DI-UMONS : Dépôt institutionnel de l’université de Mons

Recherche transversale
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2013-06-30 - Colloque/Présentation - poster - Anglais - 1 page(s)

Hambye Stéphanie , Ansseau Eugénie , Carminati Aurélie, Vanden Eynde Jean-Jacques, Belayew Alexandra , Blankert Bertrand , "Strategies for the development of a screening test in myotonic dystrophy type 1. Application to a new library of synthetized pentamidine analogs" in 24th International Symposium on Pharmaceutical and Biomedical Analysis, Bologne, Italie, 2013

  • Codes CREF : Chimie analytique (DI1314), Biologie moléculaire (DI3111), Sciences pharmaceutiques (DI3400)
  • Unités de recherche UMONS : Biologie moléculaire (M122), Analyse pharmaceutique (M130), Chimie organique (S836)
  • Instituts UMONS : Institut des Sciences et Technologies de la Santé (Santé)

Abstract(s) :

(Anglais) Myotonic dystrophy type I (DMI) or Steinert disease is an autosomal dominant inherited disorder, characterized by a variety of symptoms including myotonia and muscular dystrophy. The expansion of a CTG trinucleotide repeat in a non-coding region of the DMPK (dystrophia myotonica protein kinase) gene on chromosome region 19q13 is the cause of this genetic disease. Transcription of the repeat region leads to aggregates of long tracts of (CUG)n in nuclear foci which can sequestrate the MBNL1 protein, a splicing factor. This process results in the loss of function of MBNL1 and the missplicing of several pre-mRNA [1]. As recently described, Pentamidine (PTMD), an aromatic diamidine derivative used as antiprotozoal agent, could release the sequestrated splicing factor by competition for binding to CUG repeats [2]. Once MBNL1 released, the correct alternative splicing of the implicated mRNAs could partially be restored. Unfortunately, the required PTMD concentration is far too toxic for its use in vivo. The laboratory of Organic Chemistry (UMONS) is specialized in the synthesis of new, potentially less toxic analogs of PTMD. The aim of the present work is to develop a new and rapid test to evaluate the ability of new analogs to disrupt the complex of MBNL1 with CUG repeats in DMI cells. Two strategies are currently considered. The first one involves fluorescence microscopy as previously described [1]. HeLa cells are co-transfected with an expression vector coding for a MNBL1 protein fused to the green fluorescent protein (GFP-MNBL1) and with another plasmid expressing exons 11-15 of the DMPK gene containing 960 (CTG) triplets. After transfection, foci formation is observed by fluorescence and corresponds to the co-localisation of the GFP-MBNL1 protein with the CUG repeats. After addition of PTMD or analogs, the presence or absence of foci is used to evaluate the interruption of the RNA-MBNL1 complex. The conditions of transfection have been optimized and the screening of PTMD and analogs at different concentrations is in progress. Preliminary results show that the addition of 75 µM of Pentamidine reduces the total number of foci and the number of foci per nucleus. The results of the microscopy will be compared with those obtained by the other strategy envisaged, the competition dialysis. The CUG probe, corresponding to the DMPK gene with 960 CUG repetitions is synthetized by in vitro transcription. This one is added to a small device with a dialysis membrane and is dialyzed against a solution containing Pentamidine or analogs to be tested. When equilibration is reached, the amount of studied compound bound to the RNA is determined by ultraviolet (UV) or fluorescence spectrophotometry.