Doctoral Degrees (Pharmaceutical Sciences)

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The antibiotic treatment of gram negative bacteremia-pharmacokinetics and dynamics.
Rheeders, Malie.; Miller, Raymond Martin.
Abstract available in PDF file.
Beta-lactamase mediated resistance in Escherichia Coli isolated from state hospitals in KwaZulu-Natal.
(2008) Mocktar, Chunderika.; Essack, Sabiha Yusuf.; Sturm, Adriaan Willem.
Escherichia coli, one of the most common pathogens causing urinary tract infections, has shown increased resistance to commonly used antibiotics. In this study we analyzed the β-lactamase profiles of 38 inhibitor-resistant E. coli isolates obtained from public hospitals at three different levels of healthcare in KwaZulu-Natal, selected on the basis of their resistance profiles to the three antibiotic/inhibitor combinations, viz., amoxicillin/clavulanate, ampicillin/ sulbactam and piperacillin/ tazobactam. The isolates were subjected to MIC determinations, IEF analysis, plasmid profile analysis, PCR of the different β-lactamase genes and sequencing thereof to detect the possible mechanism/s of resistance. A range of β-lactamases including two novel inhibitor-resistant TEM β-lactamases, TEM-145 and TEM-146 were detected in two isolates whilst a novel plasmid-mediated AmpC-type β-lactamase, CMY-20 was detected in three isolates. Other β-lactamases included OXA-1, TEM-55, SHV-2, CTX-M-l and TEM-1. Changes were detected in the chromosomal AmpC promoter/attenuator regions in one isolate. Diverse β-lactamase genes and plasmid profiles inferred extensive mobilization of β-lactamase genes causing the concern of limited therapeutic options in the face of increasing resistance.
The binding of Alzheimer’s amyloid beta peptides with a candidate drug molecule, 12-crown-4, and a biological membrane : insight from molecular dynamics simulations.
(2018) Agrawal, Nikhil.; Skelton, Adam Arnold.
Abstract available in PDF file.
Bio-computational evaluation of Cryptosporidium inosine monophosphate dehydrogenase as a viable target in the attenuation of cryptosporidiosis.
(2021) Omolabi, Kehinde Foluke.; Soliman, Mahmoud Elsayed Soliman.
Cryptosporidiosis caused by Cryptosporidium species is an enteric disease infecting vertebrate. The disease is associated with poor living conditions, such as contamination of recreational water facilities with infective oocysts. Transmission can be zoonotic or anthroponotic through direct contact or faeco-oral routes. Cryptosporidiosis is a significant opportunistic parasitic organism of immune-compromised patients. Globally, The World Health Organisation (WHO) reported three million infections annually, mostly in developing countries. Notably, cryptosporidiosis self-resolve in immune-competent individuals after few days of watery diarrhea. However, infection is often treated by nitazoxanide, a thiazolide class of drug. Previous studies have reported nitazoxanide's ineffectiveness in immune-compromised patients (such as pregnant women, children, and HIV/AIDS individuals). Hence, there is a need for continuous research on effective drugs against cryptosporidiosis in individuals with a challenged immune system. This study investigates the pooled prevalence of Cryptosporidium infection in the southern Africa region and also the molecular mechanism of action of potential anticryptosporidials. Meta-analysis was conducted by screening literature database (Google Scholar, PubMed, Ovid Medline, AJOL, and Web of Science) between 2000 and 2020 to estimate the recent pooled prevalence of Cryptosporidium infection in southern Africa. This thesis investigated the inhibitory dynamics of the promising anticryptosporidial drug P131 on Cryptosporidium parvum inosine monophosphate dehydrogenase (CpIMPDH) compared to the orthologous mouse protein (mIMPDH). Crucial moieties of P131 were identified and subsequently adopted to create a pharmacophore model for virtual screening in the ZINC database through the per residue energy decomposition approach. This was done to mine for compounds that could be as effective as or more effective than P131. The potential inhibitory mechanism of these compounds was probed using molecular dynamics simulation and Molecular Mechanics Generalized Poisson Boltzmann Surface Area (MM/PBSA) analyses. In addition, a dedicated library of 107,000 natural compounds available in the ZINC database was virtually screened against CpIMPDH –NAD+ binding site to determine the compounds that have the best complementarity to the binding site. Cryptosporidium infection in southern Africa shows the pooled prevalence of 16.8% (95%CI 9.7-25.3), with subgroup analyses revealing the highest pooled prevalence of 25.2% in HIV/AIDS patients. The high prevalence of Cryptosporidium spp. infections among immune-compromised patients in southern Africa showed that the pathogen is of significant importance in this region. The relatively high-affinity interactions occurring at the CpIMPDH-NAD+ site were majorly mediated by SER22, VAL24, PRO26, SER354, GLY357 and, TYR358 located on chain D of CpIMPDH. These residues are unique to the parasite IMPDH and not in their eukaryotic host, thereby explaining the selective action of P131. Three compounds ZINC46542062, ZINC58646829, and ZINC89780094, which contained the pharmacophore of P131, showed a respective, favorable docking score of -8.3kcal/mol, -8.2 kcal/mol, and -7.5kcal/mol in CpIMPDH-NAD+ site. Results revealed that one of the hits (ZINC46542062) exhibited a higher binding free energy of -39.52kcal/mol than P131, which had -34.6 kcal/mol. Conformational perturbation induced by the binding of the identified hits to CpIMPDH was similar to P131, suggesting a similarity in inhibitory mechanisms. The top three natural compounds identified with the best complementarity to the CpIMPDH–NAD+ binding site included ZINC5225833, ZINC4258873, and ZINC3841381. The latter (ZINC3841381) had the best binding free energy of -58.43kcal/mol. The high prevalence of Cryptosporidium infections among immune-compromised patients in southern Africa revealed that cryptosporidiosis is of significant importance in this region. The molecular dynamics approaches presented revealed positive prospects toward the identification of novel anticryptosporidials. Identified ZINC compounds from both inorganic and natural sources could serve as the basis for further experimental investigations, optimization for improved selectivity, thereby providing several therapeutic options of treatment.
Bioavailability studies on various dosage forms of the anorectic, diethylpropion hydrochloride.
(1984) Dangor, Cassim Mahomed.; Veltman, A. M.
The stereo-chemistry, structure activity relationships and the metabolism of the anorectic drug, diethylpropion hydrochloride, have been reviewed briefly, together with the analytical methods for the determination of this drug and its metabolites in biological fluids. In addition, the physico-chemical properties, mode of action, pharmacology and uses of the metabolites have been presented. A comprehensive review on general principles of salivary excretion of drugs and their therapeutic drug monitoring in saliva with relevant published data on saliva/plasma drug concentration relationships has been outlined. Sensitive and specific assay procedures, based on gas-liquid chromatography for the identification, separation and determination of diethylpropion and its two major metabolites i.e. ethylaminopropiophenone (11) and diethylnorpseudoephedrine (IV) in aqueous and biological fluids, have been developed. These methods were used to study the urinary excreUon as well as saliva and plasma levels of the two major metabolites and, where possible, the unchanged drug, in man. Sustained release pellets with diffusion rate-controlled membranes were employed to control the rate of input into the body by oral or rectal route of administration. Urinary excretion data and plasma levels of metabolites 11 and IV in volunteers, where the urine was controlled at an acidic pH, were used for the evaluation of the bioavailabilities of different dosage forms of diethylpropion hydrochloride. The concentrations of metabolites 11 and IV were also measured in saliva and in plasma after administration of the drug in different doses and dosage forms: relationships between saliva and plasma concentrations (S/P) and between urinary excretion rates and plasma concentrations (U/P) were developed for each of the two metabolites during plateau levels after oral administration of the sustained release pellets (Lot R 7773). The potential use of salivary excretion of the metabolites as an index to monitor their plasma levels and bioavailabilities, was examined. The distinct advantage of using a subdivided controlled release system (i. .e. sustained release pellets) to a single unit sustained release tablet (erosion-core type) in relation to influence of the physical presence of food on the rate and extent of absorption has been demons t rated . It was found that the route of administration (oral or rectal) did not significantly affect the bioavailability of the sustained release pellets. The study also involved the investigation of the release of the drug from the pellets. Because the release control step was diffusion, no significant influences on dissolution rates were observed with the use of different dissolution test models and agitation intensities. The influence of the concentration and composition (presence of cations viz. Na+ and K+ i~r anions viz . phosphate and borate) of the dissolution medium on the release of the drug from sustained release pellets, was also studied. Any potential changes in the dissolution pattern on storage of the pellets under different conditions (4°C, room temperature and 37°C) ovrr, a period of at least one year, were investigated. The in vitro and in vivo correlations of two lots of sustained release pellets, each exhibiting different dissolution profiles, and administered rectally and orally, were developed: the in vitro data on the free drug were related to the sum of the urinary excretion data of metabolites II and IV. An attempt to use an empirical approach to predict urinary excretion rate profiles of metabolite II after oral administration of the sustained release pellets, was promising; the calculated profiles were reasonably comparable with those of in vivo studies. However, the complete validity of such equations needs further investigations.
