Conception, synthèse et vectorisation d'inhibiteurs potentiels de la protéine bactérienne TonB

La multiplication des résistances aux antibiothérapies actuelles et l utilisation potentielle de bactéries pathogènes dans le cadre d attentats bioterroristes rendent nécessaire la recherche de nouvelles cibles biologiques et la découverte de nouvelles stratégies antibiotiques. Dans ce contexte, les mécanismes d assimilation du fer chez les bactéries à Gram négatif sont des cibles particulièrement prometteuses. Le fer est en effet un élément essentiel à la vie, mais peu biodisponible. Les bactéries ont donc développé des mécanismes efficaces pour subvenir à leurs besoins en fer. Ces mécanismes de transport nécessitent un apport d énergie fourni par une machinerie bactérienne complexe, la machinerie TonB. La protéine TonB, qui joue un rôle central dans le fonctionnement de cette machinerie, est la cible de notre approche. Nous souhaitons séquestrer cette protéine dans le périplasme grâce à des composés peptidiques fonctionnalisés par des hétérocycles de type isoindole ou 1,2,4-triazine. La conception et la synthèse de ces molécules sont présentées dans ce manuscrit, ainsi que leurs perspectives de vectorisation en utilisant une stratégie dite du "cheval de Troie". Notre contribution à la mise au point d un test d affinité in vitro est également abordée.The increasing resistances to the current antibiotherapies, and the potential use of pathogenic bacteria as biological weapons led us to the absolute necessity of discovering new biological targets and new antibiotic strategies. In this context, iron uptake pathways of Gram negative bacteria are promising targets. Indeed, iron is an essential nutrient, but it has a low bioavailability. Bacteria have developed efficient iron uptake pathways in order to proliferate. Iron is transported in the bacterial cell by specific outer membrane transporters and thanks to the energy provided by a complex molecular machinery, called TonB. The TonB protein, which is the keystone of this machinery, is a key target for the development of new antibiotics. We would like to sequester this protein in the periplasm thanks to molecules constituted of a peptidic moiety and a heterocyclic moiety such as isoindole or 1,2,4-triazine. The conception and the synthesis of these compounds are presented in this document, as well as their possibilities to be vectorized using a Trojan Horse strategy. Our contribution to the development of an in vitro test of affinity is presented as well.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

Vectorisation d'antibiotiques par des sidérophores et synthèse d'inhibiteurs potentiels de l'assimilation du fer chez Pseudomonas aeruginosa et Burkholderia cepacia

La pyochéline est un siderophore commun à Pseudomonas aeruginosa et Burkholderia cepacia, bactéries responsables d infections graves chez les patients atteints de mucoviscidose. Le système d assimilation du fer pyochéline-dépendant, faisant intervenir plusieurs protéines dont son récepteur spécifique FptA, est une cible de choix dans le cadre de nouvelles stratégies antibiotiques. Dans ce contexte, nous avons synthétisé une pyochéline fonctionnalisée en position N3" qui se lie au récepteur FptA et transporte le fer en utilisant la voie de transport pyochéline-dépendante. Dans une stratégie prodrogue de type cheval de Troie , cette pyochéline fonctionnalisée a ensuite été utilisée comme motif d adressage d'antibiotiques de la famille des fluoroquinolones. Malgré une activité moindre que l antibiotique testé seul, la synthèse de conjugués entre la pyochéline fonctionnalisée et le groupement fluorescent 4-nitro-2,1,3-benzoxadiazole (NBD) a montré que la pyochéline synthétique est capable de vectoriser efficacement un xénobiotique en utilisant le système de transport du fer pyochéline-dépendant. En parallèle, nous avons conçu des inhibiteurs potentiels de la protéine membranaire FptA. Plusieurs de ces composés se lient fortement à la protéine cible mais sans parvenir à inhiber le transport du fer. Ces molécules ont cependant permis d améliorer nos connaissances sur les relations structure-activité liant la pyochéline à son récepteur de membrane externe, notamment grâce à la synthèse de l énantiopyochéline, un sidérophore de Pseudomonas fluorescens, qui a mis en évidence les fondements structuraux de la stéréospécificité de reconnaissance du récepteur FptA.The pyochelin is a common siderophore of Pseudomonas aeruginosa and Burkholderia cepacia , two pathogenic Gram negative bacteria responsible for severe infections in the case of cystic fibrosis affected patients. The pyochelin-dependant iron uptake, involving several proteins like its specific receptor FptA, is a promising target to develop new antibiotic strategies. In this context, we synthesized a pyochelin analog, functionalized on the N3" position, which binds to the FptA receptor and efficiently promotes iron uptake using the pyochelin-dependant iron uptake. The functionalized pyochelin was then conjugated to fluoroquinolone antibiotics in a Trojan horse prodrug strategy. Despite a lower biological activity compared to the antibiotics tested alone, the synthesis of conjugates between the N3" functionalized pyochelin and the 4-nitro-2,1,3-benzoxadiazole (NBD) fluorophore proved that our synthetic siderophore analogue is able to efficiently transport a xenobiotic molecule using the pyochelin-dependant iron uptake system. In parallel, we designed potential inhibitors of FptA receptor. Several molecules were able to efficiently bind to the target receptor without inhibiting the iron uptake. Nevertheless, these molecules brought invaluable information on the structure-activity relationships between the siderophore and its specific outer membrane receptor. In this context, the synthesis of enantiopyochelin, a siderophore produced by Pseudomonas fluorescens, brought new insights on the structural basis underlying the high stereospecificity of the FptA receptor binding site.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Synthesis of conjugates between oxazolidinone antibiotics and a pyochelin analogue

