Identification of therapeutic small molecule compound for cystic fibrosis by targeting pseudo exon-inclusion

[Speaker] Saiko Shibata:1
[Co-author] Masahiko Ajiro:1, Masatoshi Hagiwara:1,2
1:Department of Drug Discovery Medicine, Graduate school of Medicine, Kyoto University, Japan, 2:Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Japan

Background: Cystic fibrosis (CF) is an autosomal recessive rare genetic disease with approximately 70,000 patients in the world. The pathogenesis of CF is attributable to ~2,000 of known mutation within the CFTR gene, which interfere with the processing or integrity of the CFTR protein. One of the deep intronic mutations, IVS22+12191C>T, that causes CF by creating pseudo exon within CFTR intron 22, is the most common splicing-type mutation found in ~2% of CF patients. The objective of this study is to provide novel therapeutic compounds for CF by targeting pseudo exon created by IVS22+12191C>T.

Methods: We conducted screening of the focused library of splicing modulating small molecule compounds to evaluate suppression of CFTR pseudo exon-inclusion by IVS22+12191C>T mutation, taking advantage of the dual-fluorescent splicing reporter vector system. The reporter vector consists of CFTR exon 22 through exon 23 with the IVS22+12191C>T mutation, which produces mRFP if CFTR pseudo exon is included, and EGFP if skipped. Identified hit compounds were further evaluated for their mechanism of action (MoA) on CFTR pseudo exon recognition in the background of IVS22+12191C>T mutation.

Results: Focused compound library screening with dual fluorescent reporter vector of CFTR harboring IVS22+12191C>T successfully identified cdc-like kinase (CLK) inhibitors as hit compounds. CLK is known as activator of serine/arginine-rich (SR) proteins through phosphorylation of RS domain, which promotes spliceosomal recognition of suboptimal splice sites. Through point-mutation analysis and RNA binding assays, we identified suboptimal acceptor sites of CFTR pseudo exon is dependent on specific SR proteins bound at exonic splicing enhancer (ESE) within pseudo exon. We confirmed CLK inhibitor suppresses CFTR pseudo exon recognition, by functionally inhibiting those SR proteins in the mutant background.

Conclusion: We found CLK inhibitor as a promising therapeutic molecule for CF caused by the deep intronic mutation IVS22+12191C>T, by targeting SR protein function employed for the recognition of suboptimal splice site of CFTR pseudo exon.

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