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Doctoral Degrees (Pharmaceutical Sciences)

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    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.
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    The in vitro and in vivo efficacy of novel metallo-β- lactamase inhibitors co-administered with meropenem to target CREs.
    (2022) Reddy, Nakita.; Naicker, Tricia.
    The evolution and phenotypic expression of metallo β-lactamase genes across the world has led to the escalated transmission rates of carbapenem resistance. The effect has crippled the already impaired healthcare system, with the emergence of COVID-19 exacerbating the crisis further. Our plight for a solution to combat antimicrobial resistance has not been greater. One strategy to tackle this non-susceptibility is the development of metallo-β-lactamase inhibitors that can neutralize the metallo-β-lactamase enzyme, thereby allowing the carbapenem antibiotic to elicit its function on the microorganism. Currently, there is no FDA-approved metallo-β-lactamase inhibitor to meet the clinical challenges of drug resistance. In a desperate need to find a candidate drug, research has been initiated into the discovery and development of biologically active inhibitors. Therefore, this thesis focuses on the advances made by our research group, the Catalysis and Peptide Research Unit, in developing novel β-lactam derived inhibitors; NOTA, NO3PY, BP- 1, 6,10 and 14, that re-sensitize the microbe to the efficacy of meropenem. The in vitro and in vivo activities of the initial chelators, NOTA and NO3PY, were evaluated as potential metallo-β-lactamase inhibitors (MBLIs) against metallo-β-lactamase (MBL) resistant bacteria. Time-kill studies showed that NOTA and NO3PY restored the efficacy of meropenem against all bacterial strains tested. A murine infection model was then used to study both metal chelators’ in vivo pharmacokinetics and efficacy. NO3PY displayed poor bioavailability at the selected doses using a validated LC-MS/MS method, therefore discouraging the in vivo efficacy evaluation. NOTA showed good bioavailability; hence, the in vivo efficacy was determined in a murine thigh infection model. The co-administration of meropenem and NOTA (100 mg/ each) significantly decreased the colony-forming units of K. pneumoniae NDM over an eight-hour treatment period. The findings suggested that chelators, such as NOTA, hold strong potential for use as an MBLI in treating CRE infections; however, further preclinical development was needed to improve the pharmacokinetic properties of these agents to increase their bioavailability and tissue distribution. With this information, our group derivatized NOTA by coupling it to a β-lactam to create the BP series of novel MBLIs. The results generated by the BP compounds have proven to interact synergistically with meropenem, by restoring the MIC of meropenem to therapeutically acceptable concentrations (< 2 mg/L) that concur with the breakpoints outlined by CLSI. In addition, the bactericidal activity of the re-sensitized meropenem was evident in the time-kill study over 24 hours. Cytotoxicity assays were further conducted to study the inhibitors, with an outcome in favor of safe administration in vivo. The metallo-β-lactamase inhibitors reported herein have demonstrated good potency against NDM-1 and VIM-2 metallo-β-lactamases with a Ki of 25-97μM. Since the BP compounds are metal chelators that function as metallo-β- lactamase inhibitors, it was important to determine the binding specificity of the BP compounds to a physiologically relevant zinc-harboring enzyme, glyoxylase II. At concentrations of up to 500 μM of BP, the activity of glyoxylase II remained unhindered. This confirmed the hypothesis of BP specificity to be exclusive to NDM-1 and VIM-2 metallo-β-lactamases. These findings prompted further interest in the binding exhibited by BP and led to additional studies to address the binding interactions of BP with the metallo-β-lactamases through quenching and computational experiments. Fluorescent quenching experiments investigating the Ka of BP indicated that a higher binding affinity was noted for NDM-1 compared to VIM-2 MBLs, thus implying a stronger interaction with NDM-1. Molecular docking and dynamic simulation experiments shed light on the BPs’ mode of action, showing the interaction of the chelators’ carboxylic moiety with the Zn 2+ ions in the MBLs structure. In favor of this BP series as functional inhibitors, in vivo efficacy was explored in a murine infection model (BP1 and BP10). In Klebsiella pneumoniae NDM infected mice, BP co-administered with meropenem was efficacious in reducing the bacterial load by > 3 log10 units’ post-infection, compared to meropenem monotherapy. These findings validate our strategy for derivatizing NOTA into the series of the BPs, as the bioavailability of NOTA, when coupled to a cephalosporin, improved the overall in vivo efficacy, and allowed the drug to be quantified in plasma under the same conditions previously used. This study clearly indicated the influence of the BP compounds in reducing the bacterial burden and the success of employing combination therapy as a treatment alternative. Moreover, the outcome of this preclinical development represents a solid foundation, whereby we can build on our existing knowledge. In aligning with our research goals of alleviating the threat of antimicrobial resistance, coupling β-lactams to a cyclic zinc chelator offers a safe and efficacious solution to meet the calamity that plagues our healthcare system.
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    In silico insights into the effect of mutation on peramivir resistance against influenza H7N9 virus and the development of potential inhibitors.
    (2022) Mtambo, Sphamandla Enock.; Kumalo, Hezekiel Mathambo.
    Influenza A virus infections causes substantial population illness with consequent healthcare and economic problems. There has been an outbreak of novel influenza A (H7N9) virus strains on the Chinese mainland as of March 2013. As a result of their fast geographical spread and genomic variety, the ongoing circulation of H7N9 virus poses a pandemic threat. At present, available anti-influenza drugs are mainly directed at the viral M2 ion-channel (amantadine and rimantadine), neuraminidase (oseltamivir, zanamivir, laninamivir, and peramivir), or polymerase (baloxavir marboxil), and emerging anti-viral resistance against these inhibitors is a concern. The development of safe and effective anti-influenza drugs is essential to a balanced strategy against seasonal influenza. The use of computational approaches for designing and developing new antiinfluenza drugs has proven beneficial in response to resistance to current therapies. The use of computer-aided drug design (CADD) is crucial to the development of novel drugs. It has been shown over the years that CADD plays a vital role in the drug design process, accelerating the discovery of possible drug candidates at a lower cost. CADD approaches are able to investigate protein-ligand interactions at the atomic level, which provides insights that can be used to improve drug design. As a result, the studies presented in this thesis employed CADD approaches in order to explore molecular mechanisms of action of new therapeutic approaches designed to combat H7N9 viral infections. The aim of this study was to offer an indepth understanding of the effect of H7N9 mutation on neuraminidase inhibitor resistance and discover the fundamentals for the design and development of more potent anti-viral drugs. Clinical studies demonstrated that the peramivir resistance to extremely pathogenic influenza H7N9 viruses is caused R292K mutation. As such, we used numerous molecular dynamics methods to assess the effect of neuraminidase-R292K mutation towards peramivir resistance in influenza H7N9 viruses. We found that a R292K mutation caused peramivir orientation to be altered in the binding site of the peramivir-R292 mutant complex, consequently hampering the mutant's ability to bind peramivir. In contrast to its wildtype counterpart, R292K mutant decreased the interaction