communication of both proteins in the presence of mutations. Protein-protein docking was then applied to calculate plausible SRA-ApoL1 C-terminal wild-type complex structures to further elucidate the nature of SRA-mediated infection. Through MD simulations, twelve SRA-ApoL1 dimeric structures were narrowed down from five to two energetically sound complexes. The two feasible SRA-ApoL1 complexes (1 and 2) exhibited favourable communication observed through DRN analysis, including the retaining key communication residues identified in prior monomer DRN calculations. ApoL1 C-terminal variants were additionally incorporated into SRA-ApoL1 complexes 1 and 2 for further complex dynamics analysis This investigation into the nature of SRA-ApoL1 binding resulted in five primary outcomes: 1) highlighting the intramolecular effects ApoL1 variants have on the stability of the protein, 2) the identification of crucial SRA and ApoL1 communication residues in both monomeric or dimeric form, 3) the isolation of feasible SRA-ApoL1 complexes determined through global and local structural analyses 4) identification of residues crucial to the complex formation and maintenance of SRA-ApoL1, overlapping with those identified in (1), and 5) the minimal dissociative role of the G1 mutations in the complex, but compounding effect of the G2 deletion mutation. Computational modelling and drug repurposing were employed to achieve the thesis's second aim as they drastically cut down the costs involved in drug discovery and provide a more time-efficient screening method through numerous drug candidates. Using high throughput virtual screening, a subset of 2089 approved DrugBank compounds were screened against TbPTR1. The outputs were filtered to 24 viable compounds in 54 binding poses using binding energy and molecular interactions. Through subsequent MD simulations of 200ns, thirteen potential hit compounds were identified. The resultant hit compounds were subjected to further blind docking against TbDHFR and molecular dynamics to identify compounds with the potential for dual inhibition. The filtered subset was also tested in in vitro single concentration and dose-response bioassays to assess inhibitory properties against Trypanosoma brucei, complementing in silico findings. Post-molecular dynamics, four compounds exhibited high stabilities and molecular interactions with both TbPTR1 and TbDHFR, with two presenting favourable results in the in vitro assays. Three compounds additionally shared common structural moieties. In all, the in silico repurposing highlighted drugs characterised by favourable interactions and stabilities in TbPTR1, thus providing (1) a framework for further studies investigating anti-folate HAT compounds and (2) modulatory scaffolds based on identified moieties that can be used for the design of safe anti-folate trypanosomal drugs. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2023
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- Date Issued: 2023-10-13
Computer aided approaches against Human African Trypanosomiasis
- Authors: Kimuda, Magambo Phillip
- Date: 2020
- Subjects: African trypanosomiasis , African trypanosomiasis -- Chemotherapy , Genomics , Macrophage migration inhibitory factor , Trypanosoma brucei , Pteridines , Tetrahydrofolate dehydrogenase , Adenylic acid , Molecular dynamics , Principal components analysis , Bioinformatics , Single nucleotide polymorphisms , Single Nucleotide Variants , Candidate Gene Association Study (CGAS)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/142542 , vital:38089
- Description: The thesis presented here is divided into two parts under a common theme that is the use of computer based tools, genomics, and in vitro experiments to develop innovative ways of tackling Human African Trypanosomiasis (HAT). Part I of this thesis focused on the human host genetic determinants while Part II focused on the discovery of novel chemotherapeutics against the parasite. Part I is further sub-divided into two parts: The first involves a Candidate Gene Association Study (CGAS) on an African population to identify genetic determinants associated with disease and/or susceptibility to HAT. The second involves studying the effects of missense Single Nucleotide Variants (SNVs) on protein structure, dynamics, and function using Macrophage Migration Inhibitory Factor (MIF) as a case study. Part II is also sub-divided into two parts: The first involves a computer based rational drug discovery of potential inhibitors against the Trypanosoma the folate pathway; particularly by targeting Trypanosoma brucei Pteridine Reductase (TbPTR1) which is an enzyme used by trypanosomes to overcome T. brucei Dihydrofolate Reductase (TbDHFR) inhibition. Lastly the derivation of CHARMM force-field parameters that can be used to accurately model the geometry and dynamics of the T. brucei Phosphodiesterase B1 enzyme (TbrPDEB1) bimetallic active site