Thesis (doctoral)--München, 1982.

Gold compounds have been used for the treatment of rheumatoid arthritis since the mid 20th century as a disease modifying anti-rheumatic drug. Auranofin, an oral anti-rheumatic drug, has been used for many years in the treatment of rheumatoid arthritis (RA). Although the drug has been successful in treating the symptoms of RA, many patients discontinue its use due to severe toxicity over long periods of continued treatment. Since the introduction of auranofin in 1985 there has been no new clinically approved gold drug. Drug discovery research is directing focus on overcoming these toxicity problems. Much of the problems related to the toxicity related to auranofin are due to its lipophilicity. As a result, three compounds (Asa-fin, Mpta-fin and Pta-fin) with varying substituents were synthesised and hence the lipophilic- hydrophilic balance was modulated. All compounds including auranofin were tested against normal cells to determine its toxicity as well as its anti-inflammatory activity. Three novel auranofin derivatives were compared to auranofin with regards to lipophilicity, toxicity and anti-inflammatory properties The lipophilicity of the three compounds were compared to auranofin using the octanol-water partition coefficient method. All the novel compounds showed variable lipophilicity compared to auranofin, with Pta-fin and Mpta-fin being more hydrophilic than auranofin. The cytotoxicity of these novel gold compounds Asa-fin, Mpta-fin and Pta-fin were compared to auranofin using primary porcine hepatocytes and chicken embryo fibroblasts cultures. A metabolic assay based on the reactivity of 3-[4,5-dimethylyhiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) with viable cells was done to measure the effect of the drugs on the growth of cultures. All three novel compounds proved less toxicity at comparable concentrations in primary porcine hepatocytes and in fibroblast proliferation, Asa-fin and Mpta-fin proved less toxic than Auranofin. The Anti-inflammatory activity of the experimental compounds was determined by testing the effects of the experimental compounds on human lymphocyte proliferation. The MTT assay was used to measure the effect of the drugs on the growth of the cell cultures. All three compounds inhibited the proliferation of human lymphocytes with Pta-fin having the least effect. The effect of these drugs was also evaluated on the reactive oxidant production by chemiluminescence and flow cytometry on resting, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) and Phorbol Myristate Acetate (PMA) stimulated human neutrophils. Oxidant production by neutrophils was measured after a 45-minute incubation period with luminol enhanced chemiluminescence. Treatment of neutrophils with auranofin and the three compounds showed that auranofin, Asa-fin and Mpta-fin had a biphasic activity on hydrogen peroxide production with higher concentrations decreasing hydrogen peroxide production, possibly leading to the anti-inflammatory action of these drugs. With Pta-fin no decrease in hydrogen peroxide was observed. Using flow cytometry three dyes specific to different reactive oxygen species were used. 2’, 7’-Dichloroflourescein diacetate (DCFH) is specific for detecting nitric oxide, Dihydrorhodamine 123 (DHR) is specific for detecting hydrogen peroxide and Hydroethidine (HE) is specific for detecting superoxide. Oxidant production was measured after a 30 minute incubation period with the relative dyes on a flow cytometer. Auranofin and Asa-fin decreased hydrogen peroxide and superoxide production. None of the drugs had an effect on nitric oxide production. The expression of the â2-integrin adhesion molecule, CR3, on resting and PMA stimulated neutrophils treated with the experimental compounds was measured by flow cytometry. CR3 expression by neutrophils was measured after 10 minute incubation in the dark with CD11b FITC monoclonal antibody. Treatment of neutrophils with auranofin and the three experimental compounds showed a decrease in CR3 expression on resting and stimulated neutrophils, however the effect was more marked in stimulated neutrophils. The Anti-inflammatory activity of the experimental compounds was determined by testing the effects of the experimental compounds on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX 2) in resting and lipopolysaccharide (LPS) stimulated human monocytes. COX 1 and 2 production was measured by flow cytometry. Treatment of monocytes with the experimental compounds showed a decrease in COX 2 production in stimulated monocytes but an increase in COX 2 production in resting monocytes. No effect on COX 