between neighboring amino acid residues, as evidenced by a high degree of flexibility in the radius of gyration. The mutation altered hydrogen bond-mediated interactions with peramivir and resulted in a greater accessibility of water molecules nearby the K292 mutated amino acid residue. Based on the energy binding calculations, it was determined that the R292K mutation caused a reduction of 17.28 kcal/mol in the peramivir binding affinity compared to the peramivir-wildtype complex. As a result, the peramivir was oriented differently in the binding site and the overall conformation of the peramivir-mutant complex changed. Experimental investigations have been conducted into the mutation of E119 in neuraminidase. Contrary to this, there is insufficient information regarding the impact of E119V mutation towards peramivir at the intermolecular level. Therefore, a thorough understanding of the protein-ligand intermolecular interactions is crucial to understanding its inhibition. In the present study, we explored the intermolecular mechanism and dynamics associated with the susceptibility of peramivir to influenza H7N9 virus containing E119V mutation. We utilized molecular dynamic simulations and a wide range of post-molecular dynamic analysis for comprehensive insights into the impact of the E119V mutation and the conformational of the peramivir-E119V mutant complex. Based on the post-molecular dynamic analysis, the peramivir-E119V mutant complex showed relative stability. For the peramivir-wildtype complex, the calculated binding free energy (ΔGbind) is -49.09 ± 0.13 kcal/mol, while for E119V mutant it is -58.55 ± 0.15 kcal/mol. The increase in binding free energy by 9.46 kcal / mol is in accordance with other post-molecular dynamic analyses, which found that the E119V mutation increases protein stability. These findings could play a crucial role in developing new antiinfluenza drugs and controlling the avian influenza H7N9 virus. New and re-emerging diseases like influenza are challenging to treat due to the lengthy development process and high failure rate. To develop potential therapies against the H7N9 virus, we repurposed FDA-approved drugs using an in silico drug repurposing method. A total of 2,568 drugs were screened for potential inhibitors. A DrugBank database virtual screening identified the compounds promacta, tucitanib, and lurasidone as promising hits. The calculations of MM-GBSA suggest that tucitanib (-54.1 kcal/mol) and promacta (-56.2 kcal/mol) occupy the active site of neuraminidase with a higher binding affinity than the standard drug peramivir (- 49.09 kcal/ mol). Based on the results of Molecular dynamics (MD) simulation, the C-α atom backbones of the complexes of tucatinib and promacta neuraminidase remained stable during the simulation time. Absorption, distribution, metabolism, and excretion (ADME) analysis revealed that the hit compounds have a high gastrointestinal absorption (GI) and lack properties that allow them to cross the blood-brain barrier (BBB). Based on the in silico toxicity prediction, promacta is not cardiotoxic, while lurasidone and tucatinib are only weakly inhibitory. We, therefore, propose to test these compounds experimentally against influenza H7N9. To bring these compounds to clinical settings, further investigation, and validation of these potential H7N9 inhibitors are necessary. In summary, this study has established that the R292K mutation decreases peramivir binding affinity and distorts peramivir optimum position in the binding site of neuraminidase. In contrast, the E119V mutation contributed to relative stability of the peramivir-neuraminidase complex. Promacta and tucatinib could be used as lead compounds to combat the H7N9 influenza virus. Insights gained from this study will enhance future drug development and help in combating the avian influenza H7N9 virus. Nonetheless, the concept of multi-target drugs, quantum mechanics (QM) method such as density functional theory (DFT) and hybrid quantum mechanics/molecular mechanics (QM/MM) for effective design of neuraminidase inhibitors should be widely explored.
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    A novel library of hybrids as potential antimalarial agents: design, synthesis, characterization and in vitro biological evaluation.
    (2021) Kayamba, Francis.; Rajshekhar, Karpoormath.; Nyamori, Vincent Onserio.
    Malaria continues to be a fundamental health threat worldwide due to the rise of resistance in frontline medications, including artemisinin-based combination therapy (ACT). The condition requires new, innovative drug candidates to overcome existing challenges in the current treatment. The synthetic alteration involving existing pharmacophoric antimalarial drugs and natural products were utilized to construct an antimalarial candidate that could serve as a potent and effective lead compound for potential optimization. In continuing our ongoing research and the need to discover newer antimalarials, we endeavored to synthesize a novel library of antimalarial hybrids analogues comprising 4,6-diphenylpyrimidine core, a pharmacophoric moiety inspired by pyrimethamine and chalcone. Pyrimidine is a versatile heterocyclic building block of many drugs with vast medicinal properties such as antimicrobial, anti-inflammatory, analgesic, anticonvulsant, anticancer, and antioxidant. Fifty-three hybrids consisting of pyrimidine core fused with other significant antimalarial moieties, namely quinoline, cinnamoyl and 1,2,3-triazole through an alkane diamine linker, particularly piperazine, were synthesized. All the newly synthesised compounds were identified based on physicochemical and spectral data (IR, 1H NMR, 13C NMR and HRMS) per their predicted structures and preliminarily screened for antimalarial (in vitro) activities. Also, the binding affinities of two essential cytosolic Plasmodium (P) falciparum heat shock protein 70 homologues (PfHsp70-1 and PfHsp70-z) were examined. Pyrimidine-quinoline hybrids (Chapter 3) demonstrated antimalarial ranging from 0.32 to 83 μM. Of this series, compounds 7a and 7b were the most potent with IC50 value of 0.32 ± 0.06 μM and IC50 1.62 ± 1.14 μM, respectively with a safety profile of 9.79 to human kidney epithelial (HEK293) cells for 7a. Equally, compounds 7a and 7b presented the highest binding affinity of two essential cytosolic P. falciparum heat shock protein 70 homologues; PfHsp70-1 and PfHsp70-z, with KD in a lower nanomolar range (4.4-11.4 nM) Pyrimidine-cinnamoyl hybrids (Chapter 4) exhibited antimalarial activity from 0.18 to 50 μM. Compounds 8a and 8l were the most active with IC50 value of 0.18 ± 0.02 μM and IC50 0.21 ± 0.00 μM with the safety profile of 18.59 and 16.75 to human kidney epithelial (HEK293) cells, correspondingly. Compounds 8a and 8l showed the highest binding affinity of two essential cytosolic P. falciparum heat shock protein 70 homologues; PfHsp70-1 and PfHsp70-z, with KD in a lower nanomolar range (9.69-10.8 nM). Pyrimidine-1,2,3-triazole hybrids (Chapter 5) showed antimalarial activity from 0.04 to 2.41 μM. Compounds 8c, 8e and 8t were the most promising with IC50 values ranging from 0.18 to 0.29 μM with a safety profile tenfold compared to human kidney epithelial (HEK293) cells. Similarly, compounds 8c, 8e and 8t displayed the highest binding affinity of two essential cytosolic P. falciparum heat shock protein 70 homologues; PfHsp70-1 and PfHsp70-z, with KD in a lower nanomolar range (11.3-90.0 nM). The pyrimidine-1,2,3-triazole hybrid library demonstrated the most promising family as it had eight more potent compounds with IC50 values less than 0.50 μM, followed by the pyrimidine-cinnamoyl and lastly, pyrimidine-quinoline. All families exhibited PfHsp70-1 and PfHsp70-z enzyme activity in nanomolar concentration with a safety of 10-fold against human kidney epithelial (HEK293) mammalian cells.
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    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.
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    Synthesis and antimicrobial evaluation of novel 2,4-dihydro-3h-pyrazol-3-one hybrids: a new class of antibiotics.
    (2019) Jain, Kavita.; Karpoormath, Rajshekhar.
