High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines.

Abstract

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.

• Authors: Ami Ketley, Catherine Z Chen, Xin Li, Sukrat Arya, Thelma E Robinson, Javier Granados-Riveron, Inyang Udosen, Glenn E Morris, Ian Holt, Denis Furling, Soraya Chaouch, Ben Haworth, Noel Southall, Paul Shinn, Wei Zheng, Christopher P Austin, Christopher J Hayes, J David Brook
• Journal: Human molecular genetics (Hum Mol Genet) Vol. 23 Issue 6 Pg. 1551-62 (Mar 15 2014) ISSN: 1460-2083 [Electronic] England
• PMID: 24179176 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• CELF1 Protein
• CELF1 protein, human
• Indoles
• MBNL1 protein, human
• Peptide Library
• RNA-Binding Proteins
• Chromomycin A3
• Sarcoplasmic Reticulum Calcium-Transporting ATPases
• ATP2A1 protein, human
• Ro 31-8220

Topics

• Alternative Splicing
• Animals
• CELF1 Protein
• Cell Nucleus (drug effects, pathology)
• Cells, Cultured
• Chromomycin A3 (pharmacology)
• Disease Models, Animal
• Gene Expression Regulation
• High-Throughput Screening Assays
• Humans
• Indoles (pharmacology)
• Myotonic Dystrophy (pathology)
• Peptide Library
• RNA-Binding Proteins (genetics, metabolism)
• Sarcoplasmic Reticulum Calcium-Transporting ATPases (genetics, metabolism)
• Signal Transduction (drug effects)
• Zebrafish