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In vitro biological investigation of novel anti-tubercular compound classes and the development of improved research tools

Book - Dissertation

Despite advances in modern medicine and healthcare, tuberculosis (TB) still poses a major threat worldwide. With approximately 10.0 million new cases and the attributable cause of death of an estimated 1.3 million people, TB is part of the top ten leading causes of death worldwide and has been declared the world’s deadliest infectious disease for several years. In addition, the ever-increasing emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains places an enormous strain on current anti-TB chemotherapy. Accordingly, the World Health Organization (WHO) has set up several strategies to improve TB management, in which the discovery and development of novel anti-TB drugs is key. Although the pipeline for novel anti-TB drugs is steadily filling up with novel candidates, the current arsenal is still limted and likely not sufficient due to high attrition rates. Therefore, there is still an urgent need for new anti-TB compounds. To support and broaden the early discovery phase with knowledge on novel compound classes, the present thesis primarily reports on the in vitro biological evaluation of a series of triazenes, quinones and thiazolidinedione-hydroxamates. The studied compounds were synthesized by chemist of the University of Antwerp (Belgium), University of Ghent (Belgium), University of Ljubljana (Slovenia) and Palacký University (Czech Republik). To evaluate the small compound libraries for antimycobacterial hits and provide a primary evaluation of their in vitro anti-TB properties at the Laboratory for Microbiology, Parasitology and Hygiene (LMPH), an anti-TB drug screening platform was set up. Using this cascade of state-of-the-art assays, several triazene and quinone hits were described in terms of extra- and intracellular antimycobacterial activity, acute cytotoxicity, early genotoxicity and metabolic stability. In addition to the phenotypically identified hits, a small library of target-based synthesized thiazolidinedione-hydroxamates was evaluated. In vitro anti-TB properties of the discovered hits were described and compared to their activity results in an enzymatic assay, which was performed by the Palacký University (Czech Republik). Secondly, the present thesis reports on the improvement of compound assessment methodologies and development of new research tools as these are equally important as the discovery and development of novel anti-TB drugs. In an effort to improve the standard biosafety level two (BSL2) TB infection model, calcium-alginate encapsulation of Mtb H37Ra was envisaged. Although micro-bead encapsulation was successful, the in vivo course of infection was not enhanced during both the acute and chronic phase of infection. Moreover, the generation of inflammation during infection was not influenced as well. Though, experiments confirmed the disputed statement that Mtb H37Ra is attenuated in mice, rather than avirulent. Further, a next-generation bioluminescent Mtb H37Ra reporter model was developed to facilitate future in vitro and in vivo anti-TB drug screening at the LMPH. Upon optimization of the bioluminescent reaction, the selected reporter was determined to be a sensitive and robust alternative for the prokaryotic luciferase reporter strain currently used in the LMPH’s in vitro anti-TB screening platform. Finally, proof-of-concept studies indicated the presence of a bioluminescent signal in living BALB/c mice, illustrating the novel bioluminescent reporter strain might be used to develop a bioluminescent imaging model (BLI). This would in turn facilitate in vivo anti-TB drug screening at the LMPH.
Number of pages: 286
ISBN:978-90-5728-631-5
Publication year:2019
Keywords:Doctoral thesis
Accessibility:Open