Combinatorial in-silico modeling and bioinformatics analysis of immune proteins and small-molecular weight inhibitors: a potential for cancer chemotherapy.
(2020) Soremekun, Opeyemi Seun.; Soliman, Mahmoud Elsayed Soliman.
The immune system carries out pivotal functions in the protection of the body from damaging substances, microbes, and cellular alteration that could affect the health of an individual. The component of the immune system comprises of proteins, cells, and diverse organs. Individuals remain in a good state of health and wholeness if the immune system is working optimally, but if it becomes incapacitated toward fighting off germs or other harmful foreign substances, a diseased state set in. The innate and adaptive systems are the two sub-categories of the immune system. They work synergistically in the defence of the body and fighting off germs that triggered an immune response. Several proteins have been discovered to play pivotal roles in immune evasion and have therefore become attractive targets. Three of these proteins form the core of this thesis. Programmed death-ligand 1 (PD-L1), is an immune checkpoint protein which upon binding with another inhibitory checkpoint protein programmed cell death protein 1 (PD-1), elicit a cascade of reaction that leads to the reduction of proliferating antigen-specific T cells. The upregulation of PD-L1 can therefore, lead to evasion of the immune system by cancer cells. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is made up of three parts (a transmembrane part, extracellular part, and a cytoplasmic part). CTLA-4 has been implicated in the downregulation of immune response and blocking CTLA-4 activity results in a surge in immune functions. It has also been found that CTLA-4 negatively controls the T-cells. Natural killer group 2, member D (NKG2D) is located on the surface of immune cells where it acts as an activating receptor and regulator of the adaptive and innate immune system upon binding to its constitutive ligands such as UL16-binding protein (ULBP6). ULBP6 is a highly polymorphic protein, hence, the interaction between NKGD2 and ULBP6 is often altered. This effect has a great impact on the function of NKGD2 as a regulator of the immune system. Various humanized antibodies have been developed to specifically target PD-L1 and CTLA4. Some have been approved while others are at different phases of clinical trial. Ipilimumab and tremelimumab are designed as CTLA-4 inhibitors. Durvalumab and atezolimab are designed as anti-PD-L1 inhibitor. However, due to the limitations that have characterized the use of humanized antibodies inhibitors such as production cost, instability, and low tumour penetration, etc, small molecule inhibitors have been considered as a better alternative to humanized antibodies inhibitors. This thesis explored the mechanism of inhibition of newly synthesised PD-L1 inhibitors (BMS- 1166 and BMS-1001). Also, per-residue based virtual screening was employed to predict potential CTLA-4 inhibitors. Computational methods such as molecular docking, molecular dynamic simulation, virtual screening, and SNPinformatics were employed. These computational techniques revealed that BMS-1166 and BMS-1001 caused a motional movement in the monomers of PD-L1 to form a dimer, thereby preventing PD-L1-PD-1 interaction. Although the PD-L1 monomers have the same residues, their affinity for the BMS compounds differ. Two compounds ZINC04515726 and ZINC08985213 were identified as possible targets of CTLA-4. These two compounds elicited favourable interaction with CTLA- 4 facilitated by some crucial residues. Furthermore, the non-synonymous Single Nucleotide Polymorphism (nsSNPs) associated with ULBP6 were identified, and the effect of these nsSNPs on the interaction between NKGD2 and ULBP6 was also investigated. The first study (Chapter 4) investigates the structural dynamics and also provides insights into the mechanism of inhibition of BMS-1166 and BMS-1001 on PD-L1. The second study (Chapter 5) determines the binding site landscape of CTLA-4 and also employs binding site similarities between unrelated proteins to repurpose an inhibitor to target CTLA-4. The third study (Chapter 6) identifies deleterious polymorphisms associated with ULBP6. The effect of these polymorphisms on NKGD2-ULBP6 binding as a consequent on immune response is also explored in this chapter. Chapter 7 gives a detailed report on how the use of bioinformatics tools and strategies have aided and advanced the field of cancer immunotherapy. This study provides a thorough insight into the in-silico design, development and mechanism of action of small molecule inhibitors of PD-L1 and CTLA-4. Furthermore, this study gives insight into the polymorphic nature of ULBP6. Thence, the work presented in this study would serve a s a platform towards the design of small molecule inhibitors of CTLA-4 and PD-L1 with high therapeutic and less toxicity.
Comparative study of covalent & non-covalent drug inhibitory mechanism investigation: targeting HSP72 protein in cancer therapy using molecular modelling techniques.
(2021) Aljoundi, Aimen Khalefa Misbah.; Soliman, Mahmoud Elsayed Soliman.
Cancer is the most complicated and diverse disease that has been menacing human beings worldwide. Up to date, important advancement has been done to improve the existing therapeutic interventions in the treatment and management of cancer. However, the side effect of these drugs that are mostly associated with the “off-target” effects is a perpetual failure in cancer drug development. Therefore, efficient regimen with minimal toxicities and high drug target selectivity should be achieved. Covalent inhibition is an emerging field in drug discovery and a very distinct category of therapeutics that reduces adverse side effects and possible interactions that lead to drug resistance due to its attainable reactivity and high selectivity. The Heat shock proteins (HSPs) play a crucial role in the clearance of damaged proteins by encouraging proteotoxicity and proteins acclamation. This process occurs by avoiding unsuitable stress-induced protein aggregation, ensure suitable refolding of denatured proteins, and promoting their degradation; thus, the involvement of this enzyme in many human diseases, including cancer. In this study, we delve into the structural features of one of the most crucial enzymatic targets of the stress proteins, the Heat shock proteins72. In drug development, the integration of computational techniques including molecular dynamic simulations, docking and molecular modelling has allowed drug developers to screen and syntheses millions of compounds and thus screen out possible lead drugs. Computer-Aided Drug Design has been validated as a cost-effective strategy to fast trace the drug discovery process due to these in silico methods. One of the characteristics of the HSP72 is its ability to be targeted either covalently or non-covalently through small drug molecules. Therefore, the above-mentioned methods, amongst several other computational tools were employed out in this study to provide insights into conformational changes that explain potential covalent and non-covalent inhibitory mechanisms, binding sites assessment features leading to promising small molecule inhibitor candidates. These combinatorial computational studies offer an inclusive in silico perspective to fill the gap in drug design studies about targeting protein degradation, thus providing insights toward the structural characteristics of the pivotal target and describing promising drug developments.
Complexation strategies for the design of novel nano-drug delivery systems to treat bacterial infections caused by superbugs.
(2019) Hassan, Daniel.; Govender, Thirumala.
Abstract available in the PDF.
A computational perspective of influenza a virus targets : neuraminidase and endonuclease.
(2016) Singh, Ashona.; Soliman, Mahmoud Elsayed Soliman.
Through the ages the viruses have plagued mankind claiming the lives of millions, pre-dating any advancements in the medicinal sciences. One such pathogenic virus is influenza A, which has been implicated in the 1918-Spanish flu, the 2006-avian flu outbreak and the 2009-swine flu pandemic. It is a highly sophisticated species, alluding efforts to thwart the spread of disease and infection. One of the main reasons influenza has survived this long is simple evolution. Natural mutation within the genome of virions expressed in proteins, enzymes or molecular structure render us unable to predict or take preventative measures against possible infection. Thus, research efforts toward the competitive inhibition of biological pathways that lead to the spread of disease, have become attractive targets. The influenza A virus has a number of chemotherapeutic targets, such as: 1) The surface antigens, hemagglutinin and neuraminidase, 2) RNA-dependent RNA polymerase, and 3) The M2 proton channel. Influenza RNA polymerase is composed of three large segments encoding polymerase acidic protein (PA), polymerase basic protein 1 (PB1) and polymerase basic protein 2 (PB2). The PA protein is an N-terminal domain subunit which contains the endonuclease activity. The influenza virus is incapable of synthesizing a 5’-mRNA cap, so it has adapted a cap-snatching mechanism whereby the PB2 subunit binds to the 5’-end of host mRNA, after which 10-14 nucleotides downstream the PA-subunit (aka PAN) cleaves the strand forming a primer for viral mRNA synthesis which is catalysed by the PB1 subunit. Influenza target identification is based primarily on evidence suggesting sequence conservation of each entity and its selective expression in the virus and not the host. In this thesis two enzymatic targets were investigated, the PA protein of RNA polymerase and neuraminidase. The studies focussed on using computational tools to: 1) provide insight into the mechanism of drug-resistance, 2) describe the conformational structure of the protein in the presence of point mutations and in complex with an inhibitor, 3) determine the essential binding pharmacophoric features to aid the design of new drug therapies. An array of computational techniques were employed in the studies, such as: molecular dynamics (MD) simulation, structure-based and ligand-based in silico screening, principal component analysis, radius of gyration analysis, binding free energy calculations and solventaccessible surface area analysis. The first study (Chapter 5) determined the mechanism of drug-resistance in influenza A neuraminidase as a consequence of antigenic variations. Two distinct mutations in the enzyme sequence that were investigated are H274Y and I222K. The active site residues of neuraminidase are conserved among the subtypes of influenza A. However, it was discovered that the occurrence of resistance to the drug oseltamivir, in the H1N1 species was different to the H5N1 virus. Although both systems shared a loss in hydrophobicity of the active site, the conformational distortion of the active site pocket distinguished the enzyme of the two viral entities, from one another. The discoveries made in the first study laid the foundation for the second study (Chapter 6), which was based on the in silico design and screen of potential neuraminidase inhibitors. As a result 10 characteristic molecular scaffolds were suggested as potential inhibitors. The pharmacophore design was constructed with consideration to the new conformational structure of the active site pocket. Chapter 7 is the third study of this thesis. The active site pocket enclosing the endonuclease activity of the PA subunit was investigated. Using molecular dynamics simulations and postdynamic analyses, a description of the protein conformation was offered. Subsequently, a pharmacophore was proposed as a potential scaffold to which endonuclease inhibitors may be modelled upon. It is my belief that the impact of the results derived from the above mentioned studies would greatly contribute to the development of new and effective anti-influenza drugs.
