The hepatitis C virus (HCV), with a global prevalence of roughly 2%, is among the most serious diseases today. Among the more promising HCV targets is the NS3 protease, for which several drug candidates have entered clinical trials. In this work, computational methods have been developed and applied to the design of inhibitors of the HCV NS3 protease. Comparative molecular field analysis (CoMFA) modeling and molecular docking are the two main computational tools used in this work. CoMFA is currently the most widely used 3D-QSAR method. Methodology for improving its predictive performance by evaluating 6120 combinations of non-default parameters has been developed. This methodology was tested on 9 data sets for various targets and found to consistently provide models of enhanced predictive accuracy. Validation was performed using q2, r2pred and response variable randomization. Molecular docking was used to develop SARs in two series of inhibitors of the HCV NS3 protease. In the first series, preliminary investigations indicated that replacement of P2 proline with phenylglycine would improve potency. Docking suggested that phenylglycine-based inhibitors may participate in two additional interactions but that the larger, more flexible phenylglycine group may result in worse ligand fit, explaining the loss in potency. In the second series, β-amino acids were explored as α-amino acid substitutes. Although β-amino acid substitution may reduce the negative attributes of peptide-like compounds, this study showed that β-amino acid substitution resulted in reduced potency. The P3 position was least sensitive to substitution and the study highlighted the importance of interactions in the oxyanion hole. Finally, docking was used to provide the conformations and alignment necessary for a CoMFA model. This CoMFA model, derived using default settings, had q2 = 0.31 and r2pred = 0.56. Application of the optimization methodology provided a more predictive model with q2 = 0.48 and r2pred = 0.68.

Infection by the hepatitis C virus (HCV) leads to inflammation of the liver, i.e. hepatitis. The acute infection often progresses to a chronic phase during which the liver function is gradually impaired. Approximately 20% of these chronic cases develop liver cirrhosis, with an ensuing increased risk of liver cancer. Global estimates of the total number of chronic cases range from 123–170 million. Yet, neither specific anti-HCV drugs nor vaccines are available. When drugs become available for daily clinical use, rapid development of drug-resistant strains is expected, making resistance an important issue. One of the most studied targets for specific anti-HCV drugs is the NS3 protease. The main objectives of the work presented in this thesis were to design and synthesise peptidomimetic inhibitors of this enzyme, and to establish the structure–activity relationships (SARs) regarding the inhibition of the wild type as well as of the known resistant variants A156T and D168V. Substituted prolines are common P2 residues in HCV NS3 protease inhibitors. To decrease the peptide character of the inhibitors, the non-coded phenylglycine was evaluated as a proline replacement in combination with known and novel P3 and P1 residues and P2 substituents. The results confirmed that phenylglycine is a promising P2 scaffold, with a possible π-stacking interaction with histidine 57 of the active site. However, to benefit from its full potential, additional optimisation is required. A 2(1H)-pyrazinone-based scaffold was introduced as P3 residue. Utilising the scope of the method developed for the pyrazinone scaffold synthesis, the phenylglycine side-chain was transferred to the scaffold. In combination with an aromatic P1 building-block, this design yielded achiral, peptidomimetic inhibitors, three times more potent than the tripeptide lead. The SARs for the inhibition of the resistant variants A156T and D168V were investigated for compounds based on either P2 proline or phenyl­glycine. It was concluded that the vulnerability of the inhibitors to alterations in the enzyme depends on the P2 and the P1 residue, not only on the P2 as previously suggested. These results provide important information for the design of a new generation of inhibitors with improved properties.

