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.
Subject(s)
Cell Nucleus/drug effects , Chromomycin A3/pharmacology , Indoles/pharmacology , Myotonic Dystrophy/pathology , RNA-Binding Proteins/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Alternative Splicing , Animals , CELF1 Protein , Cell Nucleus/pathology , Cells, Cultured , Disease Models, Animal , Gene Expression Regulation , High-Throughput Screening Assays , Humans , Peptide Library , RNA-Binding Proteins/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Signal Transduction/drug effects , ZebrafishABSTRACT
Antagonists of the chemokine receptor, CCR5, may provide important new drugs for the treatment of HIV-1. In this study we have examined the mechanism of action of two functional antagonists of the chemokine receptor CCR5 (UK-396,794, UK-438,235) in signalling and internalisation assays using CHO cells expressing CCR5. Both compounds were potent inverse agonists versus agonist-independent [(35)S]GTPgammaS binding to membranes of CHO cells expressing CCR5. Both compounds also acted as allosteric inhibitors of CCL5 (RANTES) and CCL8 (MCP-2)-stimulated [(35)S]GTPgammaS binding to CHO-CCR5 membranes, reducing the potency and maximal effects of the two chemokines. The data are consistent with effects of the allosteric inhibitors on both the binding and signalling of the chemokines. Both compounds inhibited CCR5 internalisation triggered by chemokines. When CHO-CCR5 cells were treated with either of the two compounds for prolonged periods of time (24 h) an increase (approximately 15%) in cell surface CCR5 was detected.