1 production was observed with the experimental compounds. Prostaglandin E2 (PGE2) was measured with a Prostaglandin E2 Enzymeimmunoassay (ELISA) kit on human macrophages. Auranofin, Asa-fin, Mpta-fin and Pta-fin inhibited the production of PGE2. Auranofin and Asa-fin inhibited the PGE2 directly proportional to the drug concentration. The effect of these drugs was also evaluated on various inflammatory cytokines using an inflammatory cytokine kit and measured on a flow cytometer. The cytometric bead array (CBA) human inflammation kit was used to quantitatively measure interlukin-8(IL-8), interlukin-1â (IL-1â), interlukin-6 (IL-6), interlukin-10 (IL-10), tumour necrosis factor alpha (TNFá) and interlukin-12p70 (IL-12p70). Auranofin and Asa-fin decreased IL 10, TNFá, and IL1â in stimulated cells. No effect was observed on IL 8, IL-12p70 and IL 6. With Mpta-fin and Pta-fin, no significant effect was observed in the cytokines tested. Drug toxicity was evaluated in mice using all four compounds in BALB/c inbred mice. Aspartate transaminase (AST), gamma glutamine transferase (GGT), urea and creatine levels were measured in the test mice. The group receiving the highest dose of Asa-fin showed the greatest elevation of AST . The lowest dose of the auranofin treatment group showed the greatest elevation in GGT, however this increase was not seen in the subsequent higher dosing groups. None of the treatment groups indicated an increase in urea levels. Mpta-fin and Pta-fin showed no increase in the liver enzymes or in urea and creatine. The results of this work are indicative that novel gold compounds could play a promising role in anti arthritic applications. Asa-fin exhibited similar anti-inflammatory activity to auranofin but in vivo toxicity was high. Mpta-fin showed slightly inferior anti-inflammatory activity to auranofin but in vivo toxicity profiles were much more promising. Pta-fin showed the least anti-inflammatory activity of the three novel compounds tested with a similar in vivo toxicity profile as Mpta-fin. / Dissertation (MSc)--University of Pretoria, 2009. / Pharmacology / unrestricted

Thesis (Ph. D.) in Chemistry--University of Maine, 2000. / Includes vita. Includes bibliographical references (leaves 145-152).

Thesis (MSc.(Pharmacology)--Faculty of Health Sciences)-University of Pretoria, 2008. / Includes bibliographical references.

Selenoproteins are unique proteins in which selenocysteine is inserted into the polypeptide chain by highly specialized translational machinery. They exist within all three kingdoms of life. The functions of these proteins in biology are still being defined. In particular, the importance of selenoproteins in pathogenic microorganisms has received little attention. We first established that a nosocomial pathogen, Clostridium difficile, utilizes a selenoenzyme dependent pathway for energy metabolism. Following this initial characterization, we demonstrate that this pathway is linked to production of toxins by this organism. Finally, we show that interruption of selenium metabolism is a viable pathway for development of antimicrobials against this, and other selenoprotein dependent pathogens. We investigated whether Stickland reactions (paired amino acid fermentation) might be at the heart of C. difficile's bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases; glycine and D-proline reductase. These selenoenzymes were expressed upon addition of the corresponding Stickland acceptor (glycine, proline or hydroxyproline). Purification of the selenoenzyme D-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys containing) proteins. D-proline reductase utilized only D-proline but not L-hydroxyproline, even in the presence of an expressed and purified proline racemase. The enzyme was found to be independent of divalent cations, and zinc was a potent inhibitor. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of D-proline reductase may differ significantly from similar enzymes from non-pathogenic species. C. difficile pathogenesis is due to the production of toxins, A and B, members of the large clostridial cytotoxin family. Previous studies have shown that toxin production by this organism is influenced by the composition of the growth medium. We examined the impact of Stickland acceptor amino acids (Stickland acceptors; glycine, proline and hydroxyproline) on growth kinetics and yield, protein synthesis, toxin production and gene expression. Although addition of Stickland acceptors moderately increases growth yield and total protein synthesis, there does not appear to be a clear impact on entry into stationary phase. Glycine dramatically increases the