A Computational perspective on the concerted cleavage mechanism of the natural targets of HIV-1 protease.
(2018) Lawal, Monsurat Motunrayo.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Honarparvar, Bahareh.
One infectious disease that has had both a profound health and cultural impact on the human race in recent decades is the Acquired Immune Deficiency Syndrome (AIDS) caused by the Human Immunodeficiency Virus (HIV). A major breakthrough in the treatment of HIV-1 was the use of drugs inhibiting specific enzymes necessary for the replication of the virus. Among these enzymes is HIV-1 protease (PR), which is an important degrading enzyme necessary for the proteolytic cleavage of the Gag and Gag-Pol polyproteins, required for the development of mature virion proteins. The mechanism of action of the HIV-1 PR on the proteolysis of these polyproteins has been a subject of research over the past three decades. Most investigations on this subject have been dedicated to exploring the reaction mechanism of HIV-1 PR on its targets as a stepwise general acid-base process with little attention on a concerted model. One of the shortcomings of the stepwise reaction pathway is the existence of more than two TS moieties, which have led to varying opinions on the exact rate-determining step of the reaction and the protonation pattern of the catalytic aspartate group at the HIV-1 PR active site. Also, there is no consensus on the actual recognition mechanism of the natural substrates by the HIV-1 PR. By means of concerted transition state (TS) structural models, the recognition mode and the reaction mechanism of HIV-1 PR with its natural targets were investigated in this present study. The investigation was designed to elucidate the cleavage of natural substrates by HIV-1 PR using the concerted TS model through the application of computational methods to unravel the recognition and reaction process, compute activation parameters and elucidate quantum chemical properties of the system. Quantum mechanics (QM) methods including the density functional theory (DFT) models and Hartree-Fock (HF), molecular mechanics (MM) and hybrid QM/MM were employed to provide better insight in this topic. Based on experience with concerted TS modelling, the six-membered ring TS structure was proposed. Using a small model system and QM methods (DFT and HF), the enzymatic mechanism of HIV-1 PR was studied as a general acid-base model having both catalytic aspartate group participating and water molecule attacking the natural substrate synchronously. The natural substrate scissile bond strength was also investigated via changes of electronic effects. The proposed concerted six-membered ring TS mechanism of the natural substrate within the entire enzyme was studied using hybrid QM/MM; “Our own N-layered Integrated molecular Orbital and molecular Mechanics” (ONIOM) method. This investigation led us to a new perspective in which an acyclic concerted pathway provided a better approach to the subject than the proposed six-membered model. The natural substrate recognition pattern was therefore investigated using the concerted acyclic TS modelling to examine if HIV-1 (South Africa subtype C, C-SA and subtype B) PRs recognize their substrates in the same manner using ONIOM approach. A major outcome in the present investigation is the computational modelling of a new, potentially active, substrate-based inhibitor through the six-membered concerted cyclic TS modelling and a small system. By modelling the entire enzyme—substrate system using a hybrid QM/MM (ONIOM) method, three different pathways were obtained. (1) A concerted acyclic TS structure, (2) a concerted six-membered cyclic TS model and (3) another sixmembered ring TS model involving two water molecules. The activation free energies obtained for the first and the last pathways were in agreement with in vitro HIV-1 PR hydrolysis data. The mechanism that provides marginally the lowest activation barrier involves an acyclic TS model with one water molecule at the HIV-1 PR active site. The outcome of the study provides a plausible theoretical benchmark for the concerted enzymatic mechanism of HIV-1 PRs which could be applied to related homodimeric protease and perhaps other enzymatic processes. Applying the one-step concerted acyclic catalytic mechanism for two HIV-1 PR subtypes, the recognition phenomena of both enzyme and substrate were studied. It was observed that the studied HIV-1 PR subtypes (B and C-SA) recognize and cleave at both scissile and non-scissile regions of the natural substrate sequences and maintaining preferential specificity for the scissile bonds with characteristic lower activation free energies. Future studies on the reaction mechanism of HIV-1 PR and natural substrates should involve the application of advanced computational techniques to provide plausible answers to some unresolved perspectives. Theoretical investigations on the enzymatic mechanism of HIV-1 PR— natural substrate in years to come, would likely involve the application of sophisticated computational techniques aimed at exploring more than the energetics of the system. The possibility of integrated computational algorithms which do not involve partitioning/restraining/constraining/cropped model systems of the enzyme—substrate mechanism would likely surface in future to accurately elucidate the HIV-1 PR catalytic process on natural substrates/ligands.
Computer-aided approaches in drug design: the exigent way forward: dynamic perspectives into the mechanistic activities of small molecule inhibitors toward antiviral, antitubercular and anticancer therapeutic interventions.
(2021) Agoni, Clement.; Soliman, Mahmoud Elsayed Soliman.
The crucial role of CADD in the drug design process is now indisputable and has proven over the years that it can accelerate the discovery potential drug candidates while reducing the associated cost. Using knowledge and information about biological target or knowledge about a ligand with proven bioactivity, CADD, and its techniques can influence various drug discovery pipeline stages. The ability CADD approaches to elucidate drug-target interactions at the atomistic level allows for investigations of the mechanism of drugs' actions, revealing atomistic insights that influence drug design and improvement. CADD approaches also seek to augment traditional in vitro and in vivo experimental techniques and not replace them since CADD approaches can also allow modeling complex biological processes that hitherto seemed impossible to explore using experimental methods. According to the World Health Organization (WHO), featuring prominently in the top ten causes of death are cancer, lower respiratory tract infection, tuberculosis (TB), and viral infections such as HIV/AIDS. Collectively, these diseases are of global health concerns, considering a large number of associated deaths yearly. Over the years, several therapeutic interventions have been employed to treat, manage, or cure these diseases, including chemotherapy, surgery, and radiotherapy. Of these options, small molecule inhibitors have constituted an integral component in chemotherapy, thereby undoubtedly playing an essential role in patient management. Although significant success has been achieved using existing therapeutic approaches, the emergence of drug resistance and the challenges of associated adverse side effects has prompted the need for the drug design processes against these diseases to remain innovative, including combining existing drugs and establishing improved therapeutic options that could overcome resistance while maintaining minimal side effects to patients. Therefore, an exploration of drug target interactions towards unraveling mechanisms of actions as performed in the reports in this thesis are relevant since the molecular mechanism provided could form the basis for the design and identification of new therapeutic agents, improvement of the therapeutic activity of existing drugs, and also aid in the development of novel therapeutic strategies against these diseases of global health concern. Therefore the studies in this thesis employed CADD approaches to investigates molecular mechanisms of actions of novel therapeutic strategies directed towards some crucial therapeutics implicated in viral infections, tuberculosis, and cancer. Therapeutic targets studied included; SARS-CoV-2 RNA dependent RNA polymerase (SARS-CoV-2 RdRp), Human Rhinovirus B14 (HRV-B14) and human N-myristoyltransferases in viral infections, Dihydrofolate reductase (DHFR) and Flavin-dependent thymidylate synthase (FDTS) in TB, human variants of TCRCD1d, and Protein Tyrosine Phosphatase Receptor Zeta (PTPRZ) in cancer. The studies in this thesis is divided into three domains and begins with a thorough review of the concept of druggability and drug-likeness since the crux of the subsequent reports revolved around therapeutic targets and their inhibitions by small molecule inhibitors. This review highlights the principles of druggability and drug-likeness while detailing the recent advancements in drug discovery. The review concludes by presenting the different computational, highlighting their reliability for predictive analysis. In the first domain of the research, we sought to unravel the inhibitory mechanism of some small molecule inhibitors against some therapeutic targets in viral infections by explicitly focusing on the therapeutic targets; SARS-CoV-2 RdRp, HRV-B14, and N-myristoyltransferase. Therapeutic targeting of SARS-CoV-2 RdRp has been extensively explored as a viable approach in the treatment of COVID-19. By examining the binding mechanism of Remdesivir, which hitherto was unclear, this study sought to unravel the structural and conformational implications on SARS-CoV-2 RdRp and subsequently identify crucial pharmacophoric moieties of Remdesivir required for its inhibitory potency. Computational analysis showed that the modulatory activity of Remdesivir is characterized by an extensive array of high-affinity and consistent molecular interactions with specific active site residues that anchor Remdemsivir within the binding pocket for efficient binding. Results also showed that Remdesivir binding induces minimal individual amino acid perturbations, subtly interferes with