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Item The ligandin activity of Schistosoma 26-kDa and 28-kDa glutathione transferases towards 17β-Hydroxyandrost-4-ene-3-one from a biophysical perspective(University of the Witwatersrand, Johannesburg, 2023) Makumbe, Hattie Hope; Achilonu, Ikechukwu AnthonySchistosomiasis, caused by helminth worms, ranks second amongst parasitic diseases and accounts for over 220 million fatalities globally. Statistics show that in South Africa, schistosomiasis (bilharzia) has infected approximately 4 million individuals. Currently, there are parasite resistance challenges with the sole available remedy. The World Health Organisation (WHO) acknowledges the need for new effective drugs. The 26-kDa Schistosoma bovis/haematobium (Sbh26GST) and 28-kDa Schistosoma haematobium (Sh28GST) are parasite Glutathione S-transferases (GSTs) which consist of two identical subunits that perform a vital role in mitigating the adverse effects of harmful electrophilic substances within the parasite since the parasite is devoid of the neutralizing cytochrome P-450. This automatically renders these parasite GSTs as potential therapeutic targets for schistosomiasis. Testosterone, the major hormone responsible for sexual characteristics and growth in males, can be repurposed as a drug target against schistosomiasis. In this study, we examined the structural, stability and functional interactions between the parasite GSTs and testosterone. After confirmation of inhibition, IC50 experiments were performed. The enzymes were overexpressed in Escherichia coli (E.coli) and then purified through a single-step nickel ion-immobilized metal affinity chromatography (IMAC). Extrinsic fluorescence spectroscopy was also done to provide evidence for the binding of the recombinant GSTs with testosterone. The GST activity was measured by employing 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate. Additionally, we investigated if the enzyme activity was influenced by the presence of testosterone. To analyse the stability of the enzymes, a SYPRO Orange-based thermal shift assay was used in the presence and absence of testosterone. In addition to empirical investigations, computational modelling, molecular docking, and molecular dynamic simulations were used to provide complementary insights to show binding affinities, prediction of binding modes and stability of the GST-testosterone complex. The secondary structural composition was found to be predominantly alpha-helical. Insights into tertiary structure analysis revealed the presence of buried solvent exposed tryptophan residues. The findings from spectroscopy with 8-anilino-1-naphthalenesulfonate (ANS) indicated that both Human GST-mu and parasite GSTs bound to ANS. Enzyme kinetic studies show that testosterone is a potent inhibitor of the parasite GSTs, with a specific activity that decreases from 16 μmol min-1mg-1 to 0.03 μmol min-1mg-1 and IC50 in the nanomolar range of 20 µM for Sh28GST. Sbh26GST exhibited a specific activity that decreased from 20 μmol min-1mg-1 to 0.14 μmol min-1mg-1, and a testosterone IC50 of 23 µM. The thermal stability assay confirmed Sh28GST to be more stable than Sbh26GST, and this stability of Sh28GST intensified when the enzyme bound to testosterone and GSH. Steady state kinetics towards glutathione (GSH) revealed a Km of 4.2mM and 6.6 mM for Sh28GST and Sbh26GST respectively. The present study has practical implications for novel application of the enzymes to serve as a basis for future studies aimed at development of inhibitors with potential therapeutic benefits through rational drug design.Item Evaluating the in vitro anti-metastatic effects of silver(I) phosphine complexes on malignant breast cancer cell lines(University of the Witwatersrand, Johannesburg, 2023-08) Ferreira, Mizan; Engelbrecht, Zelinda; Cronje, Marianne JacquelineBreast cancer is the most diagnosed cancer type among females worldwide. Metastasis, the spread of cancer cells from the primary tumour and establishment of macroscopic secondary tumours, is regarded as the most dangerous characteristic of cancer cells as it is responsible for over 90% of cancer-related deaths. Globally there is a lack of drugs available to specifically target or prevent either the dissemination of cells from the primary tumour or the establishment of distant metastases. The purpose of this study was to ascertain whether a series of silver(I) phosphine complexes, which have previously been shown to display anti-cancer properties in vitro, are also effective as anti-metastatic compounds. The migration, invasion and adhesive abilities of two malignant breast cancer cell lines, MCF-7 and