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2017-05-22 - Colloque/Présentation - poster - Anglais - 1 page(s)

Neaga Ioan-Ovidiu, Hambye Stéphanie , Bodoki Ede, Ansseau Eugénie , Blankert Bertrand , Oprean Radu, "Affinity capillary electrophoresis as fast screening method for the identification of potentially active compounds for DM1 treatment" in The 45th International Symposium on High Performance Liquid Phase Separations and Related Techniques, Prague, République Tchèque, 2017

  • Codes CREF : Chimie analytique (DI1314), Sciences pharmaceutiques (DI3400), Techniques séparatives (DI2729)
  • Unités de recherche UMONS : Analyse pharmaceutique (M130)
  • Instituts UMONS : Institut des Sciences et Technologies de la Santé (Santé)

Abstract(s) :

(Anglais) Myotonic dystrophy type 1 (DM1) is a genetic disorder caused by abnormal expansion of a CTG triplet in a non-coding region of DMPK gene. Its transcription produces RNA (CUG)n hairpins, able to sequestrate proteins and other small molecules. One of them, MBNL1, is a splicing factor which is essential for the maturation of some pre-mRNAs. Even though currently no clinically validated treatment for DM1 exists, several hypothetical approaches are considered. One theory postulates the use of ligands to bind either the (CTG)n triplet at the DMPK gene site, or the (CUG)n chains of RNA, which eventually could block of their harmful effects. In this work, the use of affinity capillary electrophoresis (ACE) as a new screening method is investigated in order to identify novel potentially active compounds for DM1 treatment. The simple and straightforward screening method relies on a ligand concentration-dependent increase of the migration time as an indicative of tested ligand - target probe interaction. As target probes, specific DNA and RNA chains as disease models were produced. The DNA probes were amplified in E. coli using the pSP72 vector and digested after extraction using the proper restriction enzymes. The development of the affinity capillary electrophoresis method was performed using pentamidine as model ligand compound. Because of pentamidine’s high affinity for the silica surfaces, several coated capillaries were tested, where eventually PEO coating offered the best EOF suppression and minimized wall interactions. In all the experiments, HEPES buffer (pH 7.4) ensured the physiological pH. The optimized ACE experiments are able to supply data for the quick and cost-effective estimation of both the binding constant and stoichiometry of the studied ligands. In the following several small molecules, positively charged at physiological pH, as ligands were tested. The ACE experiments highlighted three active molecules: neomycin, a pentamidine analogue and pentamidine itself, confirming some of the results already reported in the literature. In further experiments the screening of a much wider number of ligands is envisaged comparing the obtained affinity parameters to the ones generated by other in vitro methods from literature. In conclusion, our work could improve the workflow of ligand screening, saving time and reagents due to the inherent features of capillary electrophoretic techniques. Moreover, such works could set further example and promote the application of CE methods in the high throughput screening of other new potentially active compounds for DM1 treatment.