deviations of C-α atoms, and restricts the systematic transition of SARS-CoV-2 RdRp from the “buried” hydrophobic region to the “surface exposed” hydrophilic region. Based on observed high-affinity interactions with SARS-CoV-2 RdRp, a pharmacophore model was generated, which showcased the crucial functional moieties of Remdesivir. The pharmacophore was subsequently employed for virtual screening to identify potential inhibitors of SARS-CoV-2 RdRp. The structural insights and the optimized pharmacophoric model provided would augment the design of improved analogs of Remdesivir that could expand treatment options for COVID-19. The next study sought to explore the therapeutic targeting of human rhinoviruses (HRV) amidst challenges associated with the existence of a wide variety of HRV serotypes. By employing advanced computational techniques, the molecular mechanism of inhibition of a novel benzothiophene derivative that reportedly binds HRV-B14 was investigated. An analysis of the residue-residue interaction profile revealed of HRV upon the benzothiophene derivative binding revealed a distortion of the hitherto compacted and extensively networked HRV structure. This was evidenced by the fewer inter-residue hydrogen bonds, reduced van der Waals interactions, and increased residue flexibility. However, a decrease in the north-south wall's flexibility around the canyon region also suggested that the benzothiophene derivative's binding impedes the “breathing motion” of HRV-B14; hence its inhibition. The next study in the first domain of the research investigated the structural and molecular mechanisms of action associated with the dual inhibitory activity of IMP-1088. This novel compound reportedly inhibits human N-myristoyltransferase subtypes 1 and 2 towards common cold therapy. This is because it has emerged that the pharmacological inhibition of Nmyristoyltransferase is an efficient non-cytotoxic strategy to completely thwart the replication process of rhinovirus toward common cold treatment. Using augmentative computational and nanosecond-based analyses, findings of the study revealed that the steady and consistent interactions of IMP-1088 with specific residues; Tyr296, Phe190, Tyr420, Leu453, Gln496, Val181, Leu474, Glu182, and Asn246, shared within the binding pockets of both HNMT subtypes, in addition to peculiar structural changes account for its dual inhibitory potency. Findings thus unveiled atomistic and structural perspectives that could form the basis for designing novel dualacting inhibitors of N-myristoyltransferase towards common cold therapy. In the second domain of the research, the mechanism of action of some small molecule inhibitors against DHFR, FDTS, and Mtb ATP synthase in treating tuberculosis is extensively investigated and reportedly subsequently. To begin with, the dual therapeutic targeting of crucial enzymes in the folate biosynthetic pathway was explored towards developing novel treatment methods for TB. Therefore, the study investigated the molecular mechanisms and structural dynamics associated with dual inhibitory activity of PAS-M against both DHFR and FDTS, which hitherto was unclear. MD simulations revealed that PAS-M binding towards DHFR and FDTS is characterized by a recurrence of strong conventional hydrogen bond interactions between a peculiar site residue the 2-aminov decahydropteridin-4-ol group of PAS-M. Structural dynamics of the bound complexes of both enzymes revealed that, upon binding, PAS-M is anchored at the entrance of hydrophobic pockets by a strong hydrogen bond interaction while the rest of the structure gains access to deeper hydrophobic residues to engage in favorable interactions. Further analysis of atomistic changes of both enzymes showed increased C-α atom deviations and an increase C-α atoms radius of gyration consistent with structural disorientations. These conformational changes possibly interfered with the enzymes' biological functions and hence their inhibition as experimentally reported. Additionally, in this domain, the therapeutic targeting of the ATP machinery of Mtb by Bedaquiline (BDQ) was explored towards unravelling the structures and atomistic perspectives that account for the ability of BDQ to selectively inhibits mycobacterial F1Fo-ATP synthase via its rotor c-ring. BDQ is shown to form strong interaction with Glu65B and Asp32B and, consequently, block these residues' role in proton binding and ion. BDQ binding was also revealed to impede the rotatory motion of the rotor c-ring by inducing a compact conformation on the ring with its bulky structure. Complementary binding of two molecules of BDQ to the rotor c-ring, proving that increasing the number of BDQ molecule enhances inhibitory potency. The last study in this research domain investigated the impact of triple mutations (L59V, E61D, and I66M) on the binding of BDQ to Mtb F1F0 ATP-synthase. The study showed that the mutations significantly impacted the binding affinity of BDQ, evidenced by a decrease in the estimated binding free energy (ΔG). Likewise, the structural integrity and conformational architecture of F1F0 ATP-synthase was distorted due to the mutation, which could have interfered with the binding of BDQ. The third domain of the research in this thesis investigated some small molecule inhibitors' inhibitory mechanism against some therapeutic targets in cancer, specifically PTPRZ and hTCRvi CD1d. Studies in the third domain of the research in the thesis began with the investigation of the investigation of the inhibitory mechanism of NAZ2329, an allosteric inhibitor of PTPRZ, by specifical investigating its binding effect on the atomic flexibility of the WPD-loop. Having been established as crucial determinant of the catalytic activity of PTPRZ an implicated protein in glioblastoma cells, its successfully therapeutic modulation could present a viable treatment option in glioblastoma. Structural insights from an MD simulation revealed that NAZ2329 binding induces an open conformation of the WPD-loop which subsequently prevents the participation of the catalytic aspartate of PTPRZ from participating in catalysis hence inhibiting the activity of PTPRZ. A pharmacophore was also created based of high energy contributing residues which highlighted essential moieties of NAZ2329 and could be used in screening compound libraries for potential inhibitors of PTPRZ. A second study in this domain sought to explore how structural modification could improve a therapeutic agent's potency from an atomistic perspective. This study was based on an earlier report in which the incorporation of a hydrocinnamoyl ester on C6’’ and C4-OH truncation of the sphingoid base of KRN7000 generated a novel compound AH10-7 high therapeutic potency and selectivity in human TCR-CD1d and subsequently results in the activation of invariant natural killer T cells (iNKT). The hydrocinnamoyl ester moiety was shown to engage in high-affinity interactions, possibly accounting for the selectivity and higher potency of AH10-7. Molecular and structural perspectives provided could aid in the design of novel α-GalCer derivatives for cancer immunotherapeutics. Chapter 3 provides theoretical insights into the various molecular modeling tools and techniques employed to investigate the various conformational changes, structural conformations, and the associated mechanism of inhibitions of the studied inhibitors towards viral, tuberculosis, and cancer therapy. Chapter 4 provided sufficient details on druggability and drug-likeness principles and their recent advancements in the drug discovery field. The study also presents the different computational tools and their reliability of predictive analysis in the drug discovery domain. It thus provides a comprehensive guide for computational-oriented drug discovery research. Chapter 5 provides an understanding of the binding mechanism of Remdesivir, providing structural and conformational implications on SARS-CoV-2 RdRp upon its binding and identifying its crucial pharmacophoric moieties. Chapter 6 explains the mechanism of inhibition of a novel benzothiophene derivative, revealing its distortion of the native extensively networked and compact residue profile. Chapter 7 unravels molecular and structural bases behind this dual inhibitory potential of the novel inhibitor IMP-1088 toward common cold therapy using augmentative computational and cheminformatics methods. The study also highlights the pharmacological propensities of IMP- 1088. Chapter 8 unravels the molecular mechanisms and structural dynamics of the dual inhibitory activity of PAS-M towards DHFR and FDTS. Chapter 9 reports the structural dynamics and atomistic perspectives that account for the reported ability of BDQ to halt the ion shuttling ability of mycobacterial c-ring. Chapter 10 presents the structural dynamics and conformational changes that occur on Mtb F1F0 ATP-synthase binding as a result of the triple mutations using molecular dynamics simulations, free energy binding, and residue interaction network (RIN) analyses. Chapter 11 explored the impact of NAZ2329, a recently identified allosteric inhibitor of Protein Tyrosine Phosphatase Receptor Zeta (PTPRZ), on the atomic flexibility of the WPD-loop, an essential loop in the inhibition of PTPRZ. The study also presents the drug-likeness of NAZ2329 using in silico techniques and its general inhibitory mechanism. Chapter 12 provides atomistic insights into the structural dynamics and selective mechanisms of AH10-7 for human TCR-CD1d towards activating iNKT cells. The studies in this thesis collectively present a thorough and comprehensive in silico perspective that characterizes the pharmacological inhibition of some known therapeutic targets in viral infections, tuberculosis, and cancer. The augmentative integration of computational methods to provide structural insights could help design highly selective inhibitors of these therapeutic targets. Therefore, the findings presented are fundamental to the design and development of next generation lead compounds with improved therapeutic activities and minimal toxicities.
Design and development of novel nanocomposite based electrochemical sensors for quantification of antimalarial drugs and early detection of malaria.
(2021) Nate, Zondi.; Karpoormath, Rajshekhar.