MDA-MB-231, in response to silver(I) phosphine treatment were evaluated. In addition, the colony-forming abilities of cells under both anchorage-dependent and -independent conditions were investigated. Furthermore, the effects of silver(I) phosphine treatment on the expression and activities of key metastatic proteins, matrix metalloproteinases (MMPs), were studied. Of the nine complexes evaluated, all of them showed the ability to reduce one or more metastatic steps namely cell migration, invasion through collagen towards a chemoattractant or adhesion to collagen. In addition, a selected number of complexes reduced the colony-forming abilities of MCF-7 and/or MDA-MB-231 cells in culture plates as well as in soft agar. Moreover, three of these complexes increased the in vitro invasion and colony formation of breast cancer cells. Further investigation into complexes showing anti-metastatic abilities revealed that, apart from one complex on MDA-MB-231 cells, anti-metastatic effects were not achieved through a reduction in MMP levels or activities. The findings presented here show the potential for silver(I) phosphine complexes to reduce the in vitro metastatic abilities of breast cancer cells, warranting further investigations into these complexes for their use as anti-metastatic drugs.Item Elucidating the Structure-Function Relationships of Enterococcus faecium Nicotinate-Nucleotide Adenylyltransferase through X-Ray Crystallography, Computational Modelling and Binding Studies(University of the Witwatersrand, Johannesburg, 2024) Jeje, Olamide Adetomi; Pandian, Ramesh; Achilonu, Ikechukwu A.Nicotinate nucleotide adenylyltransferase (NNAT) is a vital enzyme at the heart of NAD biosynthesis, catalysing a crucial reaction that leads to the formation of pyridine dinucleotides. NAD+ is an essential coenzyme in numerous metabolic processes, DNA repair, and cellular signalling. Given its pivotal role, NNAT has emerged as a compelling drug target, particularly for its potential to disrupt the survival mechanisms of bacterial pathogens. By inhibiting NNAT, it is possible to undermine the metabolic integrity of these pathogens, making NNAT a promising focal point in the fight against bacterial infections and antibiotic resistance. However, understanding the structure-function relationship of Enterococcus faecium NNAT (EfNNAT) has remained elusive. Hence, this study aimed to address this gap bycharacterising EfNNAT and validating its potential as a druggable target. EfNNAT was overexpressed and purified using the Escherichia coli system and IMAC purification technique. Subsequently, biophysical characterisation was performed, followed by the determination of the three-dimensional structure in both apo and liganded forms using X-ray crystallography. High-throughput virtual screening, along with SP and XP docking, was conducted using a library of synthesizable flavonoids. Molecular dynamic simulation and fluorescence studies were employed to establish and validate the binding of identified inhibitors to EfNNAT. Successful expression and purification of EfNNAT yielded approximately 101 mg per 7.8 g of wet E. coli cells, with a purity exceeding 98%. High-resolution crystal structures of EfNNAT in native, adenine-bound, and NMN-bound forms were determined at 1.90 Å, 1.82 Å, and 1.84 Å, respectively. These structures provided insights into EfNNAT's substrate preference and revealed a potential allosteric site at the dimer interface of the NMN-bound structure. Virtual screening identified quercetin 3-O-beta-D-glucose- 7-O-beta-D-gentiobioside as the only potential inhibitor from the flavonoid library used. A 500 ns atomistic molecular dynamics simulation showed the compound interacted through hydrogen bonding and water bridges, albeit unstable within the receptor. ANS and mant-ATP fluorescence spectroscopy confirmed quercetin binding, while thermal shift assay revealed minimal impact of the inhibitor on the protein stability and structure. This study establishes a pipeline from expression and purification to structure solution and potential inhibitor identification for EfNNAT, validating its druggability. The mechanistic insights offer a foundation for advancing drug discovery efforts targeting EfNNAT and other bacterial NNAT enzymes.Item Investigating FOXP2 dynamics, stability, and DNA-binding capabilities(University of the Witwatersrand, Johannesburg, 2024) Perumal, Cardon; Fanucchi, SylviaAll forkhead box (FOX) transcription factors share a conserved DNA-binding domain called the forkhead domain. They regulate gene expression in different organisms and have widespread biological roles, ranging from embryogenesis to immune regulation. The FOXP