    The rise in multidrug resistance (MDR) pathogenic microbes has emerged as a critical global health burden. To address this problem, the scientific community and Pharmaceutical industries worldwide are focused in developing newer, safer and cost-effective antimicrobial agents. Design and development of potential antimicrobial agents has been one of the focus areas of the Synthetic and Medicinal Chemistry Research Group (SMCRG), UKZN. In continuation of the ongoing research in SMCRG and the need to discover newer antimicrobials, I envisaged to synthesize novel antimicrobial agents containing 2,4-Dihydro-3H-pyrazol-3-one (pyrazolone) as a core pharmacophoric moiety. Pyrazolones are versatile heterocyclic building blocks and is a core scaffold in several marketed drugs such as for analgesic and antipyretic (phenazone, metamizole, propyphenazon, and remifenazone); neuroprotective agent (edaravon); antispasmodic (dipyron); anti-inflammatory agents (famprofazone, phenylbutazone, and remifenazone) and more recently there have been several reports on the pyrazolone analogs as potential antimicrobial agents. In this research work I have synthesized a series of potential pyrazolone hybrids containing substituted 1,3,4-thiadiazole, thiazolidinone, triazole, oximes, and chalcones as antimicrobial agents (Figure 1). The work in this thesis is divided into 7 chapters:
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    Structural and functional characterization of the egress and invasion machinery of the Malaria parasite: proposing a new way forward in malaria therapeutics from an atomistic perspective.
    (2019) Munsamy, Geraldene.; Mahmoud Elsayed Soliman, Soliman.
    The past decade has witnessed numerous efforts to control the invasive tactics of the malarial parasite, including focused research towards selective malarial inhibitors of Plasmodium falciparum, the most lethal strain of the Plasmodium species. The recent discovery of the key mediators of egress and erythrocyte invasion of the malaria parasite has opened a new avenue that may be harnessed for the development of effective therapeutics that may permanently eradicate the malaria virus. These new parasitic targets of P. falciparum are PIX and PX and have gained considerable attention in drug discovery pipelines however, the absence of crystal structures of these enzymes evidenced a lack in structural information, as there is currently little known regarding the structural dynamics, active site domains and the mechanism of inhibition of these enzymes. This has therefore led to the modeling of the 3D protein structure of each enzyme to gain a fundamental understanding regarding the structural and functional characteristics that may be visualized from an atomistic perspective. The emergence of new drug targets has led to the integral use of computational techniques including molecular modeling, molecular docking, virtual screening protocols and molecular dynamic simulations which allow chemists to evaluate and assess millions of compounds and thus funnel out potential lead drugs. These in silico techniques further justify the current use of Computer-Aided Drug Design as a cost-effective approach to fast track the drug discovery process. The above-mentioned techniques, amongst a vast range of other computational tools were integrated in this study to provide insight into conformational changes that elucidate potential inhibitory mechanisms, identification of the active site cleft, characterization and pharmacophoric features leading to novel small molecule inhibitors. This study focused on analysing the flap dynamics specific to the aspartic protease family of enzymes using a defined set of parameters to map out the binding domain for the design of potential antimalarial drugs. To gain a molecular perspective of the conformational binding of two proposed experimental drugs which showed substantial inhibitory activity against PIX and PX molecular dynamic simulations were performed and further evaluated employing in silico thermodynamic analysis to provide insight into the proposed binding of mode of each inhibitor, highlighting the key moieties required for binding. A pharmacophoric model was also generated using in silico tools to screen for tailored inhibitors specific to PIX. The aim of this study was to generate fundamental insight into the structural and functional characterization of two prominent targets that play an indispensable role in survival of the malaria virus. The implementation of thebinformation extracted from this study, may provide a structural outline for molecular biologists, and pharmaceutical scientists to aid in the design of novel antimalarial therapeutics.
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    Synthesis and antimicrobial evaluation of novel 2,4-dihydro-3h-pyrazol-3-one hybrids: a new class of antibiotics.
    (2019) Jain, Kavita.; Karpoormath, Rajshekhar.
    ABSTRACT The rise in multidrug resistance (MDR) pathogenic microbes has emerged as a critical global health burden. To address this problem, the scientific community and Pharmaceutical industries worldwide are focused in developing newer, safer and cost-effective antimicrobial agents. Design and development of potential antimicrobial agents has been one of the focus areas of the Synthetic and Medicinal Chemistry Research Group (SMCRG), UKZN. In continuation of the ongoing research in SMCRG and the need to discover newer antimicrobials, I envisaged to synthesize novel antimicrobial agents containing 2,4-Dihydro-3H-pyrazol-3-one (pyrazolone) as a core pharmacophoric moiety. Pyrazolones are versatile heterocyclic building blocks and is a core scaffold in several marketed drugs such as for analgesic and antipyretic (phenazone, metamizole, propyphenazon, and remifenazone); neuroprotective agent (edaravon); antispasmodic (dipyron); anti-inflammatory agents (famprofazone, phenylbutazone, and remifenazone) and more recently there have been several reports on the pyrazolone analogs as potential antimicrobial agents. In this research work I have synthesized a series of potential pyrazolone hybrids containing substituted 1,3,4-thiadiazole, thiazolidinone, triazole, oximes, and chalcones as antimicrobial agents (Figure 1). The work in this thesis is divided into 7 chapters.
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    Effects of naringenin on metformin disposition in a diabetic rat model.
    (2018) Mofo Mato, Edith Pascale.; Owira, Peter Mark Oroma.; Essop, M Faadiel.
    Diabetes mellitus (DM) is one of the largest global health emergencies of the 21st century. It is a major cause of blindness, kidney failure, cardiovascular diseases, lower limb amputation and accounted for 10,7 % of global all-cause mortality among people aged between 20 and 79 years old. Metformin is currently the most widely prescribed anti-diabetic drug. It exists as a hydrophilic cation at physiological pH. As such, membrane transporters play a substantial role in its oral absorption, hepatic uptake, and renal elimination. Among these transporters, organic cation transporters OCT 1 (SLC22A1) and OCT 2 (SLC22A2) are known to be important determinants of the pharmacokinetics of metformin. Naringenin, which is a plant-derived compound found in citrus fruits and vegetables, has been presumed to interact with conventional drugs and influence their disposition by modification of drug-metabolizing enzymes and transporters. The aim of this study was to investigate the effects of naringenin on organic cations transporters OCT1 and OCT2 protein expression and subsequently on metformin disposition in streptozotocin- induced diabetic rats. Methods Forty-nine male Sprague Dawley rats 250–300 g body weight (BW) were randomly divided into 7 experimental groups (n = 7). They were orally treated daily with 3.0 ml/kg body weight (BW) of distilled water (group 1) or 250 mg/kg BW of metformin (groups 3, 6 and 7) or 60 mg/kg BW of naringenin (groups 2, 5 and 7) dissolved in distilled water. Groups 4, 5, 6 and 7 were given a single intraperitoneal injection of 60 mg/kg BW of streptozotocin to induce diabetes. Animal body weights and water intake were recorded daily. Fasting blood glucose (FBG) and glucose tolerance tests (GTT) were subsequently done. Urine samples were collected from rats kept in individual metabolic cages for 24 hours, to determine output, electrolytes, albumin, creatinine and metformin levels. Thereafter, the animals were sacrificed by halothane overdose and blood was collected via cardiac puncture. Liver and kidneys were excised, rinsed in normal saline, blotted dry, weighed, snap frozen in liquid nitrogen and stored at -80°c for analysis of OCT 1 and OCT 2 protein expression by Western blot. OCT 1 and OCT 2 proteins were extracted and separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Then, the gel was blotted electrophoretically onto a nitrocellulose membrane which was then probed with a primary antibody and ultimately an enzyme conjugated secondary antibody and substrate to visualize the bands representing the target proteins. Results Diabetic rats treated with naringenin and metformin either alone or in combination exhibited weight gain, improved creatinine clearance and reduced polydipsia, albuminuria, serum creatinine and blood urea nitrogen compared to untreated diabetic rats. By contrast, metformin with/without naringenin did not significantly ameliorate hyperglycemia in diabetic rats. Treatment with naringenin increased hepatic uptake and renal clearance of metformin in diabetic rats compared to untreated groups. In addition, naringenin significantly increased lactate concentrations and metabolic acidosis in rats treated with metformin compared to those that were not treated with metformin. Furthermore, diabetic rats exhibited lower OCT1 and OCT2 protein expressions but naringenin treatment significantly increased hepatic OCT1 and renal OCT2 protein expressions in the presence of metformin. Conclusion Collectively, our data suggest that metformin disposition could be affected by naringenin through the upregulation of OCT1 and OCT2 protein expressions. Upregulation of OCT1 expression may be associated with metformin-induced lactic acidosis while increased renal OCT2 expression might facilitate metformin excretion and reduce the risk of lactic acid. However, increased renal excretion of metformin by naringenin may not be sufficient to avert metformin-induced lactic acidosis.