Malaria is still a major disease in sub-Saharan Africa and South-East Asia. This is despite different interventions by the World Health Organization (WHO) such as the use of insecticide-treated mosquito net, antimalarial drugs, indoor residual spraying, and rapid diagnostic tools. In 2018, the mortality rate due to malaria was estimated to be 405 000, with children under the age of 5 accounting for 67% of all malaria deaths. Malaria can be prevented and treated using different strategies as recommended by WHO. However, the lack of rapid diagnostic tools with good selectivity and sensitivity is still a challenge. Another problem is the high prevalence of counterfeit antimalarial drugs. These drugs are dominant in most African countries since the cost of medicine is high and some countries lack good quality control and verification processes. Therefore there is a need to develop rapid, low-cost, and portable analytical methods for the quantification of malaria and antimalarial drugs. This review focuses on the role of malaria biomarkers in diagnosis; Plasmodium falciparum Lactate Dehydrogenase (PfLDH), Plasmodium aldolase, Plasmodium falciparum Histidine-Rich Protein 2 (PfHRP2) and Plasmodium falciparum Glutamate dehydrogenase (PfGDH) and Hemozoin. Recent developments in nanomaterial-based electrochemical and colorimetric biosensors for malaria diagnosis are discussed. Also, electrochemical sensors for qualitative and quantitative analysis of different antimalarial compounds (Quinoline-related compounds, artemisinin derivatives, antifolates, and antibiotics), which have been approved by the World Health Organization are covered. Finally, the closing remarks and future perspectives of electrochemical sensors and biosensors conclude the review.
Design and synthesis of novel pH-responsive fatty acid-based lipids for the development of nano-delivery systems to enhance Vancomycin activity against Methicillin-resistant Staphylococcus aureus (MRSA).
(2020) Makhathini, Sifiso Sakhiseni.; Govender, Thirumala.
The ability of antimicrobials to prevent and treat infections caused by a range of microorganisms, including bacteria, is threatened by the emergence of drug-resistant microorganisms that is associated with high mortality rates globally. Novel nano-drug delivery systems, including lipidbased drug delivery systems, represent an alternative therapeutic approach to combat antimicrobial resistance resulting from conventional dosage forms. Since bacteria are associated with an acidic environment and the bacterial envelope is made up of lipid bilayer, the application of pHresponsive lipid-based nanomaterials for targeted antibiotic delivery is recognized as an active area of research. The aim of this study was to design and synthesize fatty acid-based pH-responsive lipids ( FAL, OLA-SPDA and DMGSAD-lipid) and explore their potential for the preparation of pH-responsive nano-based vancomycin (VCM) delivery systems to treat infectious diseases caused by methicillin-resistant Staphylococcus aureus (MRSA) infections. All the lipids were synthesized, and its structures were confirmed by FTIR, 1H NMR, 13C NMR and HR-MS. The nontoxic nature of the synthesized lipids was demonstrated by cell viability results above 75% on all tested mammalian cell lines using the MTT assay. After the synthesis and characterization, the novel fatty acid-based lipids were employed to formulate three pH-responsive lipid-based nanodrug delivery systems (liposomes, micelles and lipid polymer hybrid nanoparticles) for efficient and targeted delivery of VCM for the treatment S. aureus and MRSA infections. These systems were characterised for their physicochemical properties (Zetasizer), in vitro drug release (dialysis bag), morphology (HR-TEM), in vitro cell viability studies (flow cytometry), in vitro cytotoxicity (MTT assay), in vitro antibacterial activity (broth dilution method) and in vivo antibacterial activity (mice skin infection model). The four formulated pH-responsive liposomes had a mean size ranging from 86.28 ± 11.76 to 282 ± 31.58 nm, with their respective PDI’s ranging from 0.151 ± 0.016 to 0.204 ± 0.014 at pH 7.4 and 6.0 respectively. The ZP values were negative at physiological pH (7.4) and shifted towards positivity with a decrease in pH (6.0). The encapsulation efficiency (%EE) and loading capacity were in the range of 29.86 ± 4.5% and 44.27 ± 9.2%, The drug release profiles of all formulations at both pH 7.4 and 6.0 were sustained throughout the studied period of 72 h. Enhanced in vitro antibacterial activity at pH 6.0 was observed for the DOAPA-VAN-Liposome and DLAPA-VANLiposome formulations. Flow cytometry studies indicated a high killing rate of MRSA cells using DOAPA-VAN-Lipo (71.98%) and DLAPA-VAN-Lipo (73.32%) using the MIC of 1.59 µg/ml. In vivo studies showed reduced MRSA recovery from mice treated with liposome formulations (DOAPA-VAN-Lipo and DLAPA-VAN-Lipo) by 4- and 2-folds compared to bare VCM-treated mice respectively. The pH-responsive oleic acid-based dendritic lipid amphiphile self-assembled into stable micelles with particle size, PDI, ZP and %EE of 84.16 ± 0.184 nm, 0.199 ± 0.011 and -42.6 ± 1.98 mV and 78.80 ± 3.26%, respectively. The micelles demonstrated pH-responsiveness with an increase in particle size to 141.1 ± 0.070 nm at pH 6.0. The drug release profiles of formulations at both pH 7.4 and 6.0 were sustained throughout the studied period of 72 h. The in vitro antibacterial efficacy of VCM-OLA-SPDA-micelle against MRSA was 8-fold better when compared to bare VCM, and the formulation was 4-fold better at pH 6.0 when compared to the formulation’s MIC at pH 7.4. The MRSA viability assay showed that the micelles had a high percentage killing of 93.39% when compared to bare VCM (58.21%) at the same MIC (0.98 µg/ml). The in vivo mice skin infection model also demonstrated an enhanced antibacterial effect, showing 8-fold reduction in MRSA burden on skin treated with VCM-OLA-SPDA-micelles when compared with the skin sample treated with bare VCM. The optimized pH responsive lipid polymer hybrid nanoparticles (LPHNPs) formulations, RH40_VCM_LPHNPs had a particle size, PDI and ZP of 64.05 ± 0.64 nm, 0.277 ± 0.057 and 0.55 ± 0.14Vm, respectively, whereas SH15_VCM_LPHNPs displayed a size of 73.41 ± 0.468 nm, PDI of 0.487 ± 0.001 and ZP of -1.55 ± 0.184 Vm at pH 7.4. There was a significant change in particle size and ZP to 113.6 ± 0.20 nm and 9.44 ± 0.33 Vm for RH40_VCM_LPHNPs, respectively, whereas for SH15_VCM_LPHNPs, there was no change in is size but a significant change in surface charge switch to 9.83 ± 0.52 Vm at pH 6.0. The drug release profiles of formulations at both pH 7.4 and 6.0 were sustained throughout the studied period of 72 h. The VCM release profile, together with release kinetic study on LPHNPs, demonstrated the influence of pH on the high rate of VCM release at pH 6.0 as compared to pH 7.4. The LPHNPs a had better antibacterial activity against S. aureus and MRSA at both pH conditions when compared to bare VCM. Furthermore, the MIC of LPHNPs against MRSA was better by 8-fold at pH 6.0 than at 7.4. In summary, synthesized novel lipid materials showed superior biosafety profiles and potential in the development of lipid-based pH-responsive nanoantibiotic delivery systems against bacterial infections and other disease types characterized by low pH. The data from this study has resulted in three first-authored research publications, one co-authored research publication and one coauthored review article.
Design of advanced materials and nano delivery approaches for enhancing activity against Methicillin resistant Staphylococcus aureus.
(2018) Omolo, Calvin Andeve.; Govender, Thirumala.; Mocktar, Chunderika.
Infectious diseases, including bacterial infections, continue to be a significant cause of morbidity and mortality globally, antimicrobial resistance has further made them fatal. Limitations of conventional dosage forms have been found to be one of the contributing factors to antimicrobial resistance. Novel nano delivery systems are showing potential to combat antimicrobial resistance. The search for novel materials for efficient delivery of antibiotics is an active research area. The aim of the study was to design and synthesize advanced materials and explore nano-based strategies for preparations of novel drug delivery systems to treat SA and MRSA infections. In this study two novel materials; a linear polymer dendrimer hybrid star polymer (3-mPEA) comprising of a generation one poly (ester-amine) dendrimer (G1-PEA) and copolymer of methoxy poly (ethylene glycol)-b-poly(ε-caprolactone) (mPEG-b-PCL) and oleic acid based quaternary lipid (QL) were synthesized and characterized and Poloxamer 188 (P188) material available in the market were employed to formulate three nano drug delivery systems for efficient and targeted delivery of antibiotics. The synthesized materials and the drug delivery system were found to be biosafe after exhibiting cell viability above 75% in all the cell lines tested on using MTT assay. The formulated nano based systems were evaluated for sizes, polydispersity indices (PDI), zeta potential (ZP), surface morphology, drug release, in vitro and in vivo antibacterial activity. Nanovesicles were formulated from 3-mPEA and they had sizes, PDI, ZP and entrapment efficiency of 52.48 ± 2.6 nm, 0.103 ± 0.047, -7.3 ± 1.3 mV and 76.49 ± 2.4%. respectively. QL lipid was employed to formulate vancomycin (VCM) loaded liposomes with Oleic acid based ‘On’ and ‘Off’” pH responsive switches for infection site and intracellular bacteria targeting. They were found to have the size of 98.88 ± 01.92 at pH 7.4. and exhibited surface charge switching from negative at pH 7.4 to positive charge accompanied by faster drug release at pH 6.0. Fusidic acid nanosuspension (FA-NS) with size, PDI and ZP of 265 ± 2.25 nm, 0.158 ± 0.026 and -16.9 ± 0.794 mV respectively was formulated from P188. The drug release profile from both the nanovesicles and liposomes was found to have sustained release. In vitro antibacterial activity for the nanovesicles, FA-NS and liposomes showed 8, 6 and 4-fold better activity at pH 7.4, while the liposome being a pH responsive antibacterial system at pH 6 showed 8- and 16- fold better activity against both Methicillin susceptible (MSSA) and resistant Staphylococcus aureus (MRSA) respectively when compared with the bare drugs. An in vivo BALB/c mice, skin infection model revealed that treatment with VCM-loaded nanovesicles, liposomes and FA-Ns significantly reduced the MRSA burden compared to bare drugs and untreated groups. There was a 20, 6.33 and 76-fold reduction in the MRSA load in mice skin treated with nanovesicles, liposomes and FA-NS respectively compared to those treated with bare VCM and fusidic acid. In summary, synthesized material showed to be biosafe and potential for the development of nano-based drug delivery systems of antibiotics against bacterial infections. The data from this study has resulted in one book chapter and 3 first authored and 3 co-authored research publications.
