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Development of new antibiotics against Bacillus anthracis
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Resumo(s)
The rise of bacterial resistance has driven the search for new antimicrobials employing not only innovative mechanisms of action, but also new characteristics such as high selectivity or multitargeting capabilities, whilst the search for new biological targets continues. Past work demonstrated the potential of carbohydrate chemistry, with a new family of surface-active deoxy sugars showing promising antimicrobial activity and surprising selectivity towards the Bacillus genus. These characteristics, and the absence of correlation between MIC and CMC values for the most active compound, motivated the creation of a small library of compounds with the optimal structural features for these alkyl deoxy glycosides. Simultaneously, the referred antimicrobial and surfactant characteristics showed remarkable differences when compared to the literature of similar alkyl glycosides, hence the study of a possible mechanism of action, independent of the expected surfactant effect was undertaken. The synthesis of antimicrobial derivatives was target oriented, and the methodology applied successfully provided the desired analogs in good yields. The evaluation of antimicrobial activity allowed to establish the essential structural features of this alkyl deoxy glycoside family, leading to dodecyl 2,6-dideoxy-α-L-arabino-hexopyranoside (1) as the ideal compound for the biological studies. This compound showed to be ineffective in the disinfection of surfaces and lack sporicidal activity, however, bacterial mutants resistant to the main families of antibiotics revealed unaltered susceptibility (MIC=16 μg/mL), and no resistance development was observed after continuous exposure. The study of the mode of action was initiated by kinetic characterization of the bactericidal action, followed by potential target assessment by measuring the differential metabolic response when exposed to compound 1, defining the main metabolic pathways affected. By establishing a hierarchical relationship model between them, the most probable targets were identified: ABC and PTS transport systems and the amino acyl t-RNA biosynthesis. The creation of mutant libraries helped to understand that the existence of a specific molecular target is very unlikely, since no significant alteration of the susceptibility of all mutant libraries to compound 1 was observed. Hence, the importance of the bacterial ultrastructures to the antibacterial activity was assessed and the susceptibility of protoplasts and spheroplasts revealed that, for Gram-negative bacteria, the ultrastructures enveloping the cytoplasmic membrane are responsible for the resistance to this compound, while in Gram-positive bacteria they have no effect. This also means that the specificity observed to Bacillus spp., here exemplified S. aureus, results from the characteristics of the bacterial membrane. The impact of compound 1 in the cellular envelope was achieved using AFM techniques, unequivocally demonstrating that these compounds cause alterations of cell surface morphology and cellular damage, even at sub inhibitory concentrations. Additionally, an opportunity emerged to apply this expertise in carbohydrate synthesis to develop new tools for early Alzheimer’s disease diagnosis and possible therapeutics, aiming to improve solubility and bioavailability of known bioactive molecules while providing additional selectivity. A simple, low cost synthetic strategy was paramount to the challenge proposed: the glucoconjugation of fluorescein, quinolone, curcumin and cyclohexylpiperazine derivatives. The methodologies chosen resulted in the successful glucosilation, particularly for the latter. In summary, the multidisciplinary work here reported denotes the capabilities and importance of carbohydrate chemistry for all life sciences. Allowed to successfully establish a correlation between the structure and the bioactivity of alkyl deoxy glycosides, fully characterized the exhibited bactericidal action and provided essential information regarding the peculiarities of the mechanism of action, to be used as guidelines for future work. Moreover, it demonstrated potential in assisting the development of new tools against AD.
Descrição
Tese de doutoramento, Química (Química Orgânica), Universidade de Lisboa, Faculdade de Ciências, 2018
Palavras-chave
Teses de doutoramento - 2018