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    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.
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    A simple yet novel strategies for the synthesis of pharmacologically versatile benzoxazole and benzothiazole scaffolds via transamidation.
    (2022) Kumar, Vishal.; Karpoormath, Rajshekhar.; Singh, Parvesh.
    Transamidation is a popular way for amide transformation from another amide despite conventional amide bond formation methods. Acid and acid chloride were utilised as starting ingredients in previous procedures. Transamidation has shifted the perspective of researchers on amide synthesis. Nowadays, many methodologies have recently come to light for amide transformation, using non-metal and metalmediated techniques. On the other hand, a few approaches involving amide bond activators have been reported. Moreover, N-Boc, Tosyl, and Mesyl groups are commonly utilised as amide bond activators. As a result, many amides transformations have been synthesised and published in the literature over the last few decades. Based on previous research, we have attempted to develop cost-effective and novel techniques of transamidation for unactivated amides, which could serve as a potential alternative to synthesising amide bond containing compounds from lab to plant scale. We also demonstrated a novel approach for synthesising 2-substituted benzoxazole/benzothiazole compounds using this transformation. The history and development of unactivated amides transformation are briefly described in Chapter 2. This chapter also discusses different transamidation methods for primary, secondary, and tertiary unactivated amides that are transformed into other amides without the need for an amide bond activator. The development of a novel green, efficient catalyst-free, one-pot synthetic methodology to synthesise amides is described in Chapter 3. Under the optimisation investigations, the effects of various acids, solvents, temperatures, and time intervals were also described. The reaction was catalysed by hydrochloride, which activated the carbonyl bond, and then attacked the carbonyl carbon with an additive amine as a nucleophile. Finally, the amine as a byproduct is removed, resulting in the desired converted amide. Furthermore, this new transamidation process allows for a wide range of amide types, including primary, secondary, and tertiary. This approach also works with a variety of primary and secondary amines, including aliphatic and bulky, hetero-aromatic substituted amines. To continue the work begun in Chapter 4, we develop a new solvent-free and metal-free method for synthesising 2-substituted benzoxazole and benzothiazole in Chapter 4. In this approach, we increase the temperature and employ substituted 2-aminophenols or 2-aminothiophenols instead of amines. To lead the annulation product, the oxygen or sulphur atom of phenol or thiophenol attacks the carbonyl carbon of the transformed amide group. Furthermore, this technique works well with a variety of substituted 2-aminophenols. Additionally, the amide scope for this reaction is quite broad. Chapter 5 describes the development of a new method for the transamidation of aliphatic amides using CuCl2 as a catalyst. The effects of various catalysts and Lewis acids and the effect of different solvents were explained. Moreover, the annulated substituted 2-benzoxazole and 2-benzothiazole derivatives obtain in the case of 2-aminophenol and 2-aminothiophenol. Besides, this new versatile methodology provides a wide substrate scope for the synthesis of different functionally substituted amides and 1,3- benzoxazole scaffolds. It can be further exploited as building blocks for the synthesis of pharmaceutical drugs.
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    Nanotherapeutics to combat infections.
    (2019) Walvekar, Pavan.; Govender, Thirumala.; Mocktar, Chunderika.
    The rise of drug resistant microorganisms is threatening the ability of antimicrobials to treat infectious diseases including bacterial infections, thus becoming a significant cause for premature mortality. Limitations associated with conventional dosage forms are one of the contributing factors for increasing antimicrobial resistance. Novel nano-drug delivery systems are showing considerable potential to combat antimicrobial resistance. The application of advanced novel materials for the 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 MRSA infections. In this study, two sets of novel amphiphiles; fatty acid based pyridinium cationic amphiphiles (FCAs) and novel hyaluronic acid-oleylamine (HA-OLA) conjugates were synthesized and characterized. The synthesized novel amphiphiles were employed to formulate two nano-drug delivery systems for efficient delivery of vancomycin (VCM) to treat S. aureus and MRSA infections. The synthesized materials were found to have inherent antibacterial activity on tested bacterial strains and proven to be biosafe after exhibiting cell viability above 75% on all tested mammalian cell lines using MTT assay. The formulated nano-systems were characterized in terms of particle sizes, polydispersity indices (PDI), zeta potential (ZP), surface morphology, in vitro and in vivo (VCM loaded OCA vesicles) antibacterial activity. Oleic based cationic amphiphile (OCA) was employed to construct VCM loaded OCA vesicles, and had sizes, PDI, ZP and entrapment efficiency of 132.9 ± 2.5 nm, 0.167 ± 0.02, 18.9 ± 1.2 mV and 61.24 ± 1.8%, respectively. VCM loaded polymersomes prepared using HA-OLA6 had sizes, PDI, ZP and entrapment efficiency of 248.7 ± 3.08 nm, 0.189 ± 0.01, -17.6 ± 0.6 mV and. 43.12 ± 2.18%, respectively. The drug release from VCM loaded OCA vesicles and VCM loaded HA-OLA polymersomes (VCM-PS6) was sustained throughout the studied period of 72 h. From in vitro antibacterial studies, both FCAs and HA-OLA conjugates showed bactericidal activity against the tested bacterial strains. Both VCM loaded OCA vesicles and VCM-PS6 displayed 4-fold enhanced antibacterial activity against MRSA, when compared to bare VCM. Furthermore, synergism was observed between VCM and synthesized novel amphiphiles (FCAs and HA-OLA conjugates) in nano-formulations against MRSA. An in vivo BALB/c mice skin infection model revealed that, treatment with VCM loaded OCA vesicles significantly reduced the MRSA burden compared to bare drugs and untreated groups. There was 4.2-fold reduction in the MRSA load in mice skin treated with VCM loaded OCA vesicles compared to those treated with bare VCM. In summary, synthesized novel materials showed good biosafety, antibacterial activity and drug delivery potential via nano-systems against bacterial infections. The data from this study has resulted in one first authored review article, two first authored and one co-authored research publications.
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    Quantum plasmonic sensing with application to HIV research.
    (2020) Mpofu, Kelvin Tafadzwa.; Kruger, Hendrik Gerhardus.; Tame, Mark Simon.