Design of advanced multifunctional biomaterial-based biomimetic and pH-responsive hybrid nanocarriers for antibiotic delivery against bacterial infections and sepsis.
(2023) Elhassan, Eman Hussain Elmubarak.; Govender, Thirumala.; Omolo, Calvin Andeve.
Despite the notable improvements in the management of bacterial infections and sepsis, the mounting threat of antibiotic resistance on a global scale is leading towards a post-antibiotic era. Nano-drug delivery systems have improved the delivery and efficacy of various antibiotics. Biomimicry and stimuli-responsiveness have recently been used to improve the targetability of these nanocarriers, and enhance their localization at infected sites, thus improving overall therapeutic outcomes and reducing toxicity. Strategies such as targeting bacterial biofilms and efflux pumps can further enhance the delivery and effectiveness of antibiotics. Developing smart biomaterials with multifunctional properties to confer biomimetic, stimuli-responsive and antivirulence properties to antibiotic nanocarriers is the focus of ongoing research. Therefore, the general aim of this study was to investigate the potential of various novel multifunctional biomaterial-based hybrid nanocarriers (HNs), including biomimetic and/or pH-responsive HNs in enhancing the targeted delivery of antibiotics and modulating the proinflammatory response against bacterial infections and sepsis. In this study, two biomaterials with multifunctional activities, hyaluronic acid-lysine conjugate (HA-Lys) and tannic acid (TA), were employed to design, formulate, and extensively characterize innovative biomimetic and pH-responsive HNs for efficient and targeted delivery of antibiotics. The novel HA-Lys was synthesized and fully characterized using proton nuclear magnetic resonance (1H NMR) spectroscopy and Fourier-transform infrared spectroscopy (FT-IR). Then it was successfully employed with tocopherol succinate (TS) and Oleylamine (OLA) to fabricate biomimetic pH-responsive vancomycin-loaded hybrid nanostructured lipid carriers (VCM-HNLCs). The prepared VCM-HNLCs were spherical and had average diameters, zeta potential, polydispersity index, drug encapsulation efficiency and loading capacity of 110.77  1.69 nm, 0.11  0.02, -2.92  0.21 mV, 76.27  1.20 % and 8.36  0.25 %, respectively. Both HA-Lys conjugate and its respective nanoformulations had excellent biosafety profiles (>70 % cell viability and ˂ 1 % hemolytic effect). Possible VCM-HNLCs competitive inhibition activity to toll-like receptors 2 and 4 (TLR2 and TLR4) was demonstrated via microscale thermophoresis (MST) analysis, which showed a 5-times and 16-times lower Kd values than their natural substrates peptidoglycan (PGN) and lipopolysaccharide (LPS), respectively. VCM-HNLCs exhibited a pH-responsive drug release profile under acidic conditions, higher bacterial killing kinetics, enhanced antibacterial, anti-biofilm, and efflux pump inhibition activities over bare VCM. Also, they showed an improved activity in neutralizing reactive oxygen species (ROS) and modulating the inflammatory response induced by LPS. On the other hand, tannic acid (TA) and Oleylamine (OLA) were successfully employed to formulate biomimetic ciprofloxacin-loaded tannic acid hybrid nanoparticles (CIP-loaded TAH-NPs) to enhance the efficacy of CIP against bacterial infections and sepsis. The prepared HNs had onion-shaped morphology, with average diameters, zeta potential, polydispersity index, drug encapsulation efficiency and loading capacity of 85.65 ± 0.89 nm, 0.126 ± 0.01, +16.3 ± 0.23 mV, 68.73 ± 0.54 % and 6.86 ± 0.09 %, respectively. The hemolysis and MTT assays confirmed the biosafety and non-hemolytic activity of CIP-loaded TAH-NPs formulations (>70 % cell viability and ˂ 1 % hemolytic effect). The results of MST investigations and in-silico simulations demonstrated that TA and its nanoformulation (CIP-loaded TAH-NPs) competitively inhibited TLR4 compared to its natural substrate LPS. CIP-loaded TAH-NPs showed a diffusion-based sustained release profile at physiological pH 7.4. Also, in comparison to bare CIP, the hybrid nanovesicles demonstrated improved antibacterial, anti-biofilm and efflux pump inhibition properties, as well as faster bacterial killing kinetics. Moreover, they showed a significant neutralization of ROS and the ability to control the inflammatory responses brought on by LPS. In summary, VCM-HNLCs and CIP-loaded TAH-NPs were successfully formulated and showed significant improvement in antibiotics efficacy and overall therapeutic outcomes. This study confirmed the potential of biomimetic stimuli-responsive antibiotic hybrid nanocarriers for enhancing antibiotic efficacy against bacterial sepsis and addressing the antimicrobial resistance crisis. The data from this study has resulted in one first-authored review article, two first-authored research publications and one co-authored review article.
Design, synthesis and pharmacological evaluation of novel fused pyrimidine analogues as anticancer agents.
(2018) Cherukupalli, Srinivasulu.; Karpoormath, Rajshekhar.
Cancer is a multifaceted disease considered as the most serious health burden all over the world. Due to existing of limited anticancer drugs and detrimental side effects, the anticancer research has been challenging. An investigation on identifying novel potential drugs is highly required to treat this serious abnormal cell growth. Advanced potential anticancer drug entrants are crucially required to combat the drawbacks linked with current drugs or line of therapies. Extensive investigations are being carried out on synthetic manipulations of heterocyclic aromatic compounds (purines) for developing efficient and potent anticancer drugs. Besides, these manipulations also offer effective leads for further optimization. Therefore, this project is an effort in detecting a novel and potent anticancer leads based on bioisostere of purines called pyrazolopyrimidines. In this research project we have performed an comprehensive literature survey of structural isomers of pyrazolopyrimidines (pyrazolo[1,5-a]pyrimidine and pyrazolo[4,3-d]pyrimidine) for their synthetic approaches and biological activities with special emphasis on structure-activity relationship (SAR) studies. These SAR studies prompted us to implement the observed studies on one of the structural isomer of pyrazolopyrimidine called pyrazolo[3,4-d]pyrimidine. And further, we have synthesized some novel series of pyrazolo[3,4-d]pyrimidine derivatives with various substituents at C-4 and C-6 positions of the scaffold. A total 71 compounds comprising of phenethyl and pentane hybrids (7-43, Chapter 4), benzoyl hybrids (5a-5h, 6a-6d and 7a-7c, Chapter 5) and lastly phenylcarbamoyl acetamide hybrids (9a-9s, Chapter 6) have been synthesized by molecular hybridization approach as outlined in schemes of respective chapters. The completion of reaction and the purity of novel synthesized compounds were confirmed by chromatographic analysis. All the newly synthesized compounds displayed acceptable analysis for their anticipated structures, which were established based on physicochemical and spectral data (IR, 1 H NMR, 13C NMR and HRMS). All synthesized compounds were primarily evaluated for their in vitro anticancer activities at Laboratory of Growth Regulators, Centre of the Region Hana for Biotechnological and Agricultural Research, Palacky University & Institute of Experimental Botany ASCR, Slechtitelu 27, 78371 Olomouc, Czech Republic. From the systematic analysis of anticancer activity, results obtained following key observations were made. i. Structural isomers of fused pyrimidines have been looked upon for molecular changes in emerging drug like candidates. Pyrazolopyrimidine is a bioisostere of purines has acquired considerable importance due to its diverse, facile and general synthetic methodologies with great medicinal importance. Several analogs of this scaffold have emerged as a promising leads in the design of some novel pharmacologically active compounds with enhanced iii metabolic, pharmacokinetic and pharmacological profiles, representing that there is plenty scope for considering pyrazolopyrimidine as a structural framework for evolving effective leads. ii. Chapter 4: From the 37 novel phenethyl and alkyl pentane pyrazolo[3,4-d]pyrimidine derivatives synthesized and evaluated for CDK2/Cyclin E, Abl kinase inhibitory activity and anti-proliferative activity against K-562 (chronic myelogeneous leukemia) and MCF7 (breast adenocarcinoma) cell lines. From the tested results, compounds 11 (CDK: IC50 = 5.1 µM; Abl: ˃12.5 µM), 8 (CDK: IC50 = 7.8 µM; Abl: ˃25 µM) and 36 (CDK: IC50 = 8.8 µM; Abl: >25 µM) exhibited significant inhibitory activity. Further from this series, most of the synthesized compounds indicated prominent anti-proliferative effects with IC50 value ranging from 19.2 µM to 27.4 µM. Incorporation of monosubstituted phenyl groups at C-4 of the pyrazolo[3,4-d]pyrimidine nucleus had favored for most prominent anticancer activity. iii. Chapter 5: Among the 15 novel benzoyl hybrids synthesized and evaluated, compounds 5a and 6c displayed (CDK2: IC50 = 8.8 µM, 6.8 µM) commendable inhibitory activity and notable anti-proliferative activity ranging from 18.9 µM to 89.3 µM). Presence of heteroatom containing bicyclic moieties at C-4 of the nucleus enhanced both inhibitory and anti-proliferative activity. iv. Chapter 6: Of the 19 novel phenylcarbamoyl acetamide hybrids synthesized and tested, compounds 9a, 9c, 9g, 9m and 9p showed moderate enzymatic inhibitory activity with an IC50 value ˃12.5 µM against both CDK2 and Abl kinases while, remaining compounds of this series could not generate IC50 values due to solubility limit (IC50 = ˃25 µM to ˃100 µM).
