Doctoral Degrees (Pharmaceutical Sciences)

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Designed, synthesis and antibacterial evolution of piperazine hybrids.
(2023) Girase, Pankaj Sanjay.; Karpoormath, Rajshekhar.
Piperazine is a kind of azacycloalkane that has two nitrogen atoms at1-4 places on a six-membered ring. It is well known that molecules with the piperazine ring, a key component of the N-heterocyclicfamily of bioactive natural products, are often prevalent in biologically active substances. There are many antitumor, antibacterial, antiinflammatory, antipsychotic, antifungal, and anti-diabetic compounds based on the piperazine scaffold, which has been recognized as an active structure in drug discovery. Piperazine hybrids with different moieties such as isoniazid, coumarin, benzothiazinones, isoquinoline, triazole,pyrrole, and oxazolidinone showed good activity against mycobacterium tuberculosis and microbial strains. In this thesis we have demonstrated the synthesis of piperazine hybrid with hydrazides, hydrazines, and coumarines and tested their activity against mycobacterium tuberculosis, gram positive and gram negative microbial strains. In Chapter 2, we have covered topics related to the analogues of piperazine that have anti-tubercular efficacy. This chapter we have published as a review article in European Journal of Medicinal Chemistry. In this review, we have made a concerted effort to trace the development of anti-mycobacterial compounds during the past 50 years (1971-2019), focusing on instances where piperazine has been utilized as a key building block. In depth discussion of the design, rationale, and structure-activity relationship (SAR) of the reported potent piperazine-based anti-TB molecules will help medicinal chemists fill in the blanks, capitalize on the reported strategies, and create more effective, selective, and less hazardous anti-mycobacterial drugs. In chapter 3, we have developed and synthesized a new class of hybrids between phenylpiperazine and hydrazides (c1-c15). During the synthesis of phenyl piperazines, the formylation of piperazine was observed, a phenomenon on which we have developed a different methodology discussed in chapter 5. All of the derivatives have been tested in vitro against H37Rv, a strain of mycobacterium. In addition, we have analysed the zone of inhibition against eight different bacterial strains, including both gram-positive (methicillin resistant staphylococcus aureus (MRSA), Streptococcus pyrogens, Bacillus subtilis, Enterococcus faecium, and Staphylococcus aureus), and gram-negative (Enterobacter hormaechei, Pseudomonas aeruginosa, and Escherichia coli) bacteria. Among the derivatives tested, only compound c8 showed action against the mycobacterium strain H37Rv (MIC value of 0.39-0.78 g/ml). No zone of inhibition was seen for any of the microbiological strains when exposed to any of the synthesized compounds. The hybrids between phenylpiperazine sulphonamide and phenyl hydrazide (E1-E6) and phenylpiperazine sulphonamide and phenyl hydrazine (F7-F19) were proposed and synthesized in chapter 4. All substances were evaluated against mycobacterium tuberculosis, five gram-positive and three gram-negative bacterial strains in vitro. Derivatives E1 and E2 with an isoniazid moiety were the most effective in inhibiting the growth of the H37Rv strain of tuberculosis, with an IC50 value of 3.125 M. Of the derivatives tested, F10 showed significant action against the gram-positive bacteria Enterococcus faecium (7.81 μg/mL), whereas the others (E2, E6, F7, F9, F14) were only moderately active (250-62.5 μg/mL). Using a the molecular hybridization strategy, we were enabled to create novel analogues of coumarin-(phenylsulfonyl)piperazine and 4-methyl coumarin-(phenylsulfonyl)piperazine in chapter 5.All synthesised compounds were evaluated for their in vitro anti-mycobacterial and antimicrobial activity against H37Rvand a variety of antimicrobial gram-positive and gram-negative strains.The Compounds 6G, 6H, 10D and 10E displayed moderate inhibition against gram positive and gram negative strains with MIC values in the range of 62.5-250 (table 1) against MRSA, Bacillus subtilis, and Enterococcus faecium, and gram negative strains Enterobacter hormaechei, Pseudomonas aeruginosa, and Escherichia coli.In addition, the Structure-Activity Relationship (SAR) analysis showed that phenyl ring substituents could enhance antibacterial activity. Chapter 6 came from the process of synthesizing phenyl piperazine in chapter 1. This chapter disclosed a method for efficient synthesis of transamidation in the presence of Iodine and NH2OH.HCl which published in Chemistry Select. This method is efficient for a broad range of primary, secondary, and tertiary amides, and it enables the formylation, acylation, and benzoylation of a number of different amines. The key benefits of the present technique are that it is easy to follow, quick, does not need a metal catalyst, uses a starting material that is inexpensive, and has a low effect on the environment when the synthesis process is carried out. All of the chapters in this thesis are written in thesis by publication style, rather than the conventional style.