    The main goal of this thesis is to show how the use of quantum states of light in biosensing with surface plasmon resonance (SPR) gives an enhancement over using classical states. SPR is a highly sensitive technique for monitoring changes in the optical properties of a substance in the immediate vicinity of a sensor surface, which makes it very useful in biosensing and surface science research. We focus primarily on a bio-sensing SPR setup known as the Kretschmann con guration in which surface plasmons are excited using a bulk prism and a gold coated microscope slide. The excitation is performed by means of an evanescent eld arising from total internal re ection from the backside of the sensor surface. We show theoretically that using quantum states of light such as the Fock state, twomode squeezed vacuum and two-mode squeezed displaced state improves the precision in the estimation of kinetic parameters measured from the sensorgrams produced by the Kretschmann con guration. Quantum states of light allow us to measure the parameters more accurately in comparison to the use of classical states of light. We look at a theoretical application of quantum bio-sensing in an immobilized Bovine serum albumin (BSA) interaction with anti-BSA, a binding reaction between a phosphate-bu ered saline (PBS) solution that contains Bovine carbonic anhydrase and its inhibitor benzene-sulfonamide, and a HIV case study. In our HIV case study we look at the binding reaction between a variant of HIV-1 protease and nel navir which is an inhibitor. The thesis also looks at an experimental implementation of the Kretschmann con guration with light from a single-photon source and shows an enhancement in sensitivity. We use the spontaneous parametric down conversion process to generate our single photons which we use to study the binding kinetics of BSA on a gold slide. This experiment is done with the anticipation that it will be extended in future to a drug kinetics study with HIV protease as we look at drug development.
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    Effects of Z-venusol and other pure compounds from medicinal plants on prostate, cervical and breast cancer cells.
    (2017) Mathibe, Lehlohonolo John.; Naidoo, Strinivasen.; Botha, Julia Hilary.
    Introduction According to recent World Health Organisation (WHO) estimates, cancer causes more deaths than coronary heart diseases globally (GLOBOCAN, 2012). While communicable diseases such as HIV/AIDS continue to burden African populations, cancer is increasingly recognised as a critical public and private health problem in Africa (Igene, 2008). It is estimated that by 2030, about 112 921 new cases of cancer will be diagnosed in South Africa (Singh et al., 2015). This would represent a 50% increase of new cancer cases as compared to 2012’s estimates by the WHO. Although there is little doubt about the incidences of cancer, there are, unfortunately, divergent theories in as far as tumourigenesis and the aetiology of cancer. Some researchers hold the view that cancer originates from malignant transformation of normal tissue progenitor and stem cells (Reya et al., 2001). Others believe that cancer is as a result of mature cells that have undergone de-differentiation (Sell, 2004). Notably, latest research has shown that there is a strong association between tissue-specific cancer risk and the lifetime cumulative number of cell divisions of tissue or organ-specific stem cells (Tomasetti & Vogelstein, 2015). Although there are still differing views on the origins of cancer, it is widely accepted that this devastating disease occurs as a result of abnormal cell development and is characterised by uncontrollable cell proliferation. The majority of currently-available cancer treatments target cell proliferation. However, the effectiveness of many cytotoxic drugs, including those that were discovered from plants, is limited by their serious side-effects and cost (Abratt, 2016). Chemotherapeutic agents that were originally discovered from medicinal plants include vinblastine (isolated from Catharanthus roseus), etoposide (isolated from Podophyllum peltatum), paclitaxel (isolated from Taxus brevifolia) and topotecan and camptothecin (isolated from Camptotheca acumenata). Thus, medicinal plants continue to play a critical role in the management of diseases in the world. In Africa, decoctions, which contain extracts from various medicinal plants (Bruneton, 1995; Balunas & Kinghorn, 2005), are widely used for traditional management of many diseases including cancer. However, apart from subjective oral evidence regarding the effectiveness of extracts from various plants, the identity of ingredients, as well as the science and pharmacology of active compounds found in numerous popular concoctions and decoctions are not known. Objectives The main objectives of this study were:  To assess anti-proliferative potential of three plant-derived-compounds, i.e. hypoxoside, ent-Beyer-15-en-19-ol and Z-venusol on human cancer cells, namely DU-145 (prostate), HeLa (cervical) and MCF-7 (breast) in vitro.  To determine the type of cell death, i.e. whether a compound with potential causes apoptotic or necrotic cell death on both human cancer and normal cell lines (such as MCF-12, HMECs and dMVECs).  To investigate how a potential compound exerts its cytotoxicity. Materials and Methods Initially dimethylthiazol-diphenyltetrazolium bromide (MTT) assays were conducted to find the concentrations which may inhibit proliferation in prostate (DU-145), cervical (HeLa) and breast (MCF-7) cancer cells. Normal human cell lines, which were used for control purposes, were the primary human mammary epithelial cells (HMECs), MCF-12 and the dermal microvascular endothelial cells (dMVECs). Initially, cells were exposed for 48 hr to hypoxoside, ent-Beyer-15-en-19-ol and Z-venusol, which were isolated from Hypoxis hemerocallidea, Helichrysum tenax, and Gunnera perpensa, respectively. The concentrations ranged from 2.34 μg/mL to 2400 μg/mL, dissolved in cell specific media. In subsequent experiments, the more sensitive sulforhodamine B (SRB) methodology was used, and cells were exposed to Z-venusol for 24 hr, 48 hr and 72 hr, to much lower concentrations, which ranged from 1.9 μg/mL to 240 μg/mL dissolved in dimethyl sulphoxide (DMSO). To investigate possible pathways of observed cell death, two assays were conducted. These were the fluorescein isothiocyanate (FITC) Annexin V apoptosis detection assay (using the FACS Calibur “JO” E5637 flow cytometer for analysis), and the lactate dehydrogenase (LDH) assay. To explore possible mechanism(s) of action, the activities of interleukin-6 (IL-6) and cyclic adenosine monophosphate (cAMP) were assessed. To investigate the activity of IL-6, cells were exposed for 48 hr to various working concentrations of Z-venusol; that is, 37.5 μg/mL and 75 μg/mL. To investigate the activity of direct cAMP, cells were exposed for 48 hr to various working concentrations of Z-venusol; that is, 37.5 μg/mL, 75 μg/mL, and 150 μg/mL. Absorbance, which is inversely proportional to the concentration of cAMP in both the samples and the standards, was measured using a BioRad (Model 3550) microplate reader. Epinephrine (10 μM) and propranolol (10 μM), were used separately and in combination, added to the highest concentration of Z-venusol for comparison. Main Results & Discussion Hypoxoside resulted in a statistically significant (p < 0.001) 38% and 77% increases in proliferation in MCF-7s at concentrations of hypoxoside 1200 μg/mL and 2400 μg/mL, respectively, after 48 hr exposure. In support of the current findings, Xulu (2013) also reported that hypoxoside, and its active derivative known as rooperol, significantly increases cell proliferation of both cancer and normal mammary cells in vitro (Xulu, 2013). This was considered an undesirable finding with regards to the aim of finding a cure for cancer. Therefore, no further test were carried out on this compound beyond the initial screening stages. The highest concentration (i.e., 2400 μg/mL) of the second compound, that is ent-Beyer-15- en-19-ol, decreased proliferation in prostate cancer cells (DU-145) and in breast cancer cells (MCF-7) by 6% and 19%, respectively. Interestingly, much lower concentrations, i.e. 4.7 μg/mL and 9.4 μg/mL, of ent-beyer-15-en-19-ol significantly (p < 0.05) decreased cell proliferation in cervical cancer cells (HeLa) by 37% and 41%, respectively. The differences in expression of vimentin gene, which is over-expressed in HeLa cells and suppressed in MCF- 7s and DU-145s may explain why this compound showed significant activity only in the cervical cancer cells (Oshima, 2002; Satelli & Li, 2011). More importantly, the ability of this compound to significantly inhibit cell proliferation in the HeLa cell line by almost 50% at lower concentrations offers an opportunity for further studies. The findings with regards to the third compound, i.e. Z-venusol, were the most exciting. Hence investigations on it were developed beyond the screening stages. This compound demonstrated a statistically significant, concentration-dependent, apoptotic inhibitory effect on the proliferation of MCF-7 cells, with an IC50 of 53.7 μg/mL after 72 hr exposure, while the highest concentration (250 μg/mL) resulted in 69% inhibition. Both the FITC Annexin V and LDH results suggested that apoptosis contributed to most of the effects observed. Further, there was non-significant inhibition (20%) of HMEC proliferation observed when