amount of toxin released into the growth medium. Conversely, the addition of hydroxyproline suppresses toxin production. We examine possible mechanisms of regulation and demonstrate that CodY, a regulator of toxin gene transcription does not appear to mediate this effect. Given the importance of selenium dependent Stickland reactions to C. difficile growth and toxin production we aimed to examine the efficacy of blocking such pathways as a means of antimicrobial development. Selenide is the only known substrate for selenophosphate synthetase, the first enzyme involved in the specific incorporation of selenium into selenoproteins. We have identified a stable complex formed upon reaction of auranofin (a gold containing drug) with selenide in vitro. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium (75Se) in selenoproteins in both E. coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin-selenide adduct in the culture medium. Addition of selenium in the form of selenite or L-selenocysteine to the growth media significantly reduces the inhibitory action of auranofin on the growth of C. difficile. Based on these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. The antimicrobial potential of blocking selenium metabolism is further demonstrated in the dental pathogen Treponema denticola. We show that auranofin blocks the growth this organism which also participates in Stickland fermentation. In addition, we provide evidence that the antimicrobial action of stannous salts against T. denticola is also mediated through inhibition of the metabolism of selenium. These studies clearly show that, at least in a subset of microbes that use selenium for the synthesis of selenoproteins, the need for this metalloid can be a useful target for future antimicrobial development.

L’équilibre rédox entre les niveaux des espèces réactives de l’oxygène et de l’azote (ROS, RNS) et les espèces antioxydantes cellulaires est déterminant pour le fonctionnement normal de la cellule et sa viabilité. Le déséquilibre redox ou « stress oxydant » peut altérer les voies de signalisation cellulaires et générer des dommages sur les protéines, les lipides et l’ADN des cellules. Il est ainsi associé à de nombreuses pathologies, notamment les cancers. Les cellules cancéreuses présentent une dérégulation redox importante et un stress oxydant basal intrinsèque plus élevé par rapport aux cellules normales. Elles sont donc très dépendantes des systèmes antioxydants pour leur viabilité. Ainsi, l’administration de drogues qui i) génèrent des ROS / RNS additionnelles ou ii) inhibent les systèmes antioxydant cellulaires, permet une cytotoxicité sélective contre les cellules cancéreuses. C’est la base biologique de la « thérapie anticancéreuse basée sur la modulation de l’équilibre redox». Dans ce contexte, nos travaux ont pour but de décrypter les mécanismes redox derrière l’activité anticancéreuse de la vitamine C (VitC) et de l’auranofin (AUF), seuls ou en combinaison, dans le modèle du cancer du sein. La VitC à des concentrations pharmacologiques élevées présente des propriétés pro-oxydantes. Dans cette étude, l’activité anticancéreuse de la VitC contre les lignées du cancer du sein est associée à une génération extracellulaire et intracellulaire de peroxyde d'hydrogène (H₂O₂) accompagnée d'une oxydation intracellulaire du glutathion (GSH). L’approche protéomique «redoxome» a révélé que la VitC induit une altération de l'état rédox d’enzymes antioxydantes clés et d'un certain nombre de protéines contenant des cystéines, impliquées dans les métabolismes de l’ARN et l’ADN et dans les processus énergétiques. La VitC est également responsable d’un retard dans la progression du cycle cellulaire et d’une inhibition de la traduction. Finalement, des analyses bioinformatiques ont montré que les niveaux d'expression de la peroxiredoxine 1 (PRDX1) sont corrélés à la cytotoxicité différentielle de la VitC dans les cellules cancéreuses du sein. L'AUF, un antirhumatismal, est un inhibiteur des thiorédoxines réductases qui a reçu une attention croissante pour son activité anticancéreuse. Nos travaux montrent que l’AUF inhibe également le système antioxydant du GSH et que cette inhibition est primordiale pour son activité anticancéreuse. L’AUF modifie l'état redox de nombreuses protéines impliquées dans la prolifération et le cycle cellulaire, et provoque une déplétion des dNTPs et un arrêt du cycle cellulaire. De façon remarquable, nous avons démontré que la combinaison de l’AUF et de la VitC présente une cytotoxicité accrue, synergique, médiée par H₂O₂ dans les cellules MDA-MB-231 et d'autres lignées cellulaires du cancer du sein sans trop