Design, synthesis and screening of novel PCU-peptide/peptoid derived HIV protease inhibitors.
(2011) Makatini, Maya Mellisa.; Govender, Patrick.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Govender, Thavendran.
The AIDS epidemic in Africa has reached dramatic proportions. Of the 42 million people infected with HIV worldwide, 30 million are in Africa. Current available therapies have begun to transform this fatal disease into a chronic condition but there are still major obstacles that have resulted in a great demand for new and better drugs. The aim of this study was to synthesize novel and effective HIV protease inhibitors. This work describes the first account of pentacycloundecane (PCU)-peptide and peptoid based protease inhibitors. These inhibitors are proposed to bind the wild type C-South African HIV protease (C-SA) catalytic site via the norstatine or dihydroxylethelene type functional group of the PCU. The desired compounds were synthesized by the coupling of the peptides and peptoids to the PCU cage which resulted in a series of promising and structurally diverse HIV-1 protease inhibitors. The inhibitors were characterized by Nuclear Magnetic Resonance (NMR) and evaluated against the wild type C-SA enzyme for its ability to inhibit 50 % of the enzyme’s activity (IC50). Two of the compounds reported herein, inhibited the enzyme activity at concentrations less than 80 nM. NMR investigations indicated that the activity was related to the chirality of the PCU moiety and its ability to induce conformations of the coupled peptide side chain. Employing the new Efficient Adiabatic Symmetrized Rotating Overhauser Effect Spectroscopy (EASY-ROESY) technique enabled us to obtain vital information about the 3D structure of these small linear peptides and peptoids in solution. This technique is the first example describing the successful through space correlations of such small peptides. Furthermore, docking and a combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics MD simulation at the AM1 semi empirical level mirrored the observed NMR results and the experimental IC50 activity profile of the considered inhibitors. The combination of these experimental and theoretical methods provided a powerful insight into the interaction mode of these cage peptide and peptoid inhibitors with the enzyme.
Design, synthesis and spectral characterization of quinazoline and benzoxazine derivatives as novel DNA gyrase inhibitors.
(2020) Kushwaha, Narva Deshwar.; Karpoormath, Rajshekhar.
Tuberculosis (TB) has remained one of the leading causes of the death worldwide and recently surpassed HIV/AIDS as lethal disease caused by a single infectious agent. Further, development of resistance against frontline anti-tubercular drugs has worsened the existing alarming condition. Therefore, there is an urgent need to develop novel, more effective, inexpensive and accessible antitubercular agents possessing broad spectrum of potency short duration of drug regimen, less side effects, which can counter the drug resistant TB and reduce the burden on the society. Global scientific communities and pharmaceutical industries are aggressively involved in research to develop a novel broad spectrum anti-tubercular agent to address this rising threat to human kind. In continuation of our work, focused in developing new anti-TB agents, we have attempted to develop some potential quinazoline based DNA gyrase and topoisomerase IV inhibitors as potential anti-tubercular agents. In addition, we have developed novel green and efficient catalyst-free, mild one-pot tandem synthetic strategy to synthesize benzoxazine derivatives, that can be further exploited as building blocks for the synthesis of multifaceted molecular structures, especially for anti-tuberculosis agents. Chapter 1 gives a brief overview on drug discovery and medicinal chemistry, history of antibiotic expansion, development of resistance in bacteria, history of anti-TB drugs discovery, line of treatment for TB, anti-tubercular drugs and their specific targets, classification of TB drugs, DNA gyrase and topoisomerase IV as a key target for anti-tubercular agents, marketed drugs and recently reported DNA gyrase inhibitors, importance of benzoxazine and quinazoline scaffold as well as significance of fluorine containing heterocycles in medicinal chemistry. Chapter 2 describes, the development of a novel methodology which is green, efficient catalyst-free and is a mild one-pot, multicomponent synthetic strategy to construct substituted 3,4-dihydro-2H-benzo[b][1,4]oxazine. The reaction proceeds via in-situ formation of Schiffbase followed by base mediated alkylation with phenacyl bromide/substituted phenacyl bromide, finally leading to intramolecular cyclization to give a mixture of diastereomers with excellent diastereoselectivity (up to dr = 99:1), which were isolated as single diastereomer in moderate to excellent yields (41-92%). Besides, this new versatile methodology provides a wide scope for the synthesis of different functionally substituted benzoxazine scaffolds and can be further exploited as building blocks for the synthesis of multifaceted molecular structures, especially for pharmaceutical applications. In Chapter 3, 15 novel fluorinated quinazoline derivatives have been synthesized and characterized with Infrared (IR) and Nuclear Magnetic Resonance (NMR) spectroscopic methods. This chapter also describes the influence of various substituents on the core scaffold (Fluorinated quinazoline) on its molecular conformations, intermolecular interactions and on the photoluminescent properties. Hirshfeld surfaces was used to investigate the structure-directing effects of functional groups in controlling their solid-state behaviour. In Chapter 4, a total 21 novel quinazoline derivatives (10a-q and 17a-c) were synthesized in good to moderate yields. The synthesized compounds were well-characterized by spectroscopic studies (IR and NMR) and evaluated for their preliminary in vitro screening against Mycobacterium tuberculosis H37Rv strain, which was conducted at TB Discovery Research, Infectious Disease Research Institute (IDRI), USA. The only notable % zone of inhibition was observed against Mycobacterium tuberculosis strain H37Rv for compounds 10m which showed 33% inhibition after 24 h incubation which can be considered for further study which includes MIC, MBC etc. In Chapter 5, total 23 novel N/O-methylated quinazoline derivatives (10a-o, 16a-d and 16aaad) were synthesized in good to moderate yields. All synthesized compounds were wellcharacterized by spectroscopic studies (IR and NMR) and evaluated for preliminary in-vitro screening for anti-mycobacterium activity at 20 µM concentration against Mycobacterium tuberculosis H37Rv strain at TB Discovery Research, Infectious Disease Research Institute (IDRI), USA. The % zone of inhibition for compounds 16a, against Mycobacterium tuberculosis strain H37Rv was found to be 34% after 24 h incubation. Chapter 6 describes, the development of novel quinazoline based DNA gyrase inhibitors as potential antibacterial agents. Bacterial type II topoisomerase (DNA gyrase and topoisomerase IV) control the topological state of DNA during replication and are validated targets for antibacterial agents. Type II topoisomerase is essential in all bacteria. It is also present in eukaryotic cells but unlike the prokaryotic enzymes eukaryotic topoisomerase II is homodimeric, this difference in structure makes highly attractive targets in antibacterial drug discovery. Fluoroquinolones are an example of very active gyrase-based drugs, but the rise in bacterial resistance to these agents alarm the risk. We have replaced the central core quinolone with quinazoline ring and synthesized 22 derivatives and evaluated against DNA gyrase and topoisomerase IV enzyme of Escherichia coli. The most potent compound (10l) displayed balanced IC50 value of 0.49 and 13.22 μM for DNA Gyrase and topoisomerase IV of Escherichia coli respectively. This result is intresting for the further studies as it showed promising well-balanced duel inhibiton in the low micromolar range against DNA gyrase and topoisomerase IV in E. coli.
Designed, synthesis and antibacterial evolution of piperazine hybrids.