the concentration of Z-venusol was increased beyond 16.6 μg/mL. The highest concentration of Z-venusol used in this study resulted in a statistically significant (p < 0.001) 51% inhibition of IL-6 activity in the MCF-7 after 48 hr exposure. None of the Z-venusol concentrations, either alone or in combination with epinephrine, an agonist of the adrenergic receptors, showed any statistically significant effect on the levels of cAMP in the MCF-7s. Surprisingly, there was a significant (p ≤ 0.028) 34% elevation of cAMP levels in cells which were exposed to a combination of Zvenusol and propranolol. If Z-venusol was ever able to be used clinically, there might be a need to increase the dose high enough for the attainment of desired therapeutic effects with minimal cytotoxicity on normal cells, because its potency is much lower than that of cisplatin. Increasing Z-venusol to a therapeutically-effective concentration would be possible as there was no plateauing-off of inhibition of proliferation in MCF-7s. It was only in primary normal human mammary epithelial cells (HMECs) that formation of “plateaus” was observed. Favourably, this selective plateauing-effect might allow the ‘gold-standard’ attainment of the desired cytotoxic effect on cancer cells while preserving normal cells at higher concentrations. There are no studies with which to directly compare the findings of this study. However, reports on effects of the extracts of G. perpensa on various other cancer cell lines provide an opportunity for comparison. For instance, the results of this research support the findings of Simelane and colleagues. They recently reported that G. perpensa extracts caused an inhibition of proliferation of hepatocellular carcinoma cells (HepG2) with an IC50 of 222.33 μg/mL and human embryonic kidney 293 (HEK293) cells, with an IC50 of 279.43 μg/mL both after 48 hr of treatment (Simelane et al., 2012). Conclusion Z-venusol, unlike other compounds studied, has a firm potential to play a role in the treatment of cancer in the future. Its mechanism of action involves IL-6 signaling, which may trigger other downstream mediators and may also involve cAMP “cross-talk”. Recommendations More basic science investigations using other hormone-dependent and highly invasive breast cancer cell lines such as the triple-negative MB-231 cells are needed. In vivo studies, such as using the nude mice model, are needed to confirm the in vitro results and to provide an insight into the benefits of Z-venusol in living systems.
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    Mechanistic insights and in silico studies on selected G protein-coupled receptors implicated in HIV and neurological disorders.
    (2021) Appiah-Kubi, Patrick.; Soliman, Mahmoud Elsayed Soliman.
    G protein-coupled receptors (GPCRs) are the largest membrane protein receptor superfamily involved in a wide range of physiological processes. GPCRs form the major class of drug targets for a diverse array of pathophysiological conditions. Consequently, GPCRs are recognised as drug targets for the treatment of various diseases, including neurological disorders, cardiovascular conditions, oncology, diabetes, and HIV. The recent advancement in GPCR structure resolutions has provided novel avenues to understand their molecular basis of signal transduction, ligand recognition and ligand-receptor interactions. These advances provide a framework for the structure-based discovery of new drugs in targeting GPCRs implicated in the pathogenesis of various human diseases. In this thesis, the interactions of inhibitors at two dopamine receptor subtypes and C-C chemokine receptor 5 (CCR5) of the Class A GPCR family were investigated. Dopamine receptors and CCR5 are validated GPCR targets implicated in neurological disorders and HIV disease, respectively. The lack of structural information on these receptors limited our comprehension of their antagonists’ structural dynamics and binding mechanisms. The recently solved crystal structures for these receptors have necessitated further investigations in their ligand-receptor interactions to obtain novel insights that may assist drug discovery towards these receptors. This thesis comprehensively investigated the binding profiles of atypical antipsychotics (class I and class II) at the first crystal structure of the D2 dopamine receptor (D2DR). The class I antipsychotics exhibited binding poses and dynamics different from the class II antipsychotics with disparate interaction mechanistic at D2DR active site. The class II antipsychotics were remarkably observed to establish a recurrent and vital interaction with Asp114 via strong hydrogen bond interactions. Furthermore, compared to class I antipsychotics, the class II antipsychotics were found to engage favourably with the deep hydrophobic pocket of D2DR. In addition, the structural basis and atomistic binding mechanistic of the preferential selective inhibition at D3DR over D2DR were explored. This study investigated two small molecules (R-VK4-40 and Y-QA31) with substantial selectivity (> 180-fold) for D3DR over D2DR. The selective antagonists adopted shallow binding modes at D3DR while demonstrating a deep hydrophobic pocket binding at D2DR. Also, the vital roles and contribution of critical residues to the selective binding of R-VK4-40 and Y-QA31were identified in D3DR. Structural and binding free energy analyses further discovered distinct stabilising effects of the selective antagonists on the secondary architecture and binding profiles of D3DR relative to D2DR. Furthermore, the atomistic molecular interaction mechanism of how slight structural modification between novel derivatives of 1-heteroaryl-1,3-propanediamine (Compd-21 and - 34) and Maraviroc significantly affects their binding profiles toward CCR5 were elucidated. This study utilised explicit lipid bilayer molecular dynamics (MD) simulations and advanced analyses to explore these inhibitory disparities. The thiophene moiety substitution common to Compd-21 and -34 was found to enhance their CCR5-inhibitory activities due to complementary high-affinity interactions with residues critical for the gp120 V3 loop binding. The study further highlights the structural modifications that may improve inhibitor competitiveness with the gp120 V3 loop. Finally, structure-based virtual screening of antiviral chemical database was performed to identify potential compounds as HIV-1 entry inhibitors targeting CCR5. The identified compounds made pertinent interactions with CCR5 residues critical for the HIV-1 gp120-V3 loop binding. Their predicted in silico physicochemical and pharmacokinetic descriptors were within the acceptable range for drug-likeness. Further structural optimisations and biochemical testing of the proposed compounds may assist in the discovery of novel HIV-1 therapy. The studies presented in this thesis provide novel mechanistic and in silico perspective on the ligand-receptor interactions of GPCRs. The findings highlighted in this thesis may assist in further research towards the identification of novel drug molecules towards CCR5 and D2-like dopamine receptor subtypes.
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    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.
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    Synthesis and physiochemical characterization of new siderophore-inspired peptide-chelators with 1-hydroxypridine-2-one (1,2-HOPO).
    (2020) Alshaer, Danah Mahdi Mohammad.; Garcia de la Torre, Beatriz.; Albericio, Fernando.;
    Compounds containing hydroxamate moieties (N-hydroxyl amides) in their structure have found a vast range of therapeutic applications such as antibacterial, anti-tumour, anti-immune suppressor, and for iron overloading treatment. Hydroxamate chelators binds to Fe (III) tightly through its electron donating oxygens. The binding strength is maximized in compounds containing three hydroxamic moieties due to the so called “chelate effect”. As all microorganisms require iron for surviving, they develop endogenous siderophores to acquire iron from the surroundings. Siderophores contain hydroxamate, catecholates, α-hydroxy carboxylates groups, among others, in their structures. The acquisition of iron by siderophores in microorganisms goes through specific cycles that includes sequestration of Fe(III), recognition and uptake of the ferrisiderophore through the cell membrane, and then release of the iron in the cytoplasm. Many natural hydroxamate siderophores contain a peptidyl backbone. In this work, peptides containing one or more units of 1,2-hydroxypyridine-N-oxide (1,2-HOPO) have been synthesized. The introduction of these units on the peptides has been done by means of 4- carboxy-1-hydroxypyridil-2-one (1,2-HOPO-4-COOH) using solid-phase peptides synthesis (SPPS) protocols. The obtention of the new “siderophores” containing three 1,2-HOPO units have been done by two different approaches, sequential and convergent. The compounds have been evaluated as potential iron chelators. Thus, the pKa values and the thermodynamic constants of all ligands have been spectrophotometrically determined. The Fe(III) affinities of the two hexadentate ligands (ligand B and ligand C) have been determined by competition experiments against EDTA. The results showed that the iron complex ligand B is stronger that the iron complex EDTA since the last is not be able to replace it. Hence, this new siderophore could be a promising candidate to be used in further therapeutic applications.