subfamily, unlike other FOX transcription factors, have the capacity for dimerisation, an evolutionary fate yet to be fully understood. In particular, the FOXP2 forkhead domain (FHD) has been shown in vitro to form domain-swapped dimers. Additionally, the FOXP2 leucine zipper domain forms heterotypic associations with FOXP1, 2, and 4. These somewhat isolated structural characterisations of FOXP2 have informed the domain-specific functionality of the DNA-binding forkhead domain but the leucine zipper domain has been characterized to a lesser extent, although it is thought to be implicated in FOXP2 DNA-binding as well. Elucidating the structural and functional impact of the leucine zipper on FOXP2 DNA-binding remains challenging, as it is unclear how these motifs work together to achieve binding and whether complexation is a requirement for DNA-binding. Owing to this, the cooperative structural contributions of both the leucine zipper and forkhead domain and the effect of DNA on the structure and stability of these domains have not been considered. Consequently, the aim of this study was to gain a comprehensive understanding of the FOXP2 DNA-binding mechanism by comparing the structure, stability, and dynamics of both the leucine zipper domain and the forkhead domain in the presence and absence of DNA. Assessed here, for the first time, is the conformational dynamics of the FOXP2 leucine zipper domain and FHD flanking disordered regions using hydrogen-deuterium exchange mass spectrometry. The results confirm the binding of DNA to the FHD recognition helix as well as the change in dynamics of the interlinking loop region. Additionally, the FOXP2 folding mechanisms and stability for each domain was characterised, revealing a 2-state unfolding mechanism for the forkhead domain and a 3-state mechanism for the longer LeuZip variant. Fluorescence anisotropy studies revealed that the LeuZip variant bound DNA with a higher affinity than the forkhead domain. The findings of this study highlight the structural significance of the leucine zipper domain and unstructured regions and the functional cooperativity of the FOXP2 domains investigatedItem Computational modeling approaches to validate the druggability of the 26- and 28-kDa Schistosoma glutathione transferase enzymes using bromosulfophthalein as a benchmark ligand(University of the Witwatersrand, Johannesburg, 2024) Valli, AkeelPHARMACOPHORE MODELS are 3-D representations of the chemical and spatial features required for interaction with a drug target. These. models offer advantages in early-phase drug design by expediting screening experiments and enabling the sampling of highly specific chemical. space subsets, such as those containing quality drug-like candidates. The glutathione transferase enzyme of Schistosoma spp. (SGST) has been identified as an attractive drug target for the novel treatment of human schistosomiasis. We observed selective inhibition of SGST by bromosulfophthalein. Bromosulfophthalein was found to complex with SGST at a drug binding site in the target dimer interface, providing a suitable benchmark for the design of discriminative SGST pharmacophores. The aim of this research is to construct, deploy and evaluate pharmacophore models of the SGST drug binding site. The objectives are: to characterise the SGST drug binding site, to develop the pharmacophore models and finally to evaluate the drug-resolving ability of the models. We observed significant differences in the drug-binding character of SGST, compared to human glutathione transferase (hGST) counterparts, particularly that SGST supports binding of phenol and sulfonate moieties. Five- and four feature pharmacophores were developed for the respective 26- and 28 kDa SGST variants. Finally, the models demonstrated remarkable ability to retrieve candidates displaying drug-like qualities. In conclusion, we characterised and developed pharmacophore models of the drug binding domains from two major SGST variants. Assessment of drug-resolving power validates the capability of the models to sample drug-like chemicals. Altogether, these accomplishments enable efficient and reliable screening toward novel drug treatment for human schistosomiasisItem Moving towards landscape initiatives: an analysis of environmental frameworks, protected areas, and community perspectives in the vhembe district, limpopo province, South Africa(University of the Witwatersrand, Johannesburg, 2024) Dalziel, Alexandra; Evans, MaryConservaBon strategies have shiMed towards a landscape-centric approach, emphasising conservaBon corridors and ecological networks. However, exploraBon of these strategies within the South African context