affecter les cellules normales. In vivo, l’efficacité de la combinaison AUF/VitC a été validée sur des xénogreffes de MDA-MB-231 chez les souris sans présenter une toxicité notable, tandis que l'administration de l’AUF ou de la VitC en monothérapie n’inhibe pas la croissance tumorale. Enfin, les analyses protéomiques, bioinformatiques et fonctionnelles ont identifié la prostaglandine réductase 1 (PTGR1) comme biomarqueur prédictif de la réponse des cellules cancéreuses du sein à la combinaison AUF/VitC. En résumé, ces résultats contribuent à une meilleure compréhension des mécanismes anticancéreux de la VitC et de l'AUF, seuls et en combinaison. En particulier, la combinaison de ces deux médicaments disponibles et non toxiques pourrait être efficace contre le cancer du sein triple négatif et potentiellement d'autres cancers présentant des propriétés redox similaires. Ainsi, une évaluation préclinique et clinique de ces traitements ouvrira la voie à des nouvelles thérapies anticancéreuses basées sur la modulation de l’équilibre redox cellulaire. / Reactive oxygen and nitrogen species (ROS, RNS) homeostasis and intracellular reductive/oxidative (redox) dynamics play a key role in regulating cell fate and are critical for normal cellular functions. Oxidative stress via the disruption of redox homeostasis can lead to aberrant cell signaling and toxic oxidative damage of DNA, lipids and proteins, and is therefore associated with human pathologies such as cancers. Cancer cells experience extensive redox deregulation and generally exhibit higher intrinsic basal oxidative stress than normal cells, as a consequence, they are more dependent on their antioxidant systems for survival. Thus, the administration of a drug generating additional ROS / RNS or inhibiting cellular antioxidant systems will exert a selective cytotoxicity towards cancer cells while sparing their normal counterparts. This is the biological basis for « redox-based anticancer therapy ». The work described here aims to investigate the redox-based anticancer activity of vitamin C (VitC) and auranofin (AUF), as single drugs or in combination, in breast cancer model. VitC at high pharmacological concentrations shows pro-oxidant properties. In this study, we showed that VitC anticancer activity against breast cancer cell lines was associated to extracellular and intracellular generation of hydrogen peroxide (H₂O₂), accompanied by the oxidation of intracellular glutathione (GSH). A “redoxome” proteomics approach revealed that VitC induces alterations of the redox state of key antioxidant enzymes and a number of cysteines-containing proteins including many proteins involved in RNA and DNA metabolisms and energetic processes. Cell cycle arrest and translation inhibition are associated with VitC-induced cytotoxicity. Finally, bioinformatics analysis and biological experiments identified that peroxiredoxin 1 (PRDX1) expression levels correlate with VitC differential cytotoxicity in breast cancer cells. AUF, an antirheumatic drug and known inhibitor of thioredoxin reductases, has been repurposed recently as a potent anticancer drug. We showed that AUF acts on both the thioredoxin and GSH systems and its impact on GSH system is essential for its anticancer activity. AUF alters the redox state of a number of nucleic acid-binding proteins involved in cell proliferation, cell division and cell cycle, triggering dNTP depletion and cell cycle arrest. Importantly, we observed that the combination of AUF and VitC reveals a synergetic and H₂O₂-mediated cytotoxicity towards MDA-MB-231 cells and other breast cancer cell lines without much impact on normal cells, thus decreasing the cytotoxic concentrations of AUF or VitC single drug. The anticancer potential of AUF/VitC combinations was validated in vivo on MDA-MB-231 xenografts in mice without notable side effects, while administration of AUF or VitC as a single agent failed to suppress tumor growth. Finally, SILAC proteomics, bioinformatics analysis, and functional experiments linked prostaglandin reductase 1 (PTGR1) expression levels with breast cancer cell response to AUF/VitC combination, thus identifying a potential predictive biomarker. Overall, these results provide new insights into the anticancer mechanisms of VitC and AUF, as single drugs and in combination. In particular, this combination of two non-toxic and commonly available drugs could be efficient against triple-negative breast cancer and potentially other cancers with similar redox properties. Further assessment in preclinical and clinical studies of these drugs and combinations could open new avenues for redox-based anticancer therapy.