Molecular characterization of multi-drug resistant (MDR) gram-negative bacterial pathogens from environments, patients and staff in a teaching hospital in Ghana.
(2023) Yeboah, Esther Eyram Asare.; Essack, Sabiha Yusuf.; Owusu-Ofori, Alexander.; Agyepong, Nicholas.; Abia, Akebe Luther King.; Amoako, Daniel Gyamfi.; Mbanga, Joshua.
Multidrug resistant Gram-negative bacteria (MDR GNB) are implicated in serious infections both of community and nosocomial origin and may be disseminated in the hospital in the absence of efficient infection prevention and control (IPC) practices. The prevalence and risk factors for rectal colonization of MDR GNB among patients, the carriage of MDR GNB on healthcare workers’ (HCWs’) hands and the contamination patients’ environments with MDR GNB were investigated in a teaching hospital in Ghana. In this prospective study, conducted between April 2021 to July 2021, the phenotypic profiles of the MDR GNB isolates were determined using the VITEK 2 system. Risk factors for colonization with MDR GNB were assessed using univariate and multivariate analysis of associated data. The resistome, virulome, mobilome and genetic relatedness of MDR extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and ESBL-producing or carbapenem resistant Klebsiella pneumoniae isolates from patients and their environment were also determined using whole genome sequencing performed on the Nextseq 550 (2 x 150 bp) and bioinformatics analysis. A total of 585 samples were collected from patients, HCWs’ hands and the hospital environment within the study period. The prevalence of MDR GNB rectal colonization among patients was 50.62% on admission and 44.44% after 48 hours. MDR GNB, frequently E. coli and K. pneumoniae were isolated from 6 (5.26%) and 24 (11.54%) of HCW’s hand swabs and environmental swabs, respectively. Previous hospitalization (p-value = 0.021, OR,95% CI= 7.170 (1.345-38.214) was significantly associated with colonization by MDR GNB after 48 hours of admission while age (21-30 years) (p-value =0.022, OR, 95% CI =0.103(0.015-0.716) was significantly identified as a protective factor associated with a reduced risk of rectal MDR GNB colonization. Rectal carriage and acquisition of ESBL-producing E. coli among patients was 13.65% and 11.32% respectively. blaTEM-1B and blaCTX-M-15 were commonly associated with IncFIB plasmid replicons and co-occurred with aminoglycoside, macrolide, and sulfamethoxazole/trimethoprim resistance. Multiple virulence genes, predominantly, terC were detected in the ESBL E. coli isolates. Sequence types (STs) were diverse and included one novel ST (ST13846) present in two isolates. Phylogenetic analysis grouped the ESBL E. coli isolates into four main clusters. High genetic relatedness was observed between two carriage isolates of ST940 and between a carriage isolate and an environmental isolate of ST648. Isolates with different STs, collected at different times and locations, also showed genetic similarities. Of the ten selected MDR K. pneumoniae isolates, the β-lactamase gene, blaCTX-M-15 was observed in six isolates. Mutations were found in both ompK36 and ompK37 in all isolates (both carriage isolates and isolates from hospital environments). Genes encoding resistance to fluoroquinolone (qnrB), aminoglycosides (aadA1, aadA2, aac(3)-IIa, aac(6')-Ib-cr,aph(3'')-Ib , aph(6)-Id) sulphamethoxazole/trimethoprim (sul1, sul2, dfrA14, dfrA15) were also detected. The K. pneumoniae isolates belonged to seventeen different STs with ST39 most commonly observed and common to both carriage isolates and isolates from hospital environments. A myriad of virulence genes, including irp1, irp2, iutA, gndA, ompA, fes, fep, mrkD and fimH were detected in both carriage and isolates from the hospital environment. IncFIB was the most abundant plasmid replicon occurring in nine (four carriage isolates and five isolates from hospital environments). ESBL-producing K. pneumoniae isolates appeared to be introduced into the hospital from the community. The high colonization of MDR GNB in patients, the carriage of MDR GNB on HCW’s hands, the contamination of hospital environments and the circulation of ESBL-producing E. coli and K. pneumoniae isolates with diverse genomic characteristics, highlights the need for patient screening, and stringent infection prevention and control practices to prevent the spread of MDR GNB in hospitals. The observed clonal relatedness among isolates from patients and the hospital environment, as well as between different patients, suggests a possible transmission within and between sources, hence infection prevention and control practices need to be enhanced to prevent the dissemination and transmission of these resistant strains in the hospital. This study further highlights the usefulness of whole genome sequencing as an effective tool in AMR surveillance.