is incomplete. This research aims to address this gap by applying various methods, including a systemaBc literature review, policy analysis, spaBal analysis using GIS, and conducBng interviews and surveys, resulBng in four key contribuBons invesBgaBng the potenBal for conservaBon corridors and networks. The first is a comprehensive literature review underscoring the significance of corridors and networks while highlighBng the discourse surrounding these emerging approaches in South Africa. The findings revealed a substanBal global interest in landscape iniBaBves. However, South Africa’s contribuBon is geographically limited, with 82% of studies focusing on the KwaZulu-Natal province. Understanding the efficacy of these studies in South Africa necessitates a comprehensive examinaBon of the underlying regulatory frameworks governing protected areas and conservaBon efforts. Therefore, the second contribuBon evaluated protected areas and conservaBon legislaBon and policies to assess their alignment with landscape-focused iniBaBves. The study reveals a lack of explicit provisions supporBng landscape-based conservaBon methods. The third study draws on these findings and uses the Vhembe District in the Limpopo Province as a case study to illustrate the overlooked potenBal of landscape iniBaBves. IniBally, the protected areas in the district were evaluated. Subsequently, the analysis employed Key and CriBcal Biodiversity Areas to highlight the inadequacies within the exisBng protected area network. The findings revealed that although protected areas cover 38% of the Vhembe District, over 70% of CriBcal Biodiversity Areas and 60% of Key Biodiversity Areas lack protecBon. Over 2,000 km2 of unprotected CriBcal and Key Biodiversity Areas were idenBfied along the Soutpansberg Mountain Range and marked as ideal for a conservaBon corridor. The fourth contribuBon centres on community engagement, an essenBal consideraBon for sustainable long-term conservaBon. The results underscored strong community support for a corridor in the Vhembe District, primarily moBvated by the anBcipated socio-economic benefits. However, while landscape iniBaBves promise socio-economic improvement, such outcomes are not guaranteed. Moreover, other potenBal challenges, including development, land rights, and financial constraints, were idenBfied as possible impediments to corridors in the Vhembe District. The invesBgaBon into corridors and networks in South Africa, specifically the Vhembe District, revealed that the discourse on landscape iniBaBves is in its nascent phase. Nevertheless, when prioriBsing community engagement, these iniBaBves harbour tremendous potenBal. New legislaBon and policies that account for these factors will guide proponents of these iniBaBves and propel landscape iniBaBves into South Africa’s conservaBon toolbox.Item Parageobacillus thermoglucosidasius and the water-gas shift reaction: Investigating the influences of gas composition and H2- uptake hydrogenases(University of the Witwatersrand, Johannesburg, 2024) Mol, Michael; Maayer, DeH2 gas is an increasingly important commercial reagent in a range of industries, including as a potential fuel and energy carrier. Production of H2 gas is carried out almost exclusively reliant on fossil fuel reformation through a relatively limited set of environmentally harmful processes. Pressing environmental concerns and the shift in climate policies towards greater incorporation of cleaner H2 gas production strategies and the implementation of H2 as a more sustainable and environmentally friendly alternative energy vector have necessitated development of renewable and cleaner H2 production processes. Chapter one discusses available literature on the present applications, predominant production processes and emerging alternative production strategies of H2 gas, with particular focus on the applicability and mechanism of biological H2 gas production by the thermophile, Parageobacillus thermoglucosidasius, via the water-gas shift reaction. The application of P. thermoglucosidasius to produce H2 gas is presently in its formative laboratory-scale stage of development. Although progress has been made at a fundamental level, various aspects of P. thermoglucosidasius H2 gas metabolism, remain uncharacterised or poorly understood. In Chapter two, the potential influences of two putative H2-uptake hydrogenases, encoded on the genome of a P. thermoglucosidasius strain previously identified to conduct the hydrogenogenic biological water-gas shift reaction was explored through knock- out mutagenesis. Furthermore, in Chapter three, to establish the practical implementation of pertinent and inexpensive gas