A Tool for Evaluating Drugs Targeting MBNL

The Genesis of Drug Screening Assays

Early stage drug development programs are often predicated on the development of biochemical or cell-based assays that allow identification of candidate therapeutics that engage and modulate targets deemed to be potentially disease mitigating. To be efficient and effective such screening assays must meet accepted pharmaceutical industry criteria—guidance in development of acceptable assays has been provided as part of a preclinical research toolbox by the National Center for Advancing Translational Sciences (https://ncats.nih.gov/files/agm-factsheet.pdf).

Screening assays frequently arise from academic groups focused on both mechanistic and translational goals. While not typically in compliance with industry standards for high throughput screening of libraries that can exceed 1M compounds, such assays may provide important first steps in that direction.

A Novel Effort Towards a DM1 Drug Screening Assay

Dr. Krzysztof Sobczak and colleagues at Adam Mickiewicz University have developed a minigene-based assay to assess critical RNA binding protein sites/splicing regulatory regions in pre-mRNA that may have implications as a drug screening assay for DM1. Dr. Łukasz Sznajder, currently an MDF fellow at the University of Florida, was part of the team.

The research team first evaluated functional protein (MBNL)/RNA interactions, taking into account RNA primary and secondary structure in a design that allowed assessment by CLIP-seq or other means of transcriptome analysis. In an evaluation of the potential to assess splicing regulation, antisense oligonucleotides on two backbone chemistries (2’OMe and LNA) targeted to MBNL binding regions of Atp2a1 were able to inhibit exon 22 inclusion in the Atp2a1 transcript. The approach was validated using other transcripts regulated by MBNL.

They also designed hybrid Atp2a1 mini genes containing functional MBNL-binding motifs in introns and exons. These were used to establish that inclusion or exclusion of the MBNL motifs had the predicted effect on exon 22 splicing; results were confirmed in transfected HeLa cells. Taken together, they showed that MBNL-binding regulatory regions could be transferred from their original genetic context into a different mini gene transcript and still regulate alternative splicing.

In an initial proof of concept, the research team showed that the Atp2a1 mini gene could feasibly discern the potency of various interventions to disrupt MBNL binding. Inclusion of a reporter gene in the minigene construct may provide an effective assay for drug discovery via high-throughput screening.


Hybrid splicing minigene and antisense oligonucleotides as efficient tools to determine functional protein/RNA interactions.
Cywoniuk P, Taylor K, Sznajder ŁJ, Sobczak K.
Sci Rep. 2017 Dec 14;7(1):17587. doi: 10.1038/s41598-017-17816-x


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