This thesis focuses on the design and synthesis of inhibitors targeting Mycobacterium tuberculosis ribonucleotide reductase (RNR) and hepatitis C virus (HCV) NS3 protease; enzymes that have been identified as potential drug targets for the treatment of tuberculosis and hepatitis C, respectively. Small peptides have been recognized as inhibitors of these enzymes. However, the use of peptides as drugs is limited due to their unfavorable properties. These can be circumvented by the development of less peptidic molecules, often referred to as peptidomimetics. When this work was initiated, only a few inhibitors targeting M. tuberculosis RNR had been identified, whereas the HCV NS3 protease was an established drug target. Therefore, early peptidomimetic design strategies were applied to inhibitors of RNR while the NS3 protease inhibitors were subjected to modifications in a later stage of development. It has previously been shown that peptides derived from the C-terminus of the small subunit of M. tuberculosis RNR can compete for binding to the large subunit, and thus inhibit enzyme activity. To investigate the structural requirements of these inhibitors, different series of peptides were evaluated. First, peptides from an N-terminal truncation, an alanine scan and a designed library were synthesized and evaluated to examine the importance of the individual amino acid residues. Then, a set of N-terminally Fmoc-protected peptides was evaluated, and it was found that the N-terminal group improved the affinity of the peptides even when the length of the compounds was reduced. Furthermore, potential inhibitors of less peptidic character were generated by the introduction of a benzodiazepine-based scaffold. To further reduce the peptidic character and investigate the binding properties of HCV NS3 protease inhibitors, a series of tripeptides incorporating a β-amino acid was synthesized. Inhibition was evaluated and docking studies were performed to understand how the structural changes affected inhibitory potency. The results illustrated the importance of preserving the hydrogen bonding network and retaining electrostatic interactions in the oxyanion hole between inhibitor and protein.

<p>To ameliorate Hepatitis C Virus (HCV) therapeutic and diagnostic challenges requires robust intervention strategies, including approaches that leverage the plethora of rich data published in biomedical literature to gain greater understanding of HCV pathobiological mechanisms. The multitudes of metadata originating from HCV clinical trials as well as low and high-throughput experiments embedded in text corpora can be mined as data sources for the implementation of HCV-specific resources. HCV-customized resources may support the generation of worthy and testable hypothesis and reveal potential research clues to augment the pursuit of efficient diagnostic biomarkers and therapeutic targets. This research thesis report the development of two freely available HCV-specific web-based resources: (i) Dragon Exploratory System on Hepatitis C Virus (DESHCV) accessible via http://apps.sanbi.ac.za/DESHCV/ or http://cbrc.kaust.edu.sa/deshcv/ and (ii) Hepatitis C Virus Protein Interaction Database (HCVpro) accessible via&nbsp / http://apps.sanbi.ac.za/hcvpro/ or http://cbrc.kaust.edu.sa/hcvpro/. DESHCV is a text mining system implemented using named concept recognition and cooccurrence based&nbsp / approaches to computationally analyze about 32, 000 HCV related abstracts obtained from PubMed. As part of DESHCV development, the pre-constructed dictionaries of the&nbsp / Dragon Exploratory System (DES) were enriched with HCV biomedical concepts, including HCV proteins, name variants and symbols to enable HCV knowledge specific&nbsp / exploration. The DESHCV query inputs consist of user-defined keywords, phrases and concepts. DESHCV is therefore an information extraction tool that enables users to&nbsp / computationally generate association between concepts and support the prediction of potential hypothesis with diagnostic and therapeutic relevance. Additionally, users can&nbsp / retrieve a list of abstracts containing tagged concepts that can be used to overcome the herculean task of manual biocuration. DESHCV has been used to simulate previously&nbsp / reported thalidomide-chronic hepatitis C hypothesis and also to model a potentially novel thalidomide-amantadine hypothesis. HCVpro is a relational knowledgebase dedicated to housing experimentally detected HCV-HCV and HCV-human protein interaction information obtained from other databases and curated from biomedical journal articles.&nbsp / Additionally, the database contains consolidated biological information consisting of hepatocellular carcinoma (HCC) related genes, comprehensive reviews on HCV biology and drug development, functional genomics and molecular biology data, and cross-referenced links to canonical pathways and other essential biomedical databases. Users can retrieve enriched information including interaction metadata from HCVpro by using protein identifiers, gene chromosomal locations, experiment types used in detecting the interactions, PubMed IDs of journal articles reporting the interactions, annotated protein interaction IDs from external databases, and via &ldquo / string searches&rdquo / . The utility of HCVpro&nbsp / has been demonstrated by harnessing integrated data to suggest putative baseline clues that seem to support current diagnostic exploratory efforts directed towards vimentin.&nbsp / Furthermore, eight genes comprising of ACLY, AZGP1, DDX3X, FGG, H19, SIAH1, SERPING1 and THBS1 have been recommended for possible investigation to evaluate their&nbsp / diagnostic potential. The data archived in HCVpro can be&nbsp / utilized to support protein-protein interaction network-based candidate HCC gene prioritization for possible validation by experimental biologists.&nbsp / </p>