feedstocks for the water-gas shift reaction, we explored the influences of various industrial mimetic gas feedstock compositions on the H2 gas evolution-, metabolic- and growth-profiles of P. thermoglucosidasius. Considering the highly variable compositions of industrial off-gases, which may contain oxygen and the highly sensitive nature of this system to oxygen, we further explored the impacts of varied volumetric and temporal oxygen perturbations on the system. Aside from hydrogen production, P. thermoglucosidasius and closely related thermophilic taxa from the genera Parageobacillus and Saccharococcus represent appealing targets for various other biotechnological developments. In Chapter four, we performed a comprehensive comparative genomic and phylogenomic analysis to further explore the biotechnological potential of Parageobacillus and Saccharococcus spp. for a range of whole cell applications as well as a source of industrially relevant thermostable enzymesItem A Phenotype Prediction Framework for Classifying Colorectal Cancer Patients’ Response to FOLFOX Treatment: An Integrated Approach(University of the Witwatersrand, Johannesburg, 2024) Mashatola, Lebohang; Kaur, MandeepColorectal cancer (CRC), characterised by its prevalence and heterogeneity, poses a significant challenge in understanding drug resistance, especially in the context of FOLFOX treatment. This study presents an innovative methodology that integrates diverse data analysis approaches to address the challenge of predicting the phenotype of CRC patients resistant or sensitive to FOLFOX. The initial analysis involved dierential and co-expression analyses, identifying pivotal hub genes crucial to drug resistance in CRC, regulating intricate molecular networks. Subsequent enrichment analysis revealed their significant roles in biological functions, particularly influencing DNA repair and nuclear division. To capture inherent topological characteristics within genetic expression data, a novel technique utilising topological data analysis (TDA) was employed. By applying persistence homology to generate persistence images, the Vietoris-Rips complex was constructed using the signed-topological overlap matrix, comprehensively capturing numerous topological features, including high-dimensional Betti-1 and Betti-2. This provided valuable insights into the structural patterns of gene expression between the hub genes. Furthermore, the integration of whole-slide images enhanced understanding of tissue anatomy, which is crucial for predicting cancer stages. Using a MobileNet architecture, a deep learning model classified cancer stages, contributing to a holistic understanding of colorectal tumor microenvironments. For predictive modelling of drug resistance, a multilayer perceptron applied topological summaries generated by TDA. The developed framework, GeTopology, exhibited remarkable performance metrics, achieving an overall 83% accuracy in predicting the FOLFOX response, demonstrating a 3% improvement over a previously published phenotype prediction framework (NSCLC ) that utilised similar data modes. Robust accuracies were consistently observed in independent datasets, classifying both cancer patients and healthy individuals. The results indicated an approximate 10% increase in model prediction accuracy compared to NSCLC, emphasising the potential clinical impact of this integrative approach. In conclusion, this study advances the understanding of drug resistance in CRC by proposing a novel approach that integrates topology with histopathological images, oering transformative insights into predictive modelling and precision medicineItem Identification and characterisation of the interaction between FOXP2 and the ligand binding domain of oestrogen receptor α(University of the Witwatersrand, Johannesburg, 2023-10) Lakhi, Aasiya Yakub; Fanucchi, SylviaForkhead box P2 (FOXP2) regulates the expression of various genes and is associated with language and speech, neural development and outgrowth, and cancer. As transcription factors rarely function in isolation, this study aims to investigate whether FOXP2 directly associates with oestrogen receptor α (ER1), a nuclear receptor responsible for sexual differentiation and cancer progression and outcome. The association between ER1 and FOXP2 was first identified in MCF-7 cells using co-immunoprecipitation. Thereafter, the interaction was characterised biophysically by overexpressing the FOXP2’s DNA-binding forkhead domain (FHD) and N-terminal region (NT), and ER1’s ligand-binding domain (LBD) in E. coli cells. Isothermal titration calorimetry and fluorescence anisotropy were used to investigate the thermodynamic parameters