Synthèse des aloisines immobilisées

Des résultats récents ont montré que l aloisine A (7-nButyl-6-(4-hydroxyphenyl) [5H] pyrrolo[2,3-b]pyrazine) est un puissant inhibiteur des kinases dépendantes des cyclines. Une méthode permettant d identifier d éventuelles cibles cellulaires secondaires d un inhibiteur consiste à l immobiliser sur un support solide, afin d identifier les protéines ayant une forte affinité pour l inhibiteur par chromatographie d affinité d un extrait cellulaire. Dans ce but, l aloisine A portant un bras espaceur, ainsi que son analogue N-méthylé servant de contrôle négatif, ont été synthétisés. Nous rapportons ici la préparation de ces nouveaux dérivés de l aloisine conjugués à une chaîne de triéthylèneglycol en diverses positions de la molécule et terminés par une fonction amine permettant son immobilisation sur une matrice d agarose. Par ailleurs, la stratégie d immobilisation de la molécule via une réaction de cycloaddition [3+2] offre la perspective de synthèses de dérivés de l aloisine conjugués à diverses molécules d intérêt biologique telles que la biotine ou encore la tyrosine facilitant son transport à travers la barrière hémato-encéphaliqueRecent results have shown that aloisine A (7-nButyl-6-(4-hydroxyphenyl)[5H]pyrrolo[2,3-b] pyrazine) is a potent inhibitor of CDK. An original methodology aiming at identifying potential secondary inhibition targets relies on the immobilization of the inhibitor on solid matrix, followed by identification of proteins with high affinity for the inhibitor by affinity chromatography of a cellular extract. To this end, both aloisine A and the negative control N-methyl aloisine bearing extended linker chain have been synthesized. We present here the preparation of the new aloisine analogues bearing a triethylene glycol chain at different positions of the molecule, terminated by an amine suitable for the immobilisation on an agarose-based matrix. Furthemore, we show that conjugation of the molecule with the linker via a cycloaddition [3+2] allows efficient coupling to the matrix as well as access to aloisine derivatives conjugated with biologically relevent molecules such as biotine, or a tyrosine residue targeting LAT1 for transport of drugs through the blood-brain barrierLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Synthèse des aloisines immobilisées

Des résultats récents ont montré que l aloisine A (7-nButyl-6-(4-hydroxyphenyl) [5H] pyrrolo[2,3-b]pyrazine) est un puissant inhibiteur des kinases dépendantes des cyclines. Une méthode permettant d identifier d éventuelles cibles cellulaires secondaires d un inhibiteur consiste à l immobiliser sur un support solide, afin d identifier les protéines ayant une forte affinité pour l inhibiteur par chromatographie d affinité d un extrait cellulaire. Dans ce but, l aloisine A portant un bras espaceur, ainsi que son analogue N-méthylé servant de contrôle négatif, ont été synthétisés. Nous rapportons ici la préparation de ces nouveaux dérivés de l aloisine conjugués à une chaîne de triéthylèneglycol en diverses positions de la molécule et terminés par une fonction amine permettant son immobilisation sur une matrice d agarose. Par ailleurs, la stratégie d immobilisation de la molécule via une réaction de cycloaddition [3+2] offre la perspective de synthèses de dérivés de l aloisine conjugués à diverses molécules d intérêt biologique telles que la biotine ou encore la tyrosine facilitant son transport à travers la barrière hémato-encéphaliqueRecent results have shown that aloisine A (7-nButyl-6-(4-hydroxyphenyl)[5H]pyrrolo[2,3-b] pyrazine) is a potent inhibitor of CDK. An original methodology aiming at identifying potential secondary inhibition targets relies on the immobilization of the inhibitor on solid matrix, followed by identification of proteins with high affinity for the inhibitor by affinity chromatography of a cellular extract. To this end, both aloisine A and the negative control N-methyl aloisine bearing extended linker chain have been synthesized. We present here the preparation of the new aloisine analogues bearing a triethylene glycol chain at different positions of the molecule, terminated by an amine suitable for the immobilisation on an agarose-based matrix. Furthemore, we show that conjugation of the molecule with the linker via a cycloaddition [3+2] allows efficient coupling to the matrix as well as access to aloisine derivatives conjugated with biologically relevent molecules such as biotine, or a tyrosine residue targeting LAT1 for transport of drugs through the blood-brain barrierLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Identification of potential cellular targets of aloisine A by affinity chromatography.

International audienceAffinity chromatography was used to identify potential cellular targets of aloisine A (7-n-butyl-6-(4'-hydroxyphenyl)-5H-pyrrolo[2,3b]pyrazine), a potent inhibitor of cyclin-dependent kinases. This technique is based on the immobilization of the drug on a solid matrix, followed by identification of specifically bound proteins. To this end, both aloisine A and the protein-kinase inactive control N-methyl aloisine, bearing extended linker chains have been synthesized. We present the preparation of such analogues having the triethylene glycol chain at different positions of the molecule, as well as their immobilization on an agarose-based matrix. Affinity chromatography of various biological extracts on the aloisine matrices allowed the identification of both protein kinases and non-kinase proteins as potential cellular targets of aloisine

Discovery of Novel Human Aquaporin‑1 Blockers

Human aquaporin-1 (hAQP1) is a water channel found in many tissues and potentially involved in several human pathologies. Selective inhibitors of hAQP1 are discussed as novel treatment opportunities for glaucoma, brain edema, inflammatory pain, and certain types of cancer. However, only very few potent and chemically attractive blockers have been reported to date. In this study we present three novel hAQP1 blockers that have been identified by virtual screening and inhibit water flux through hAQP1 in <i>Xenopus laevis</i> oocyte swelling assays at low micromolar concentrations. The newly discovered compounds display no chemical similarity to hitherto known hAQP1 blockers and bind at the extracellular entrance of the channel, close to the ar/R selectivity filter. Futhermore, mutagenesis studies showed that Lys36, which is not conserved among the hAQP family, is crucially involved in binding and renders the discovered compounds suitable as leads for the development of selective hAQP1 inhibitors

Chemoenzymatic Synthesis of <i>O</i>-Mannosylpeptides in Solution and on Solid Phase

<i>O</i>-Mannosyl glycans are known to play an important role in regulating the function of α-dystroglycan (α-DG), as defective glycosylation is associated with various phenotypes of congenital muscular dystrophy. Despite the well-established biological significance of these glycans, questions regarding their precise molecular function remain unanswered. Further biological investigation will require synthetic methods for the generation of pure samples of homogeneous glycopeptides with diverse sequences. Here we describe the first total syntheses of glycopeptides containing the tetrasaccharide NeuNAcα2-3Galβ1-4GlcNAcβ1-2Manα, which is reported to be the most abundant <i>O</i>-mannosyl glycan on α-DG. Our approach is based on biomimetic stepwise assembly from the reducing end and also gives access to the naturally occurring mono-, di-, and trisaccharide substructures. In addition to the total synthesis, we have developed a “one-pot” enzymatic cascade leading to the rapid synthesis of the target tetrasaccharide. Finally, solid-phase synthesis of the desired glycopeptides directly on a gold microarray platform is described