Design of advanced multifunctional biomaterial-based biomimetic and pH-responsive hybrid nanocarriers for antibiotic delivery against bacterial infections and sepsis.
(2023) Elhassan, Eman Hussain Elmubarak.; Govender, Thirumala.; Omolo, Calvin Andeve.
Despite the notable improvements in the management of bacterial infections and sepsis, the mounting threat of antibiotic resistance on a global scale is leading towards a post-antibiotic era. Nano-drug delivery systems have improved the delivery and efficacy of various antibiotics. Biomimicry and stimuli-responsiveness have recently been used to improve the targetability of these nanocarriers, and enhance their localization at infected sites, thus improving overall therapeutic outcomes and reducing toxicity. Strategies such as targeting bacterial biofilms and efflux pumps can further enhance the delivery and effectiveness of antibiotics. Developing smart biomaterials with multifunctional properties to confer biomimetic, stimuli-responsive and antivirulence properties to antibiotic nanocarriers is the focus of ongoing research. Therefore, the general aim of this study was to investigate the potential of various novel multifunctional biomaterial-based hybrid nanocarriers (HNs), including biomimetic and/or pH-responsive HNs in enhancing the targeted delivery of antibiotics and modulating the proinflammatory response against bacterial infections and sepsis. In this study, two biomaterials with multifunctional activities, hyaluronic acid-lysine conjugate (HA-Lys) and tannic acid (TA), were employed to design, formulate, and extensively characterize innovative biomimetic and pH-responsive HNs for efficient and targeted delivery of antibiotics. The novel HA-Lys was synthesized and fully characterized using proton nuclear magnetic resonance (1H NMR) spectroscopy and Fourier-transform infrared spectroscopy (FT-IR). Then it was successfully employed with tocopherol succinate (TS) and Oleylamine (OLA) to fabricate biomimetic pH-responsive vancomycin-loaded hybrid nanostructured lipid carriers (VCM-HNLCs). The prepared VCM-HNLCs were spherical and had average diameters, zeta potential, polydispersity index, drug encapsulation efficiency and loading capacity of 110.77  1.69 nm, 0.11  0.02, -2.92  0.21 mV, 76.27  1.20 % and 8.36  0.25 %, respectively. Both HA-Lys conjugate and its respective nanoformulations had excellent biosafety profiles (>70 % cell viability and ˂ 1 % hemolytic effect). Possible VCM-HNLCs competitive inhibition activity to toll-like receptors 2 and 4 (TLR2 and TLR4) was demonstrated via microscale thermophoresis (MST) analysis, which showed a 5-times and 16-times lower Kd values than their natural substrates peptidoglycan (PGN) and lipopolysaccharide (LPS), respectively. VCM-HNLCs exhibited a pH-responsive drug release profile under acidic conditions, higher bacterial killing kinetics, enhanced antibacterial, anti-biofilm, and efflux pump inhibition activities over bare VCM. Also, they showed an improved activity in neutralizing reactive oxygen species (ROS) and modulating the inflammatory response induced by LPS. On the other hand, tannic acid (TA) and Oleylamine (OLA) were successfully employed to formulate biomimetic ciprofloxacin-loaded tannic acid hybrid nanoparticles (CIP-loaded TAH-NPs) to enhance the efficacy of CIP against bacterial infections and sepsis. The prepared HNs had onion-shaped morphology, with average diameters, zeta potential, polydispersity index, drug encapsulation efficiency and loading capacity of 85.65 ± 0.89 nm, 0.126 ± 0.01, +16.3 ± 0.23 mV, 68.73 ± 0.54 % and 6.86 ± 0.09 %, respectively. The hemolysis and MTT assays confirmed the biosafety and non-hemolytic activity of CIP-loaded TAH-NPs formulations (>70 % cell viability and ˂ 1 % hemolytic effect). The results of MST investigations and in-silico simulations demonstrated that TA and its nanoformulation (CIP-loaded TAH-NPs) competitively inhibited TLR4 compared to its natural substrate LPS. CIP-loaded TAH-NPs showed a diffusion-based sustained release profile at physiological pH 7.4. Also, in comparison to bare CIP, the hybrid nanovesicles demonstrated improved antibacterial, anti-biofilm and efflux pump inhibition properties, as well as faster bacterial killing kinetics. Moreover, they showed a significant neutralization of ROS and the ability to control the inflammatory responses brought on by LPS. In summary, VCM-HNLCs and CIP-loaded TAH-NPs were successfully formulated and showed significant improvement in antibiotics efficacy and overall therapeutic outcomes. This study confirmed the potential of biomimetic stimuli-responsive antibiotic hybrid nanocarriers for enhancing antibiotic efficacy against bacterial sepsis and addressing the antimicrobial resistance crisis. The data from this study has resulted in one first-authored review article, two first-authored research publications and one co-authored review article.