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Dipyridylamine-acetamide (Dpaa): A primary amine protecting group orthogonally cleavable under acidic conditions in the presence of t-butyloxycarbonyl (Boc) and t-butylester

International audienceWe show that dipyridylamine-acetamide (Dpaa), can be cleaved under mild acidic conditions (30% formic acid in dichloromethane). The release of the amine function is orthogonal to other acid-labile protecting groups. Calculations suggest that the ease of Dpaa cleavage relies on activation of the carbonyl function by the protonated dipyridylamine nitrogen and activation of a water molecule by a hydrogen-bond network

Chryso-lactams:Gold(I) derivatives of ampicillin with specific activity against Gram-positive pathogens

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Synthesis and antibacterial properties under blue LED light of conjugates between the siderophore desferrioxamine B (DFOB) and an Iridium(III) complex

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Vancomycin-decorated microbubbles as a theranostic agent for Staphylococcus aureus biofilms

International audienceBacterial biofilms are a huge burden on our healthcare systems worldwide. The lack of specificity in diagnostic and treatment possibilities result in difficult-to-treat and persistent infections. The aim of this in vitro study was to investigate if microbubbles targeted specifically to bacteria in biofilms could be used both for diagnosis as well for sonobactericide treatment and demonstrate their theranostic potential for biofilm infection management. The antibiotic vancomycin was chemically coupled to the lipid shell of microbubbles and validated using mass spectrometry and high-axial resolution 4Pi confocal microscopy. Theranostic proof-of-principle was investigated by demonstrating the specific binding of vancomycin-decorated microbubbles (vMB) to statically and flow grown Staphylococcus aureus (S. aureus) biofilms under increasing shear stress flow conditions (0-12 dyn/cm2), as well as confirmation of microbubble oscillation and biofilm disruption upon ultrasound exposure (2 MHz, 250 kPa, and 5,000 or 10,000 cycles) during flow shear stress of 5 dyn/cm2 using time-lapse confocal microscopy combined with the Brandaris 128 ultra-high-speed camera. Vancomycin was successfully incorporated into the microbubble lipid shell. vMB bound significantly more often than control microbubbles to biofilms, also in the presence of free vancomycin (up to 1,000 µg/mL) and remained bound under increasing shear stress flow conditions (up to 12 dyn/cm2). Upon ultrasound insonification biofilm area was reduced of up to 28%, as confirmed by confocal microscopy. Our results confirm the successful production of vMB and support their potential as a new theranostic tool for S. aureus biofilm infections by allowing for specific bacterial detection and biofilm disruption

Vancomycin-decorated microbubbles as a theranostic agent for Staphylococcus aureus biofilms

Bacterial biofilms are a huge burden on our healthcare systems worldwide. The lack of specificity in diagnostic and treatment possibilities result in difficult-to-treat and persistent infections. The aim of this in vitro study was to investigate if microbubbles targeted specifically to bacteria in biofilms could be used both for diagnosis as well for sonobactericide treatment and demonstrate their theranostic potential for biofilm infection management. The antibiotic vancomycin was chemically coupled to the lipid shell of microbubbles and validated using mass spectrometry and high-axial resolution 4Pi confocal microscopy. Theranostic proof-of-principle was investigated by demonstrating the specific binding of vancomycin-decorated microbubbles (vMB) to statically and flow grown Staphylococcus aureus (S. aureus) biofilms under increasing shear stress flow conditions (0–12 dyn/cm2), as well as confirmation of microbubble oscillation and biofilm disruption upon ultrasound exposure (2 MHz, 250 kPa, and 5,000 or 10,000 cycles) during flow shear stress of 5 dyn/cm2 using time-lapse confocal microscopy combined with the Brandaris 128 ultra-high-speed camera. Vancomycin was successfully incorporated into the microbubble lipid shell. vMB bound significantly more often than control microbubbles to biofilms, also in the presence of free vancomycin (up to 1000 µg/mL) and remained bound under increasing shear stress flow conditions (up to 12 dyn/cm2). Upon ultrasound insonification biofilm area was reduced of up to 28%, as confirmed by confocal microscopy. Our results confirm the successful production of vMB and support their potential as a new theranostic tool for S. aureus biofilm infections by allowing for specific bacterial detection and biofilm disruption.</p