Quantification of Interactions between Small Molecules and RNA Probes Representative of Myotonic Dystrophy Type 1...

Myotonic dystrophy type 1 (OMIM #160900) is a multisystemic, autosomal, and dominantly inherited pathology. It is characterized by an expansion (>50) of trinucleotides [CTG] _n on the 3' untranslated region of the dystrophia myotonica protein kinase gene, transcribed into RNA [CUG] _n , aggregating in the nucleus as foci. The most accepted pathological mechanism considers the sequestration and dysregulation of proteins, including splicing factors (muscle-blind-like splicing regulator 1 and CUG-binding protein 1) by pathological RNA. Different therapeutic strategies to overcome this defect exist, such as the use of small molecules targeting the pathological RNA to prevent sequestration. Among these small molecules, pentamidine (PTMD), an antiprotozoal drug, has previously been shown to interact with a [CUG] _n repeat. In this context, we developed a fine-tuned affinity capillary electrophoresis (ACE) method that provides highly repeatable migration times. This is a crucial point, as all subsequent calculations for apparent affinity constant (Ka_app ) determination rely on them. Afterward, we quantified the interactions between pentamidine and two RNA probes: [CUG]₉₅, representative of the disease, and [CUG]₁₄ as a negative control. The results indicated an excellent affinity between [CUG]₉₅ and PTMD. Selectivity was assessed by comparing the Ka_app values: (6.1 ± 0.4) × 10³ M^-1 and (3.8 ± 0.3) × 10³ M^-1 for the positive and negative controls, respectively. For the first time, an orthogonal UPLC-UV methodology was applied to corroborate the ACE results. No significant differences were observed between the two analytical methods. To overcome the documented toxicity related to pentamidine, different libraries of small molecules have been investigated by the ACE method. Among these, two compounds, neomycin and chloroquine, demonstrated interactions with the pathological RNA model. Therefore, their affinities toward the positive and negative controls were quantified to assess the selectivity of these potential therapeutic candidates.