and regulation of interaction between FOXP2 FHD and ER1 LBD, respectively. Electrophoretic mobility shift assay was used to investigate the effect of the interaction on FOXP2’s DNA binding ability. Following the successful overexpression and purification of all three proteins, ER1 LBD was found to interact with FOXP2 FHD but not with FOXP2 NT. The affinity of the ER1 LBD for FOXP2 FHD increases with an increase in salt concentration. ITC shows a similar trend and reveals that the interaction is enthalpically favoured at lower salt concentrations but enthalpically opposed at higher salt concentrations. Additionally, the FOXP2-ER1 LBD interaction remains unaffected by the inclusion of oestrogen, but addition of FOXP2 cognate DNA results in inhibition of the formation of the complex. This research identifies a novel interaction between ER1 LBD and FOXP2 FHD and shows that the DNA simultaneously suggesting a probable role of this interaction in regulating the transcriptional pathway of FOXP2. This study serves as a foundation for further investigation into the interaction between FOXP2 and ER1 in different cell lines and its relevance in FOXP2-mediated outcomes in cancer and neurodevelopmental disorders.Item Investigating telomere dynamics using standard and AuNP-based assays and developing an LRP-based nanoparticle drug(University of the Witwatersrand, Johannesburg, 2023) Bernert, Martin; van der Merwe, Eloise; Weiss, Stefan FTTelomere dynamics, specifically telomerase activity have been implicated in age-related diseases, such as CVD, Alzheimer’s disease, and cancer. This makes the accurate detection of telomerase activity within cell cultures and tissue samples a necessity. Conventional techniques have many drawbacks, including their very high cost. Therefore, this research aimed to develop a gold nanoparticle (AuNP)-based assay to determine telomerase activity. In the assay the extracted telomerase leads to a colour change in the solution through the addition of telomeric repeats and subsequent elongation of the synthetic telomeres attached to the AuNPs. This colour change is detectable using spectrophotometric readings and represents telomerase activity. This assay would be useful as an alternative to expensive existing telomerase activity kits as large batches of AuNPs can be synthesised inexpensively. Telomerase activity was successfully detected in both HEK-293 and WHCO-5 cells using this novel technique, although the sensitivity of the AuNP-based telomerase activity assay is currently lower than a commercially available qPCR-based telomerases activity kit. In addition, telomerase activity is directly affected by the LRP protein, a highly conserved non-integrin transmembrane receptor, which has been shown to have therapeutic effects in ageing, Alzheimer’s disease, Parkinson’s disease, diabetes, and cardiovascular disease models. Recently it has been found that overexpression of LRP::FLAG, by plasmid transfection, leads to a significant increase in telomerase activity in cell culture models. This may indicate that upregulation of LRP can be used to treat various age-related diseases, however, transfection is not a viable treatment strategy and therefore, a protein-based drug was created. For a protein-based drug, a suitable delivery system needed to be developed and nano-capsules, such as those synthesised using Poly(lactic-co-glycolic acid) (PLGA), are able to contain the therapeutic protein. The molecules contained within the nanoparticles also gain the benefit of having increased stability compared to unprotected molecules and the capsules have the capacity for surface modifications for targeted therapy. These polymer- based nanoparticles are also biodegradable and biocompatible, making them a safe delivery agent. Thus, this research further aimed to develop a PLGA-based LRP drug delivery system for the 37 kDa Laminin receptor protein. Both synthesis of the nanoparticles and encapsulation of the LRP protein were successfully optimised and the completed drug was tested in a cell culture model, where treatment increased cell viability and telomerase activity in HEK-293 cells. Therefore, this LRP drug delivery system has great potential to assist in the translation of our in vitro studies into an in vivo context. Due to the wide range of applications elevating LRP levels has in the treatment of different disorders, this could represent a safer alternative to plasmid transfection treatment and could potentially be used for the treatment of age-related diseases, through its ability to increase telomerase activityItem Biochemical and biophysical analysis of a hinge region protease variant in HIV-1 subtype C(University of