Actuellement, le traitement des pathologies infectieuses chez les bovins et les animaux de compagnie est menacé par l’accroissement de l’antibiorésistance et des bactéries multirésistantes. Il est donc primordial pour les entreprises pharmaceutiques vétérinaires de développer de nouvelles gammes d’agents antibactériens spécifiques au domaine animale. Dans ce contexte, il a été montré que certains polyaminostérols naturels tels que la squalamine possèdent un fort potentiel antimicrobien. Ainsi, l’objectif de ce travail de thèse est de répondre à la problématique présentée par la société Virbac dans le traitement des mammites (bovins) et des otites/pyodermites (chien) par le développement d’une nouvelle classe de dérivés polyaminostéroïdiens synthétiques à large spectre antimicrobien. Pour cela, nous avons constitué une chimiothèque de composés originaux préparés à partir de différents acides biliaires selon des voies de synthèse inédites. Ces composés ont été évalués in vitro pour leur cytotoxicité et leurs activités antibactériennes contre diverses bactéries à Gram positif et à Gram négatif et nous avons pu établir la preuve de concept in vitro de leur potentiel thérapeutique en tant qu’agents antibactériens ou adjuvants d’antibiotiques. De plus, nous avons montré que l’un de ces nouveaux dérivés, la claramine A1, agit sur l’intégrité physique des membranes bactériennes et sur les performances d'efflux des pompes AcrAB-TolC. Ainsi, il apparaît finalement que ces dérivés polyaminostéroïdiens au mode d’action non conventionnel pourraient constituer une nouvelle classe d’agents antibactériens pour un usage en tant que substituts d’antibiotiques en médecine vétérinaire. / Currently, the treatment of infectious pathologies in cattle and pets is threatened by the growing antimicrobial resistance and the development of multidrug-resistant bacteria. Thus it is necessary for the veterinary pharmaceutical firms to develop new lines of antibacterial agents. In this context, some natural polyaminosterols such as squalamine have gained interest due to their potent antimicrobial activities. Thus the aim of this PhD work is to provide an answer in the treatment of mastitis (cattle) and otitis/pyoderma (dog) by the development of a new class of synthetic polyaminosterols with a broad spectrum of antibacterial activity. In this purpose, a chemical library of original compounds has been prepared starting from various bile acids by using unprecedented synthesis procedures. These compounds were evaluated for their in vitro cytotoxicity on CHO cells as well as their antibacterial activities against Gram-positive and Gram-negative bacteria and we have establish the in vitro proof of concept of the therapeutic potential of this family of molecules as antibacterial agent or antibiotic adjuvant. Additional investigations were then conducted on one of these novel derivatives namely claramine A1 to deepen knowledge of its mechanism of action and showed that claramine A1 acts on the physical integrity of bacterial membranes and the efflux performance of AcrAB-TolC pumps. Based on the results of claramine A1, it finally appears that these new polyaminosterol derivatives possessing a non-classical mode of action pertain to a new class of antibacterial agents and could constitute a substitute for traditional antibiotics in veterinary medicine.

Indiana University-Purdue University Indianapolis (IUPUI) / Spore photoproduct lyase (SPL) is a radical SAM (S-adenosylmethionine) enzyme that is responsible for the repair of the DNA UV damage product 5-thyminyl-5,6-dihydrothymine (also called spore photoproduct, SP) in the early germination phase of bacterial endospores. SPL initiates the SP repair process using 5'-dA• (5'-deoxyadenosyl radical) generated by SAM cleavage to abstract the H6proR atom which results in a thymine allylic radical. These studies provide strong evidence that the TpT radical likely receives an H atom from an intrinsic H atom donor, C141 in B. subtilis SPL. I have shown that C141 can be alkylated in native SPL by iodoacetamide treatment indicating that it is accessible to the TpT radical. Activity studies demonstrate a 3-fold slower repair rate of SP by C141A which produces TpTSO2 - and TpT simultaneously with no lag phase observed for TpTSO2- formation. Additionally, formation of both products shows a Dvmax kinetic isotope effect (KIE) of 1.7 ± 0.2 which is smaller than the DVmax KIE of 2.8 ± 0.3 for the WT SPL reaction. Removal of the intrinsic H atom donor by this single mutation disrupts the rate-limiting process in the enzyme catalysis. Moreover, C141A exhibits ~0.4 turnover compared to the > 5 turnovers in the WT SPL reaction. In Y97 and Y99 studies, structural and biochemical data suggest that these two tyrosine residues are also crucial in enzyme catalysis. It is suggested that Y99 in B. subtilis SPL uses a novel hydrogen atom transfer pathway utilizing a pair of cysteinetyrosine residues to regenerate SAM. The second tyrosine, Y97, structurally assists in SAM binding and may also contribute to SAM regeneration by interacting with radical intermediates to lower the energy barrier for the second H-abstraction step.

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