The need to assess human exposures to foodborne and environmental carcinogens, particularly in populations at high risk for cancer and disease, has led to the development of chemical-specific biomarkers. Sensitive biomarkers for aflatoxin and polycyclic aromatic hydrocarbons (PAHs) have been useful in providing information on population exposure and reducing associated public health impacts. Aflatoxins are fungal metabolites found in a variety of foods. Among these toxins, aflatoxin B1 (AFB1) is the most predominant and hepatocarcinogenic. Acutely, AFB1 can cause disease and death, necessitating safe and effective intervention strategies. Inclusion of NovaSil (NS) clay in the diet represents a practical, sustainable approach. NS has been shown to prevent aflatoxicosis in multiple animal species by binding aflatoxins in the gastrointestinal tract, reducing toxin bioavailability. Co-exposure to PAHs, hazardous environmental contaminants, has been shown to increase the risk for hepatocellular carcinoma (HCC). Therefore, objectives of this research were to utilize biomarkers to assess aflatoxin and PAH exposures in susceptible populations in Ghana and the U.S. and to evaluate the safety and efficacy of NS intervention in Ghana (a population at risk for aflatoxicosis). After 3-month intervention with 3.0g NS/day, median aflatoxin M1 (an AFB1 metabolite) was significantly reduced (up to 58 percent) compared to the placebo group. Furthermore, no significant differences were found in levels of nutrient minerals between NS and placebo groups at baseline and 3-months suggesting NS can be used to effectively sorb AFB1 without affecting serum concentrations of important minerals. PAH biomarker results showed participants in Ghana were significantly exposed to high levels of PAHs based on the presence of 1-hydroxypyrene (1-OHP) in the majority of urines (98.9 percent). NS treatment had no effect on 1-OHP levels, further confirming the preferential binding of aflatoxins by NS. U.S. population data from a Hispanic community in Texas with an elevated incidence of HCC demonstrated a lower percentage and level of aflatoxin and PAH biomarkers. Aflatoxin M1 excretion, however, was associated with increased consumption of certain foods prone to aflatoxin contamination; thus, some individuals may be more vulnerable to exposure and associated interactions that increase the risk for HCC (e.g., PAHs or hepatitis infection).

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use against a wide variety of diseases. Alone, this vaccine technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity against the expressed antigen. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. This study evaluated the use of the MEA for the development of DNA vaccines. We assessed the appropriate delivery conditions for gene expression and the development of humoral immunity. We used both B. anthracis and HBV as infectious models for our experiments. Our results indicated that the MEA can enhance gene expression in a mouse model with minimal to no tissue damage. Optimal delivery conditions, based on generation of antibodies, were determined to be 125-175V/cm and 150ms with 200ug and a prime boost protocol administered on Day 0 and 14. Under these conditions, end-point titers of 20,000-25,000 were generated. Neutralizing antibodies were noted in 40-60% of animals. Additionally, we utilized a guinea pig model to assess the translation potential of this electrode. The plasmid encoding HBsAg, pHBsAg, was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in the experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to the injection only groups. Results from both experimental models demonstrate that protective levels of humoral immunity can be generated and that this electrode should translate well to the clinic.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

