Actuellement le criblage en vue de la recherche de nouveaux agents antitumoraux se base principalement sur la qualité cytotoxique d’une molécule. Le principal défaut de cette approche est qu’aucune sélection n’est faite sur le mode d’action du médicament. L’objectif de ce travail est la mise au point d’une méthode permettant un classement rapide et objectif du mode d’action de molécules à visée thérapeutique par spectroscopie infrarouge. La spectroscopie infrarouge est une technique d’absorption de la lumière fournit la signature chimique d’un échantillon. L’excellente qualité du signal rend possible son utilisation comme outil discriminant. En outre, cette technique d’analyse se démarque des autres par son caractère non destructif et la rapidité d’acquisition des données. Elle se révèlerait donc une méthode de choix pour effectuer du criblage de molécules en vue de la recherche de nouveaux agents thérapeutiques. Dans un premier temps nous avons voulu évaluer la possibilité d’utiliser la spectroscopie infrarouge pour isoler la signature spectrale du mode d’action induit par des concentrations sub-létales de ouabaïne, un composé de la famille des cardénolides, sur une lignée tumorale de prostate. Nous avons montré que cette signature évolue au cours du temps et peut-être corrélée aux données biologiques décrites dans la littérature. Nous avons également mis en évidence pour la première fois une modification de la composition lipidique de la cellule. Cette altération a été caractérisée au cours du temps par spectrométrie de masse. Nous avons ensuite voulu définir les limites de la méthode. La littérature souligne la diversité des modes d’action que peut induire un agent thérapeutique selon sa concentration. Nous avons montré que cette diversité se reflète sur le spectre infrarouge de cellules tumorales traitées à la ouabaïne en distinguant au moins deux modes d’action distincts, dépendant de la concentration en ouabaïne. Par ailleurs, nous avons montré que la confluence pouvait modifier significativement le spectre infrarouge d’une cellule. Neanmoins cette signature est unique et orthogonale à celle induite par la ouabaïne. Finalement, nous avons évalué le potentiel de la spectroscopie infrarouge à distinguer des modes d’action induits par des molécules à la structure chimique proche. Nous avons montré qu’il était possible de caractériser spécifiquement chacun des modes d’action. D’autre part nous avons mis en évidence que les modes d’action de molécules issues d’une même classe d’agent thérapeutique conduisaient à des signatures spectrales similaires. Cette partie du travail souligne la possibilité d’utilisation de la spectroscopie infrarouge pour un classement objectif, uniquement basé sur leur mode d’action d’agents thérapeutiques potentiels.

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Human adenovirus (HAdV) mainly causes minor illnesses, but can lead to severe disease and death in both immunocompromised and immunocompetent patients. In such cases, the current standards of treatment often do not improve disease outcome and no approved antiviral therapy against HAdV exists. Since HAdV relies on cellular machinery to assist in the progression of the virus lifecycle, we hypothesized that small molecules targeting certain cellular proteins/pathways, without severely affecting cell health, may serve as effective anti-HAdV compounds. Thus, we aimed to identify novel inhibitors of HAdV, and investigate the molecular mechanism to determine new therapeutic targets for intervention in HAdV infection. We first examined the antiviral properties of pan-histone deacetylase (HDAC) inhibitor SAHA and found that the drug affects multiple stages of the HAdV lifecycle, resulting in significant reductions in virus yield. SAHA was effective in decreasing gene expression from clinically relevant HAdV serotypes. Subsequent investigations on the role of HDACs in HAdV infection led us to determine that class I HDAC activity, mainly HDAC2, is necessary for optimal viral gene expression. Using a wildtype-like HAdV reporter construct that allows us to monitor virus replication by fluorescence microscopy, we then designed an efficient system for screening small molecules to identify novel HAdV inhibitors. We screened over 1300 small molecules, and the screen was sensitive enough to detect compounds with both robust and modest antiviral activity. Several positive hits were validated to reduce HAdV gene expression and yield from infected cells. Further investigation on the efficacy of these compounds and the mechanism behind their inhibition of HAdV can lead to the discovery of new pharmacological targets and the development of more effective antivirals.

Introduction: Hypertension is an important public health challenge worldwide, being the leading cause of cardiovascular disease, morbidity and mortality. It is particularly prevalent in people in sub-Saharan Africa, especially in urban areas. There is an urgent need to develop strategies to prevent, detect, treat, and control hypertension effectively in the African region. Helicobacter pylori, a gram-negative bacterium responsible for many gastric disorders worldwide, has been associated with hypertension in some previous studies; where blood pressure of patients with Helicobacter pylori infection did not subside after hypertensive treatment, when compared to patients without Helicobacter pylori infections. This effect was suggested to be due to Helicobacter pylori produced and modified cardiotonic steroids that are found in elevated concentrations in hypertensive patients. Cardiotonic steroids are positive inotropic agents which are known to increase blood pressure. A sensitive analytical method is needed to detect and quantify the low concentrations of cardiotonic steroids in biological samples. Materials and Methods: An extraction method was optimised using reversed phase Solid Phase Extraction. A targeted liquid chromatography tandem mass spectrometry method using an Agilent binary series 1100/1200 LC system with a Kinetex C18 RP column (100 x 2.1 mm, 2.6 µm) coupled to a Sciex 4000QTRAP tandem mass spectrometer was developed and validated for the detection and quantitation of 9 different cardiotonic steroids in both solvent and whole blood. The method was validated according to the International Conference on Harmonization guidelines with regards to precision, accuracy, sensitivity, selectivity, linearity, range, limit of detection, limit of quantification, reproducibility, recovery, carry-over and stability. Media from Helicobacter pylori cultures and faecal samples from human and different normo- and hypertensive rat strains were analysed. Data analysis was performed with Analyst® Software (version 1.5.2) and multiple t-test and Kruskal Wallis test using GraphPad Prism 8 software. Results and Discussion: The calibration curves of tested cardiotonic steroids were linear over a concentration range of 0.1-40 ng/mL with coefficients of determination greater than 0.990 except for telocinobufagin. The analytical method was selective with an estimated limit of detection and limit of quantification between 0.02-0.5 ng/mL and 0.1-2 ng/mL respectively. All tested cardiotonic steroids showed good recovery of over 70%. Accuracy and precision were found to be within acceptable limits of 15% and 20% at lowest limit of quantification for almost all the analytes and their stability in blood and solvent at room temperature, 4°C, -20°C and -80°C was tested for a month. Cardiotonic steroids were detected in Helicobacter pylori cultures and faecal samples with the exception of ouabain and proscillaridin A which were not detected at all. Although Helicobacter pylori were shown to produce cardiotonic steroids in vitro, no evidence of the effect of Helicobacter pylori on cardiotonic steroids production was detected in different normo- and hypertensive rat groups. Conclusion: The quantitative analytical method was successfully validated, over expected in vivo concentration ranges for 8 different cardiotonic steroids. The extraction and analytical methods were both successfully applied to Helicobacter pylori cultures and faecal rat samples where cardiotonic steroids were detected. / Dissertation (MSc)--University of Pretoria 2020. / National Research Foundation Student bursary / Pharmacology / MSc (Pharmacology) / Unrestricted

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