center. The derived parameters were then used in MD simulations to characterise protein-ligand residue interactions that are important in TbrPDEB1 inhibition with the goal of targeting the cyclic Adenosine Monophosphate (cAMP) signalling pathway. In the CGAS we were unable to detect any genetic associations in the Ugandan cohort analysed that passed correction for multiple testing in spite of the study being sufficiently powered. Additionally, our study found no association of the Apo lipoprotein 1 (APOL1) G2 allele association with protection against acute HAT that has been previously reported. Future investigations for example, Genome Wide Association Studies using larger samples sizes (>3000 cases and controls) are required. Macrophage migration inhibitory factor (MIF) is a cytokine that is important in both innate and adaptive immunity that has been shown to play a role in T. brucei pathogenicity using murine models. A total of 27 missense SNVs were modelled using homology modelling to create MIF protein mutants that were investigated using in silico effect prediction tools, molecular dynamics (MD), Principal Component Analysis (PCA), and Dynamic Residue Network (DRN) analysis. Our results demonstrate that mutations P2Q, I5M, P16Q, L23F, T24S, T31I, Y37H, H41P, M48V, P44L, G52C, S54R, I65M, I68T, S75F, N106S, and T113S caused significant conformational changes. Further, DRN analysis showed that residues P2, T31, Y37, G52, I65, I68, S75, N106, and T113S are part of a similar local residue interaction network with functional significance. These results show how polymorphisms such as missense SNVs can affect protein conformation, dynamics, and function. Trypanosomes are auxotrophic for folates and pterins but require them for survival. They scavenge them from their hosts. PTR1 is a multifunctional enzyme that is unique to trypanosomatids that reduces both pterins and folates. In the presence of DHFR inhibitors, PTR1 is over-expressed thus providing an escape from the effects of DHFR inhibition. Both TbPTR1 and TbDHFR are pharmacologically and genetically validated drug targets. In this study 5742 compounds were screened using molecular docking, and 13 promising binding modes were further analysed using MD simulations. The trajectories were analysed using RMSD, Rg, RMSF, PCA, Essential Dynamics Analysis (EDA), Molecular Mechanics Poisson–Boltzmann surface area (MM-PBSA) binding free energy calculations, and DRN analysis. The computational screening approach allowed us to identify five of the compounds, named RUBi004, RUBi007, RUBi014, RUBi016 and RUBi018 that exhibited antitrypanosomal growth activities against trypanosomes in culture with IC50 values of 12.5 ± 4.8 μM, 32.4 ± 4.2 μM, 5.9 ± 1.4 μM, 28.2 ± 3.3 μM, and 9.7 ± 2.1 μM, respectively. Further when used in combination with WR99210 a known TbDHFR inhibitor RUBi004, RUBi007, RUBi014 and RUBi018 showed antagonism while RUBi016 showed an additive effect. These results indicate that the four compounds might be competing with TbDHFR while RUBi016 might be more specific for TbPTR1. These compounds provide scaffolds that can be further optimised to improve their potency and specificity. Lastly, using a systematic approach we derived CHARMM force-field parameters to accurately describe the TbrPDEB1 bi-metal catalytic center. For dynamics, we employed mixed bonded and non-bonded approach. We optimised the structure using a two-layer QM/MM ONIOM (B3LYP/6-31(g): UFF). The TbrPDEB1 bi-metallic center bonds, angles, and dihedrals were parameterized by fitting the energy profiles from Potential Energy Surface (PES) scans to the CHARMM potential energy function. The parameters were validated by means of MD simulations and analysed using RMSD, Rg, RMSF, hydrogen bonding, bond/angle/dihedral evaluations, EDA, PCA, and DRN analysis. The force-field parameters were able to accurately reproduce the geometry and dynamics of the TbrPDEB1 bi-metal catalytic center during MD simulations. Molecular docking was used to identify 6 potential hits, that inhibited trypanosome growth in vitro. The derived force-field parameters were used to simulate the 6 protein-ligand complexes with the aim of elucidating crucial protein-ligand residue interactions. Using the most potent ligand RUBi022 that had an IC50 of 14.96 μM we were able to identify key residue interactions that can be of use in in silico prediction of potential TbrPDEB1 inhibitors. Overall we demonstrate how bioinformatics tools can complement current disease eradication strategies. Future work will focus on identifying variants identified in Genome Wide Association Studies and partnering with wet labs to carry out further enzyme-ligand activity relationship studies, structure determination or characterisation of appropriate protein-ligand complexes by crystallography, and site specific mutation studies
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- Date Issued: 2020