the Witwatersrand, Johannesburg, 2023-10) Mokhantso, Tshele; Achilonu, Ikechukwu; Sayed, YasienHIV-1 protease plays a crucial role in the maturation of the virus by cleaving gag and gag-pol polyproteins. Understanding the structural and functional consequences of mutations in this enzyme is essential for developing effective anti-HIV drugs, especially in the face of emerging drug-resistant variants. This study focused on the N37T↑V+10•D25A mutant, a novel HIV-1 subtype C protease variant harbouring an insertion (↑V) and a substitution (N→T) at position 37, along with 10 naturally occurring polymorphisms. Mutations occurring distal to the active site have long been thought to contribute to drug resistance, with this in mind the study aimed to assess the impact these mutations have on the structure, stability, dynamics and drug binding of HIV-1 protease. The N37T↑V+10•D25A mutant and wild-type (WT•D25A) HIV-1 protease were overexpressed and purified from inclusion bodies formed in Escherichia coli cells using ion-exchange chromatography. Far-UV CD and SE-HPLC analysis showed that N37T↑V+10•D25A exhibited a predominantly β-sheet secondary structure (218 nm trough) and had a homodimeric size of ~23 kDa, respectively, both similar to WT•D25A. Assessment of the local tertiary structure by intrinsic tryptophan fluorescence indicated that the protease retained its tertiary structure in the presence of mutations, with partial exposure of Trp residues (Trp 6, Trp 6', Trp 42/43, and Trp 42'/43’). Overall, the insertion and substitution mutations did not significantly alter the overall structure of HIV-1 protease. However, the conformational stability of N37T↑V+10•D25A was found to be reduced compared to WT•D25A as determined by urea-induced equilibrium unfolding and thermal unfolding experiments. When denatured using urea and temperature, the mutant exhibited a two-state mechanism of unfolding without stable intermediates during the folding and unfolding process. Thermal unfolding experiment determined the melting temperature of N37T↑V+10•D25A as 58 ± 1.2 °C, which is lower than that of the wild type of 62 ± 0.9 °C. This suggests a potential decrease in dimer stability due to the mutations present in N37T↑V+10•D25A. Isothermal titration calorimetry with acetyl pepstatin as a model inhibitor was employed to examine the impact of mutations on drug binding. The enthalpy (ΔH) for WT•D25A and N37T↑V+10•D25A were 35.94 and 36.02 kJ/mol, respectively. The entropy (ΔS) for WT•D25A and N37T↑V+10•D25A was found to be 256.7 and 261.6 J.mol.K, respectively. These differences in thermodynamic parameters between the WT•D25A and N37T↑V+10•D25A proteases, may indicate altered drug-protease interactions. Induced fit molecular docking predicted the binding strengths of both WT•D25A and N37T↑V+10•D25A with nine protease inhibitors (atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir and tipranavir), revealing that specific background mutations, such as P40S present in N37T↑V+10•D25A, significantly decreased the binding energy of the HIV-1 protease to these inhibitors. Molecular dynamics simulations provided insights into the structural dynamics of N37T↑V+10•D25A, showing reduced stability and increased dynamics, particularly in the flaps and hinges of the protease. An increase in flap tip curling and involvement of the cantilever tips were observed to be related to the flap opening mechanism of HIV-1 protease. Interactions between the HIV-1 protease and inhibitors were examined and the radius of gyration and solvent accessible surface area were calculated to evaluate protein compactness and solvent accessibility of the bound inhibitors, respectively. In general, the N37T↑V+10•D25A mutant exhibited decreased compactness when bound to inhibitors, which correlated with the increased solvent exposure of PIs when bound to N37T↑V+10•D25A. This may contribute to the drug resistance mechanisms of the protease, as inhibitors would have difficulty binding the active site and exhibit weaker binding. During the N37T↑V+10•D25A and PI interactions, there was a decrease in hydrogen interactions, which form the basis for protease inhibitor drug-design and these were replaced by water-bridge interactions, which are weaker and can be easily broken. In conclusion, this study provides a comprehensive characterisation of the N37T↑V+10•D25A mutant of HIV-1 protease, shedding light on its structural alterations, conformational stability, drug binding properties, and dynamic behaviour. These findings contribute to our understanding of drug resistance mechanisms in HIV-1 protease and offer valuable insights for the design of more effective inhibitors to combat HIV-1.