<p>To ameliorate Hepatitis C Virus (HCV) therapeutic and diagnostic challenges requires robust intervention strategies, including approaches that leverage the plethora of rich data published in biomedical literature to gain greater understanding of HCV pathobiological mechanisms. The multitudes of metadata originating from HCV clinical trials as well as low and high-throughput experiments embedded in text corpora can be mined as data sources for the implementation of HCV-specific resources. HCV-customized resources may support the generation of worthy and testable hypothesis and reveal potential research clues to augment the pursuit of efficient diagnostic biomarkers and therapeutic targets. This research thesis report the development of two freely available HCV-specific web-based resources: (i) Dragon Exploratory System on Hepatitis C Virus (DESHCV) accessible via http://apps.sanbi.ac.za/DESHCV/ or http://cbrc.kaust.edu.sa/deshcv/ and (ii) Hepatitis C Virus Protein Interaction Database (HCVpro) accessible via&nbsp / http://apps.sanbi.ac.za/hcvpro/ or http://cbrc.kaust.edu.sa/hcvpro/. DESHCV is a text mining system implemented using named concept recognition and cooccurrence based&nbsp / approaches to computationally analyze about 32, 000 HCV related abstracts obtained from PubMed. As part of DESHCV development, the pre-constructed dictionaries of the&nbsp / Dragon Exploratory System (DES) were enriched with HCV biomedical concepts, including HCV proteins, name variants and symbols to enable HCV knowledge specific&nbsp / exploration. The DESHCV query inputs consist of user-defined keywords, phrases and concepts. DESHCV is therefore an information extraction tool that enables users to&nbsp / computationally generate association between concepts and support the prediction of potential hypothesis with diagnostic and therapeutic relevance. Additionally, users can&nbsp / retrieve a list of abstracts containing tagged concepts that can be used to overcome the herculean task of manual biocuration. DESHCV has been used to simulate previously&nbsp / reported thalidomide-chronic hepatitis C hypothesis and also to model a potentially novel thalidomide-amantadine hypothesis. HCVpro is a relational knowledgebase dedicated to housing experimentally detected HCV-HCV and HCV-human protein interaction information obtained from other databases and curated from biomedical journal articles.&nbsp / Additionally, the database contains consolidated biological information consisting of hepatocellular carcinoma (HCC) related genes, comprehensive reviews on HCV biology and drug development, functional genomics and molecular biology data, and cross-referenced links to canonical pathways and other essential biomedical databases. Users can retrieve enriched information including interaction metadata from HCVpro by using protein identifiers, gene chromosomal locations, experiment types used in detecting the interactions, PubMed IDs of journal articles reporting the interactions, annotated protein interaction IDs from external databases, and via &ldquo / string searches&rdquo / . The utility of HCVpro&nbsp / has been demonstrated by harnessing integrated data to suggest putative baseline clues that seem to support current diagnostic exploratory efforts directed towards vimentin.&nbsp / Furthermore, eight genes comprising of ACLY, AZGP1, DDX3X, FGG, H19, SIAH1, SERPING1 and THBS1 have been recommended for possible investigation to evaluate their&nbsp / diagnostic potential. The data archived in HCVpro can be&nbsp / utilized to support protein-protein interaction network-based candidate HCC gene prioritization for possible validation by experimental biologists.&nbsp / </p>

Since the first approved hepatitis C virus (HCV) NS3 protease inhibitors in 2011, numerous direct acting antivirals (DAAs) have reached late stages of clinical trials. Today, several combination therapies, based on different DAAs, with or without the need of pegylated interferon-α injection, are available for chronic HCV infections. The chemical foundation of the approved and late-stage HCV NS3 protease inhibitors is markedly similar. This could partly explain the cross-resistance that have emerged under the pressure of NS3 protease inhibitors. The first-generation NS3 protease inhibitors were developed to efficiently inhibit genotype 1 of the virus and were less potent against other genotypes. The main focus in this thesis was to design and synthesize a new class of 2(1H)-pyrazinone based HCV NS3 protease inhibitors, structurally dissimilar to the inhibitors evaluated in clinical trials or approved, potentially with a unique resistance profile and with a broad genotypic coverage. Successive modifications were performed around the pyrazinone core structure to clarify the structure-activity relationship; a P3 urea capping group was found valuable for inhibitory potency, as were elongated R6 residues possibly directed towards the S2 pocket. Dissimilar to previously developed inhibitors, the P1’ aryl acyl sulfonamide was not essential for inhibition as shown by equally good inhibitory potency for P1’ truncated inhibitors. In vitro pharmacokinetic (PK) evaluations disclosed a marked influence from the R6 moiety on the overall drug-properties and biochemical evaluation of the inhibitors against drug resistant enzyme variants showed retained inhibitory potency as compared to the wild-type enzyme. Initial evaluation against genotype 3a displayed micro-molar potencies. Lead optimization, with respect to improved PK properties, were also performed on an advanced class of HCV NS3 protease inhibitors, containing a P2 quinazoline substituent in combination with a macro-cyclic proline urea scaffold with nano-molar cell based activities. Moreover, an efficient Pd-catalyzed C-N urea arylation protocol, enabling high yielding introductions of advanced urea substituents to the C3 position of the pyrazinone, and a Pd-catalyzed carbonylation procedure, to obtain acyl sulfinamides, were developed. These methods can be generally applicable in the synthesis of bioactive compounds containing peptidomimetic scaffolds and carboxylic acid bioisosteres.