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

Recherche transversale
(titres de publication, de périodique et noms de colloque inclus)
2019-01-09 - Colloque/Présentation - poster - Anglais - page(s)

Leveque Mathieu , Neaga Ioan, Hambye Stéphanie , Blankert Bertrand , "Identification of small therapeutic molecules targeting specific pathological probes of Steinert’s disease by affinity capillary electrophoresis" in ITP, Toulouse, France, 2019

  • Codes CREF : Analyse et contrôle pharmaceutique (DI3450), Sciences pharmaceutiques (DI3400)
  • Unités de recherche UMONS : Analyse pharmaceutique (M130)
  • Instituts UMONS : Institut des Sciences et Technologies de la Santé (Santé)

Abstract(s) :

(Anglais) DM1, also named Steinert’s disease, is a genetic dominantly inherited degenerative disease caused by repetitions (>50) of the trinucleotide [CTG]n on the 3’UTR non-coding region of the DMPK gene (Dystrophia Myotonica Protein Kinase) located on chromosome 19 at 19q13.3. The pathogenesis of DM1 is not fully understood but is associated with RNA gain-of-function mechanism. The transcription of [CTG]n leads to the formation of a toxic RNA [CUG]n which will adopt a hairpin structure forming RNA foci in the nucleus which could sequestrate various proteins and small molecules. Among them, splicing factors such as MBLN1 and CUBP1 are particularly involved and lead to the mis-splicing of several pre-mRNAs and consequently to defective corresponding protein syntheses. Even though currently no clinical treatment for DM1 exists, several therapeutic strategies are considered. Among them, the use of small molecules (ligands) to bind either the [CTG]n triplets (blocking their transcription) at the DMPK gene or the [CUG]n RNA (blocking their harmful effects), has been widely proposed these last years1. The objective of this project is to demonstrate the fit for purpose of an affinity capillary electrophoresis (ACE) method as a powerful tool to highlight molecules which bind to specific probes of the pathology. Firstly, the synthesis and purification of DNA and RNA probes (reflecting pathological and non-pathological situations to evaluate the specificity of the binding) are needed. The DNA [CTG]n probes (n=14 or 95) are obtained by bacterial amplification of the corresponding plasmid followed by enzymatic restriction, migration on agarose gel and electrodialysis to extract DNA from the gel. The RNA [CUG]n probes ((n=14 or 95) are obtained by in vitro transcription. Secondly, the binding constants between the hereabove mentioned probes (analytes) and a various panel of therapeutic candidates (ligands; existing drugs or newly synthetized) will be assessed. This step will be performed using an affinity capillary electrophoresis (ACE) method. An Agilent G1600 capillary electrophoresis system with UV detector set at 260 nm and a bare fused silica capillary (50μm ID; 365μm OD; 40cm Lt; 31.5cm Leff) were used. The capillary was thermostated at 25°C and coated with a dynamic PEO protocol. The RNA and DNA samples were injected hydrodynamically (50mbar x 5sec) in 50mM HEPES buffer solution (pH 7.4) used as background electrolyte. The separation was performed at a constant voltage of -15kV. Ligands to test are dissolved at various concentrations in the working buffer and the interactions with the probes are highlighted by variations in their migration times and the corresponding electrophoretic mobilities. Binding constants will be therefore evaluated using a non-linear regression2. To date, the ACE method has allowed to point out 3 ligands. The biological activity of the highlighted ligands has been corroborated by a fluorescence microscopy assay2 confirming the validity of the ACE approach. Moreover, by studying the affinity for DNA and RNA probes, the CE method can offer relevant information about the unknown mechanism of action of small molecules in the treatment of DM1.