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    The role of kinins and cytokines in rheumatoid arthritis.
    (2001) Cassim, Bilkish.; Bhoola, Keshavlal Daya Narotam.
    Introduction: Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by inflammatory synovitis. The histopathological features include synovial hyperplasia, an inflammatory cell infiltration, angiogenesis and an inflammatory exudate into the synovial joint with progression to bone and joint destruction. While the exact aetiology of RA is unknown, a number of inflammatory cells and mediators have been implicated in the pathogenesis. Kinins are vasoactive peptides that have the capacity to induce the cardinal features of inflammation and considerable evidence exists for a role for the kallikrein-kinin cascade in inflammatory arthritis. The proinflarnmatory cytokines are also important mediators in rheumatoid arthritis and there is evidence for a functional relationship between the kallikrein-kinin and cytokine cascades in rheumatoid arthritis. Methods: Following approval from the Ethics Committee of the University of Natal, synovial tissue samples were obtained at arthroscopy from patients with RA and at autopsy (for controls). The tissue samples were processed for light microscopy and immunostained by the immunoperoxidase method to detect tissue kallikrein and the kinin B 1 and B2 receptors. The intensity of the immunostaining was quantified by image analysis. Blood and synovial fluid samples were obtained from patients with RA and blood from age and sex matched healthy volunteers. The RA patients were assessed clinically to determine the degree of disease activity and the presence or concomitant diseases. Disease activity was determined by the duration of morning stiffness, the twenty eight tender and swollen joint counts, pain on a visual analogue scale, patient's and physician's global assessment of disease activity (Likert scale), a local activity index, the modified Health Assessment Questionnaire (HAQ), disease activity score (DAS) and the erythrocyte sedimentation rate (ESR) and C reactive protein (CRP). In the synovial fluid (SF) samples, the functional activity of tissue kallikrein (TK) was demonstrated using an amidolytic assay and the total amount of TK was measured in a sandwich enzyme linked immunosorbent assay (ELISA). The Pearson's correlation coefficient was used to correlate the TK levels with measures of disease activity. Further, basal and generated kinins were measured in the SF by competitive ELISA, and the levels correlated with measures of disease activity. In the cytokine study, interleukin lP (IL IP) and tumour necrosis factor p (TNFP) were measured in the synovial fluid samples by ELISA, and the relationship between the cytokine levels and disease activity as well as TK, basal and generated kinins determined. Neutrophils were isolated from the blood and synovial fluid samples from the rheumatoid arthritis patients and from the blood samples from healthy volunteers. The circulating and synovial fluid neutrophils were immunostained to detect tissue kallkrein, the kinin moiety in the kininogen molecule and kinin BI and B2 receptors, and the immunofluorescence visualized by confocal microscopy. The images were digitally analysed using the Analysis 2.1 Pro system. The Kruskal Wallis and one-way ANOVA tests were used to compare the mean intensity of imrnunostaining in the control neutrophils with that present on the circulating and SF neutrophils harvested from RA patients. The intensity of labeling for these antigens was correlated with measures of disease activity. Results: 1. Synovial tissue samples: Labeling for tissue kallikrein was observed in the synovial lining and endothelial cells in control and rheumatoid tissue. There was a significant increase in the intensity of TK labeling in the endothelial cells of the rheumatoid tissue (p < 0.05). The kinin Bl and B2 receptor were visualized in the synovial lining cells, endothelial cells and the subintimal fibroblasts and macrophages in the control and rheumatoid synovial samples, with a significant increase in B 1 receptor labeling in the synovial lining cells in rhewnatoid synovial tissue (p < 0.01). 2. Tissue kallikrein activity and the total TK concentration was measured in the synovial fluid obtained from 20 patients with RA. There was no direct correlation between the between the enzymic and antigenic tissue kallikrein. There w as a significant negative correlation between the enzymic TK and the twenty eight swollen joint count (r = -0.464; p <0.05). 3. There was a significant negative correlation between the basal kinin and generated kinin levels (r= -0.454; p < 0.05). In addition, there was a negative correlation between the basal kinin levels and the C RP (r = -0.537; p < 0.05) and the disease activity score (r = -0.458; p < 0.05). In contrast, there was a positive correlation between the generated kinin levels and the twenty-eight tender and swollen joint counts (r = 0.536; p < 0.05 and r = 0.509; p < 0.05 respectively), the ESR (r = 0.598; p < 0.01), CRP (r = 0.725; p < 0.01) and the disease activity score (r = 0.676; p < 0.01). There was a significant correlation between the SF levels of IL IP and pain (r = 0.462; p < 0.05), physician's global assessment of disease activity (r = 0.549; p < 0.05), 28 tender joint count (r = 0.4 72; p < 0.05) and CRP (r = 0.530; p < 0.05). Although there appeared to be a correlation between the IL 1 p and disease activity score, this was not significant (r = 0.412; p = 0.07). In addition, the levels of synovial fluid lNF a correlated with the 28 tender joint (r = 0.458; p < 0.05) count and CRP (r = 0.653; p < 0.01). 4. There appeared to be a trend towards a negative correlation between the SF amidase TK levels and IL 1 p, however this was not significant. While there was no direct relationship between the SF levels of IL 1 P and the generated kinins, there was a positive correlation between low to moderate levels of IL 1 p and the generated kinins (r = 0.51, p < 0.05). In contrast, there was a negative correlation with higher levels of IL Ip (r = -0.5, p < 0.05). 5. Immunoreactive TK, kinin moiety and the Bl and B2 receptors were visualized on the circulating neutrophils from the healthy volunteers and the circulating and SF neutrophils from the RA patients. There was no statistically significant difference in the mean intensity of TK labeling in the circulating neutrophils from healthy volunteers (n=8) and the circulating and synovial fluid neutrophils of the RA patients n=8). However, when the intensity of labeling of the SF neutrophils (n=80) from the RA patients was compared to the circulating neutrophils (n=80) of healthy volunteers, there was a significant loss of TK labeling in the SF neutrophils of the RA patients (1-Way ANOVA, p < 0.01). In the RA patients, there was a loss of the kinin moiety from both the SF and circulating neutrophils compared to controls (Kruskal-Wallis: p < 0.05 and < 0.01 respectively). Although there was a clear increase in the intensity of labeling of the kinin BI receptor on the SF neutrophils from RA patients (n=8), when compared to the circulating neutrophils from healthy volunteers (n=8), the mean values did not reach significance (Kruskal Wallis; p > 0.05). However, a significant increase in Bl receptor labeling was observed on both the circulating and SF neutrophils of the RA patients (n=80) when compared to circulating neutrophils of the healthy volunteers (n=80) (1 Way ANOVA, p < 0.01 and < 0.05 respectively). In addition, there was a positive correlation between the immunoreactivity for the BI kinin receptor on the circulating neutrophils from the RA patients and the local activity index (r = 0.783; p < 0.05). Although there was a clear increase of the kinin B2 receptor on the circulating and SF neutrophils from the RA patients compared to circulating neutrophils from healthy volunteers, this was only significant for the circulating neutrophils from the RA patients (Kruskal-Wallis, p = 0.05). There was no correlation between the intensity of labeling of TK, the kinin moiety and the B2 receptor and measures of disease activity. Discussion and conclusions 1. This study provides the first evidence for the localization of TK and the kinin receptors in control and rheumatoid synovial tissue using antibodies specific for each protein and standard immunolabelling techniques. Synovial fibroblasts, macrophages and endothelial cells through the