Marquage métabolique des isoprénoïdes bactériens produits par la voie du méthylérythritol phosphate : un point de départ vers un nouvel inhibiteur

Isoprenoids, present in all living organisms, are synthesised according to two routes: the Mevalonate and the Methylerythritol phosphate (MEP) pathways. The MEP pathway, absent in humans, is extensively investigated as it is a target for the development of new antimicrobials. ME-N3 an azide tagged analogue of methylerythritol was synthesised and utilised for metabolic labelling studies of the MEP pathway using bioorthogonal ligation followed by LC-MS analysis. Interestingly, we found that MEP-N3, an analogue of MEP, inhibits E.coli IspD (3rd enzyme of the MEP pathway). Further inhibition kinetic studies revealed that MEP-N3 possesses the highest inhibitory activity on E.coli ispD when compared to known inhibitors. In addition, the mechanism of inhibition of E.coli ispD by MEP-N3 was found to be best described using a mixed type model. Moreover, determination of the IspD reaction mechanism has been carried out for the first time, by virtue of a bisubstrate steady state kinetic analysis.Les isoprénoïdes, présents dans tous les organismes vivants, sont synthétisés selon deux processus: la voie du Mevalonate et la voie Méthylérythritol phosphate (MEP). Cette dernière, absente chez l’humain, est très étudiée car elle représente une cible pour le développement de nouveaux antimicrobiens. Le ME-N3, un analogue du méthylérythritol portant un azoture, a été synthétisé et exploité dans des expériences de marquage métabolique de la voie MEP en utilisant un couplage bioorthogonale suivi d’une analyse par LC/MS. De façon intéressante, nous avons découvert que le MEP-N3, un analogue du MEP, inhibe l'enzyme IspD d’ E. coli (3ème enzyme de la voie MEP). Les études cinétiques ont révélé que le MEP-N3 possède la meilleure activité inhibitrice sur IspD d’ E.coli en comparaison avec les inhibiteurs connus, et que le mécanisme d'inhibition est de type mixte. Une étude détaillée du mécanisme de la réaction catalysée par IspD a été réalisée pour la première fois, en utilisant une analyse cinétique à deux substrats

Synthesis and Kinetic evaluation of an azido analogue of methylerythritol phosphate: a Novel Inhibitor of E. coli YgbP/IspD

Abstract As multidrug resistant pathogenic microorganisms are a serious health menace, it is crucial to continuously develop novel medicines in order to overcome the emerging resistance. The methylerythritol phosphate pathway (MEP) is an ideal target for antimicrobial development as it is absent in humans but present in most bacteria and in the parasite Plasmodium falciparum. Here, we report the synthesis and the steady-state kinetics of a novel potent inhibitor (MEPN3) of Escherichia coli YgbP/IspD, the third enzyme of the MEP pathway. MEPN3 inhibits E. coli YgbP/IspD in mixed type mode regarding both substrates. Interestingly, MEPN3 shows the highest inhibitory activity when compared to known inhibitors of E. coli YgbP/IspD. The mechanism of this enzyme was also studied by steady-state kinetic analysis and it was found that the substrates add to the enzyme in sequential manner

CBTF: New Amine-to-Thiol Coupling Reagent for Preparation of Antibody Conjugates with Increased Plasma Stability

Amine-to-thiol coupling is the most common route for the preparation of antibody–drug conjugates (ADC). It is usually achieved by using heterobifunctional reagents possessing an activated ester at one end and a maleimide group at the other. However, maleimide-based conjugates were recently revealed to have limited stability in blood circulation, which can compromise therapeutic efficacy of the conjugate. To address this issue, we have developed a heterobifunctional reagent, sodium 4-((4-(cyanoethynyl)­benzoyl)­oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling. It comprises a recently described 3-arylpropionitrile (APN) function in replacement of maleimide and allows for the preparation of remarkably stable conjugates. A series of antibody–dye conjugates have been prepared using this reagent and shown superior stability in human blood plasma compared to maleimide-derived conjugates

MAPN: First-in-Class Reagent for Kinetically Resolved Thiol-to-Thiol Conjugation

Thiols are among the most frequently used functional groups in the field of bioconjugation. While there exists a variety of heterobifunctional reagents that allow for coupling thiols to other functions (e.g., amines, carboxylic acids), there is no specific reagent for creating heteroconjugates using two different thiols. In response to the ever-increasing demand for bioconjugation tools, we have developed <i>p</i>-(maleimide)-phenylpropionitrile (MAPN)an efficient reagent for kinetically resolved thiol-to-thiol coupling. In a comparative study with its closest commercially available analogue, <i>p</i>-phenylenedimaleimide, MAPN has shown substantial advantages for the preparation of thiol–thiol heteroconjugates. Namely, an antibody–drug conjugate (ADC) with mertansine (DM1), conjugated to the cysteine residues of Trastuzumab, was prepared for the first time