Thesis (MSc.(Pharmacology)--Faculty of Health Sciences)-University of Pretoria, 2008.

<p>Over three million infections were reported in the United States of America in 2019. These infections were caused by either antibiotic-resistant pathogens or <i>Clostridioides difficile</i> and resulted in more than 50,000 deaths. Unfortunately, antibacterial agents are rapidly losing their ability to treat infections and the process of discovering new antibiotics is too slow to cope up with bacterial evolution. Repurposing FDA-approved drugs of well-studied safety, pharmacology and pharmacokinetics represents a faster alternative method of antibacterial drug discovery. Repurposing is more successful and less depleting method of drug discovery than classical de novo method in regard to both cost and time. In the following studies, two major pathogens are targeted, vancomycin-resistant <i>Enterococcus</i> (VRE) and <i>C. difficile</i>. Both bacteria are more prevalent in healthcare settings were more vulnerable population of elderly and immunocompromised individuals reside. In addition, healthcare settings are usually associated with higher frequency of receiving antibiotics which in turn, compromises the integrity of normal microbiota responsible for protection against invading pathogens. Furthermore, hospital stays are associated with exposure to bacterial shedding from other patients. Our aim was to identify FDA-approved drugs with novel ability to eradicate these two bacterial pathogens in the gastrointestinal tract (GIT). Notably, the GIT is considered the actual site of infection in case of <i>C. difficile while it is only a transition site for VRE where the bacteria colonize before causing true infections in other tissues. Studies against both bacteria started with an <i>in vitro</i> screening of FDA-approved drugs and clinical molecules to identify potential candidates for further investigation.</i></p><p><i>For VRE, two drugs where identified with potent inhibitory activity and favorable pharmacokinetic profiles, auranofin and ebselen. Auranofin was approved in the 1960s for the treatment of rheumatoid arthritis due to its anti-inflammatory activity. Auranofin was found to exert potent bacteriostatic activity against both vancomycin-sensitive and vancomycin-resistant <i>Enterococcus</i> strains (minimum inhibitory concentration against 90% of the strains, MIC90 = 1 µg/mL). In addition, bacteria could not develop resistant mutants against auranofin upon prolonged exposure. On the other hand, ebselen is an organoselenium compounds currently in clinical trials for several indications. Similarly, ebselen was found to be a potent inhibitor of VRE growth (MIC90 = 2 µg/mL). In addition, ebselen successfully inhibited bacterial biofilm formation and eradicated mature biofilms. In a mouse model of VRE colonization, both drugs inhibited bacterial shedding and reduced bacterial counts in the GIT of the colonized animals.</i></p><p><i>For <i>C. difficile</i>, auranofin was also found to exert potent inhibitory activity against bacterial growth (MIC90 = 2 µg/mL), toxin production and spore formation. Additionally, it was beneficial in protecting colon cells against <i>C. difficile</i> toxin-induced inflammation. Further, auranofin was found to not promote growth of VRE as seen with the current anticlostridial agents. In addition to auranofin, two more antiprotozoal drugs were found to potently inhibit <i>C. difficile</i> growth, ronidazole and secnidazole. Both drugs are 5-nitroimidazoles approved for human (secnidazole) or veterinary (ronidazole) applications. Secnidazole and ronidazole halted <i>C. difficile</i> growth at very low concentrations (MIC90 = 0.5 and 0.125 µg/mL, respectively). Furthermore, both drugs were superior to metronidazole in bacterial killing and had favorable activities against protective gut microbiota. In addition, they demonstrated efficient protection to mice in a <i>C. difficile</i> infection model. </i></p><p><i>Overall, several drugs were presented to possess favorable activities against <i>C. difficile</i> or VRE. These drugs merit more evaluation as potential candidates for the treatment of infection caused by either bacteria. </i></p><div><i><br></i></div>