release of enzymes and cytokines have the ability to mediate the inflammatory changes and cartilage and bone destruction in RA. The presence of TK and the kinin receptors in these cells therefore provides evidence for a pathogenetic role for the kallikrein-kinin cascade in RA. 2. In addition, in RA there is an exudation of fluid into the joint space. Tissue kallikrein has been previously reported in the synovial fluid obtained from RA patients, however the correlation of TK levels and disease activity has not been previously studied. The negative correlation between enzymic TK and the twenty-eight swollen joint count, an indicator of disease activity suggests that there is a consumption of TK in inflammation, presumably due to increased kininogenase activity. Similarly, the kinin generating capacity of synovial fluid obtained from RA patients has been previously reported, however, this is the first study demonstrating a link between kinin generating capacity and validated markers of disease activity. The kinin generating capacity is a complex and dynamic cascade involving the bioregulation of all the components of the kallikrein-kinin system and is therefore more likely to accurately define the role of kinins in inflammatory arthritis than are individual components of the kallikrein-kinin cascade. Measurement of tissue kallikrein and basal kinins is affected by the presence of natural inhibitors and their short half-life in biological fluids. In addition to the synovial fluid study a decrease in the urinary TK activity and an increase in urine kinin generated kinins was demonstrated, suggesting that there is a systemic activation of the kallikrein kinin cascade in RA. 3. Although there is evidence for an interaction between the kallikrein-kinin and cytokine cascades in inflammation, in this study a direct correlation between the levels of interleukin 1 J3 and tumour necrosis factor J3 in the synovial fluid and TK and generated kinin levels was not found. This may be due to the wide variations in the levels of cytokines, the presence of inhibitors and anti-inflammatory cytokines, or a complex and dynamic relationship between the two cascades. However, the correlation of both generated kinins and interleukin l J3 and tumour necrosis factor J3 with disease activity provides circumstantial evidence for a synergistic role for these mediators in inflammatory arthritis. 4. In the neutrophil study, loss of immunoreactive tissue kallkrein and kinin moiety from the neutrophils obtained from RA patients was demonstrated. This finding supports the hypothesis that kinins are released from the neutrophils by the enzymatic action of tissue kallkrein and suggests that the kallkrein-kinin system is activated both locally and systemically in patients with RA. Further, there was upregulation of the both the kinin B 1 and B2 receptors on the neutrophils from the RA patients. While the B2 receptor is thought mediate most of the actions of kinins, the correlation of the intensity of B 1 receptors and the local activity index implies that the B 1 receptor may be important in inflammation. 5. These findings provide convincing evidence for the role of the kallikrein-kinin cascade in the pathogenesis of inflammation in RA. Further development of kinin receptor antagonists may provide a novel therapeutic modality.
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    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.
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    Therapeutic strategy to end Tuberculosis (TB) world: structural and functional characterization of potential weak hotspots of Mycobacterium tuberculosis molecular targets from combinatorial in silico perspective.
    (2021) Adewumi, Adeniyi Thompson.; Soliman, Mahmoud Elsayed Soliman.
    The world has witnessed several decades of Tuberculosis (TB) pandemic and numerous advanced scientific efforts to control the invasiveness of the newly evolving Mycobacterium tuberculosis strains (Mtb) resulting in drug resistance. TB disease has killed hundreds of millions of humans and left millions maimed that need to be rehabilitated; about 10.0 million infections and 1.5 million annually in the last decade. Drug-resistant TB has remained more challenging in the previous 20 years than drug-susceptible TB and is chromosomal mutations-associated in selected genes of the Mtb. Notable mutations identified by biomarkers are related to phenotypic drug resistance, and these include; an 81 bp region in rpoB gene with > 95 % mutations in rifampicin (RIF) clinical isolates and katG gene promoter of the mabA-inhA showed to be associated with INH-resistance. Different Strategies, including the recent WHO End TB approach, have been employed to alleviate or stop TB. The current identification of the critical roles of Mtb demethylmenaquinone methyltransferase (menG) target in the survival, pathogenesis, virulence, and drug resistance created an avenue for the development of efficacious therapeutics that can eradicate TB. MenG is a member of the methyltransferase superfamily. It catalyzes one of the last steps of the menaquinone biosynthesis pathway, requires for maintenance of the Mtb cell envelope. The other two studied targets investigated in this work are N-acetylglucosamine-6-phosphate deacetylase enzyme (NagA), which represents a critical enzymatic step in the production of essential amino sugar required by Mtb for the cell wall biosynthesis and the secreted antigen 85C enzyme (Ag85C) target. The latter target catalyzes the synthesis of trehalose derivatives and attachment of mycolic acids. These targets have gained considerable attention in drug discovery pipelines. However, there is little information about menG, as it lasks structural dynamics due to the lack of crystal structure, active site regions, and amino acids of it Mycobacteria homologs. Similarly, the dynamics of the NagA and Ag85C proteins structure are still unknown. Therefore, justifications led to the modelling of the 3D Structure of menG to understand the structural and functional features that could be investigated at the atomistic level. Homology models were also created for the five (5) mycobacterial homologs. Furthermore, the inevitable need for new drugs has led to the application of in silico techniques including molecular modelling and molecular dynamics simulations, which provide opportunities for the chemists to evaluate and assess numerous compounds that can lead to potential drugs against the mycobacterial disease. Furthermore, these computational techniques justify the present incorporation of several computational tools integrated into this study to provide insights into the conformational changes that illuminate potential inhibitory mechanism, identification of the binding site amino acids, and characterization. Here, we analyze the weak hotspots dynamics specific to each of the Mtb targets, most notably the loop and active residues around or within the ligand-binding sites to obtain useful findings for the design of higher efficacious potential antitubercular drugs. Molecular dynamics simulations were performed to gain molecular standpoints of the conformational binding of the experimental drugs, which were reported to be highly effective against each respective target. Structural dynamics and motions behaviour of menG upon the binding of inhibitor (DG70, biphenyl amide compound) were estimated. Additional in silico thermodynamic analyses were further employed to explore intuitions into the binding mode of each inhibitor mainly for the proposed binding site of menG to identify the residues for binding. Sequence analysis of the homologs of Mycobacterium tuberculosis NagA and Ag85C targets, including those of smegmatis, marinum, leprae, ulcerans, were performed to obtain unique sequence similarities and differences and the structural and functional characterization upon the binding of the ligand. An experimental protocol let to the discovery of a selective covalent inhibitor, β- isomer monocyclic enolphosphorus Cycliphostin, of Ag85C SER-124. Moreover, chapter 4 also unravels the impact of the function of the non-synonymous single nucleotide polymorphisms of NagA target. The desired expectation is that the implementation of the information extricated from this study would provide the structural silhouette for pharmaceutical scientists and molecular biologists to abet in the identification and design of novel antimycobacterial drugs most especially for TB.