(2023) Girase, Pankaj Sanjay.; Karpoormath, Rajshekhar.
Piperazine is a kind of azacycloalkane that has two nitrogen atoms at1-4 places on a six-membered ring. It is well known that molecules with the piperazine ring, a key component of the N-heterocyclicfamily of bioactive natural products, are often prevalent in biologically active substances. There are many antitumor, antibacterial, antiinflammatory, antipsychotic, antifungal, and anti-diabetic compounds based on the piperazine scaffold, which has been recognized as an active structure in drug discovery. Piperazine hybrids with different moieties such as isoniazid, coumarin, benzothiazinones, isoquinoline, triazole,pyrrole, and oxazolidinone showed good activity against mycobacterium tuberculosis and microbial strains. In this thesis we have demonstrated the synthesis of piperazine hybrid with hydrazides, hydrazines, and coumarines and tested their activity against mycobacterium tuberculosis, gram positive and gram negative microbial strains. In Chapter 2, we have covered topics related to the analogues of piperazine that have anti-tubercular efficacy. This chapter we have published as a review article in European Journal of Medicinal Chemistry. In this review, we have made a concerted effort to trace the development of anti-mycobacterial compounds during the past 50 years (1971-2019), focusing on instances where piperazine has been utilized as a key building block. In depth discussion of the design, rationale, and structure-activity relationship (SAR) of the reported potent piperazine-based anti-TB molecules will help medicinal chemists fill in the blanks, capitalize on the reported strategies, and create more effective, selective, and less hazardous anti-mycobacterial drugs. In chapter 3, we have developed and synthesized a new class of hybrids between phenylpiperazine and hydrazides (c1-c15). During the synthesis of phenyl piperazines, the formylation of piperazine was observed, a phenomenon on which we have developed a different methodology discussed in chapter 5. All of the derivatives have been tested in vitro against H37Rv, a strain of mycobacterium. In addition, we have analysed the zone of inhibition against eight different bacterial strains, including both gram-positive (methicillin resistant staphylococcus aureus (MRSA), Streptococcus pyrogens, Bacillus subtilis, Enterococcus faecium, and Staphylococcus aureus), and gram-negative (Enterobacter hormaechei, Pseudomonas aeruginosa, and Escherichia coli) bacteria. Among the derivatives tested, only compound c8 showed action against the mycobacterium strain H37Rv (MIC value of 0.39-0.78 g/ml). No zone of inhibition was seen for any of the microbiological strains when exposed to any of the synthesized compounds. The hybrids between phenylpiperazine sulphonamide and phenyl hydrazide (E1-E6) and phenylpiperazine sulphonamide and phenyl hydrazine (F7-F19) were proposed and synthesized in chapter 4. All substances were evaluated against mycobacterium tuberculosis, five gram-positive and three gram-negative bacterial strains in vitro. Derivatives E1 and E2 with an isoniazid moiety were the most effective in inhibiting the growth of the H37Rv strain of tuberculosis, with an IC50 value of 3.125 M. Of the derivatives tested, F10 showed significant action against the gram-positive bacteria Enterococcus faecium (7.81 μg/mL), whereas the others (E2, E6, F7, F9, F14) were only moderately active (250-62.5 μg/mL). Using a the molecular hybridization strategy, we were enabled to create novel analogues of coumarin-(phenylsulfonyl)piperazine and 4-methyl coumarin-(phenylsulfonyl)piperazine in chapter 5.All synthesised compounds were evaluated for their in vitro anti-mycobacterial and antimicrobial activity against H37Rvand a variety of antimicrobial gram-positive and gram-negative strains.The Compounds 6G, 6H, 10D and 10E displayed moderate inhibition against gram positive and gram negative strains with MIC values in the range of 62.5-250 (table 1) against MRSA, Bacillus subtilis, and Enterococcus faecium, and gram negative strains Enterobacter hormaechei, Pseudomonas aeruginosa, and Escherichia coli.In addition, the Structure-Activity Relationship (SAR) analysis showed that phenyl ring substituents could enhance antibacterial activity. Chapter 6 came from the process of synthesizing phenyl piperazine in chapter 1. This chapter disclosed a method for efficient synthesis of transamidation in the presence of Iodine and NH2OH.HCl which published in Chemistry Select. This method is efficient for a broad range of primary, secondary, and tertiary amides, and it enables the formylation, acylation, and benzoylation of a number of different amines. The key benefits of the present technique are that it is easy to follow, quick, does not need a metal catalyst, uses a starting material that is inexpensive, and has a low effect on the environment when the synthesis process is carried out. All of the chapters in this thesis are written in thesis by publication style, rather than the conventional style.
Development of a framework for the standardization of management of diabetes and hypertension: the case of Tswanas and Zulus of South Africa.
(2019) Frimpong, Ebenezer Kwabena.; Nlooto, Manimbulu.
Introduction: In sub-Saharan Africa, available World Health Organization data indicate an increase in the prevalence rate of non-communicable diseases such as diabetes and hypertension. Dwindling economies, rising costs of orthodox conventional medicines (OCM) and a lack of health care facilities in indigenous communities have led to the patronage of Traditional Medicine (TM) by the general population, with the assistance of traditional health practitioners (THPs) to combat these chronic conditions. However, the nonexistence of standardized and formalized Tswana and Zulu diabetes and hypertension treatment guidelines for THPs indicates the need to compare their methods and treatments approaches in the two culturally diverse environments. Understanding the similarities and differences of their methods and treatment approaches could serve as the basis of affirming evidence-based practice in TM that will lead to the development of a framework to assist in managing these two diseases in indigenous African communities. This study aimed to develop a framework for the standardization of traditional health practices to manage diabetes and hypertension among Tswanas and Zulus THPs in the North-West and KwaZulu-Natal (KZN) Provinces, South Africa. Methods: A comparative cross-sectional descriptive study with mixed-method (embedded) approach was conducted in the uMgungundlovu and uThukela Districts of KZN, and the Bojanala and Dr. Ruth Sekgopomati Districts of the North-West Province. Phase 1A entailed a series of focus group discussions (FGDs) with 67 THPs in KZN and 40 in the North-West Provinces about their management of diabetes and hypertension in three geospatial areas (urban, traditional/tribal and farm areas), the data being analysed using thematic content analysis. In Phase 1B, a researcher-administered questionnaire was used to conduct face-to-face interviews about the management of diabetes and hypertension among 437 THPs in KZN and 426 in North West Province. The qualitative data was analysed using thematic content analysis while the quantitative data was examined and reported using descriptive statistics. In Phase 2, an analytical approach was used to develop a modified interventional tool that was adapted from the Ayurveda treatment guidelines for both diabetes and hypertension based on findings from Phases 1A and Phase 1B. OCM and Ayurveda health traditions experts’ opinion regarding the developed diabetes and hypertension guidelines were sought after their validation process that led to the development of a framework for the standardization of traditional health practices regarding the two conditions. Results: This study found that most Zulu and Tswana THPs acquired their knowledge and training from one of the following ways: by a family member, other person, and gift from birth, a calling without any form of training and through professional training. The majority of Zulu THPs acquired their knowledge as a gift from birth, while the majority of Tswana THPs acquired their knowledge and training via family members. Acquisition of knowledge as a gift from birth entails a call to practice via ancestral spirits initiation, while from family members involves training received from other experienced THPs in the family. There were similarities in the Zulu and Tswana THPs cultural understanding of both diabetes and hypertension. Most THPs regarded diabetes and hypertension as the same condition, as one (having diabetes) leads to the other (hypertension). The following symptoms: weight loss, sweating easily, shortness of breath, feeling dizzy, and difficulties in breathing, excessive hunger and eyesight problems, were the most commonly reported clinical features for both diabetes and hypertension by the THPs. Ethnopharmacological modalities and treatment approaches used by the Zulu and Tswana THPs to manage both diabetes and hypertension diseases were mainly the use of herbal mixtures, with Aloe vera as a major constituent. Besides, the following medicinal plants: Hypoxis hemerocallidea, Persea americana, Sutherlandia frutescens and Harpagophylum procumbens were used by both groups of THPs in its management. Commonalities in their responses from the findings in Phase 1A and Phase 1B compared to adapted Ayurveda treatment guidelines for both diabetes and hypertension led to the development of treatment guidelines for both conditions. The developed framework to manage diabetes and hypertension for use by Zulu and Tswana THPs was validated by experts in the field of OCM and Ayurveda health traditions. Conclusion: Some similarities were noted between the Zulu and Tswana THPs and OCM in terms of the description of clinical features of diabetes and hypertension. The use of several medicinal plants by the Zulu and Tswana THPs to manage the two diseases has been proving scientifically to be effective against them. The interventional tool will assist Tswana and Zulu THPs to manage diabetes and hypertension within traditional African communities. Recommendations, including signing of intellectual property (IP) agreement between the Department of Science and Technology (DST) legal experts and THPs, will help OCM researchers to obtain samples of herbal mixtures to conduct scientific research through in vivo, in vitro and randomized control trials (RCT) studies to make a meaningful contribution to updated versions of the developed guidelines.

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