Dynamics of drug resistance in environmental bacteria within an aquatic ecosystem.
(2023) Chukwu, Kelechi Benedict.; Akebe, Luther King Abia.; Essack, Sabiha Yusuf.
Recently there has been a rapid increase in the incidence and prevalence of drug-resistant bacteria and antimicrobial resistance genes in the environment, largely attributed to selection pressure from the environmental presence of antimicrobials such as antibiotics, biocides, and heavy metals, as well as other physicochemical stressors. such as Poly aromatic hydrocarbons, pH, temperature, and reactive oxygen. However, the concentrations at which these antimicrobials could elicit resistance are poorly understood. Such lack of information could hamper the development of standards for the environmental surveillance of antmicrobials with potential adverse effects on human, animal and environmental health. In this study, Water samples were collected from all the points that impact the environment directly around the Darvill wastewater treatment plant, namely the treatment plant effluent discharge point, the upstream and downstream from the effluent discharge point. Antibiotics, heavy metals, and biocides were identified and quantified from the water samples, and we ascertained the effect of environmental concentrations of some of these selected stressors on the antibiotic resistance in previously susceptible Escherichia coli. Heavy metals concentrations were determined using the United States Environmental Protection Agency (US EPA) method 200.7. Biocide and antibiotic residue concentrations were determined using validated ultra-high-performance liquid chromatography with tandem mass spectrometry-based methods. E. coli was identified and quantified using the Colilert-18TM system from IDEXX, while antimicrobial susceptibility was performed using the disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines. The concentration of antibiotics observed ranged from sulfamethoxazole (286.180 μg/L) to penicillin (2.2 μg/L); for metals, sodium (27.734 mg/L) to iron (0.001 mg/L); and for biocides, benzalkonium chloride (BAC) 12 (7.805 μg/L) to BenthEZ (0.035 μg/L). There was observed increase in the pollutant concentrations in the effluent and downstream samples compared to the upstream samples, suggesting that the WWTP might be a potential source of interest, indicating that these pollutants, were not completely removed at the WWTP. Thirty days' exposure of wholly susceptible E. coli ATCC 25922 strains, to environmental and sub-inhibitory concentrations of oxytetracycline, amoxicillin, zinc, copper, BAC 12 and DADMAC 10 was conducted but could not trigger phenotypic resistance. Genotypic analysis of the WGS on the exposed isolates, found only the macrolide resistance mdf (A) gene (which was also present in the control) and the disinfectant resistance gene sitABCD. With further analysis for single nucleotide variants (SNV), mutations were detected for 19 genes compared to the control. Only one resistance gene was detected, robA, a member of the ArcC/XylS family, that regulates the ArcAB-TolC multi-drug efflux, that contributes to multi-drug resistance. The other 18 genes we detected were tolerance conferring genes, acnB, cusA, degQ, epmA, hsmP, mlc, purH, queG, srlE, tsaB, yddh and yqhH genes, in all the exposed isolates. filA genes in only the oxytetracycline and BAC 12-exposed isolates, mutM gene in zinc exposed isolates, nudK gene in all the exposed isolates except the DADMAC 10 exposed isolates, ptsG gene in only the oxytetracycline-exposed isolates, and ompD in only DADMAC 12-exposed isolates. All the genes detected in the exposed isolates were also detected in the environmental isolates, except the robA gene. These genes detected encode for oxidative stress, DNA repair, membrane proteins efflux systems, growth and persister formations. In addition, we observed that the 30-days exposed isolates developed increased tolerance to high (25 x MIC) concentrations of ampicillin by 30 to 50% when compared to unexposed control. BAC 12-exposed isolates had the highest tolerance increase. The increased tolerance seems to emanate from multi gene induced persister cells formations, as well as tolerance gene expressions. The MSW of the exposed isolates to ampicillin and amoxicillin, also slightly increased compared to the control indicating the amplification of persister cells during the 30-day exposure but the MSW remained same to oxytetracycline. This indicates that exposure to sub-inhibitory concentrations of antibiotics, heavy metals and biocide residues, as observed in the aquatic environment, cannot induce phenotypic resistance but can encode for genes responsible for the development of persistence and tolerance in bacteria, which seems to be the pathway towards eventual antimicrobial resistance in environmental bacteria.
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.
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/kg.bw 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.
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.
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.
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.
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:
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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