Myotonic dystrophy type 1 (DM1), an autosomal dominant neuromuscular disorder affecting 1/8000 individuals, is the most common form of muscular dystrophy in adults. The disease has a wide range of multi-systemic clinical presentations, which includes myotonia, cardiac conduction defects, progressive muscle wasting, endocrine imbalance and cataracts, causing progressive disability and premature death [1
]. The genetic lesion that causes DM1 is an expansion of CTG repeats in the 3′ untranslated region (UTR) of the DM protein kinase (DMPK) gene. The size of the triplet repeat expansion ranges from hundreds to thousands of repeats and correlates with disease severity [2
]. DM represents a novel disease paradigm where the mutant RNA exerts a dominant deleterious effect that is independent of the protein it encodes. The pathogenic expanded CUG repeat (CUGexp
) RNA accumulates in nuclear foci and sequesters proteins of the muscleblind-like (MBNL) family, of which MBNL1 is the most common family member in skeletal muscle [4
]. MBNL proteins regulate alternative splicing, and their sequestration alters the splicing patterns of a myriad of transcripts. Of the affected transcripts, cardiac troponin T (cTNT), sarcoplasmic/endoplasmic reticulum Ca2+
-ATPase (SERCA1), insulin receptor (IR) and muscle-specific chloride channel (ClC-1) are the most well studied [6
The binding interaction between MBNL proteins and CUG repeat RNA is a key molecular step in the pathogenesis of DM1. Murine knockouts of MBNL1 recapitulate certain symptoms and splicing patterns of the disease [10
], while over-expression of MBNL1 in a DM1 mouse model that expresses a (CUG)250
repeat transcript reverses myotonia and RNA mis-splicing [11
]. In addition, proof of principle that blocking the CUGexp
/MBNL1 interaction in vivo
could be beneficial for DM was demonstrated by the use of morpholino oligo to disrupt MBNL1 CUGexp
binding, resulting in the correction of misregulated splicing [12
Currently, there is no treatment to halt disease progression and therapy for DM1 is limited to supportive care. Recent drug development efforts have aimed at finding ligands that bind the expanded CUG RNA using combinatorial chemistry and peptoid synthesis, as well as a targeted small molecule screen. Several compounds that are able to inhibit the interaction between expanded CUG RNA and MBNL1 protein have been found [13
]. Of these, only pentamidine has shown some in vivo
activity but the therapeutic window is narrow due to its cytotoxicity [18
]. Continued development of small molecule inhibitors to disrupt the binding interaction between MBNL1 and CUG expansion remains an attractive approach for the treatment of DM1.
Protein-RNA interactions control many aspects of RNA splicing, translation and decay. Compounds that modulate these interactions could become useful research tools as well as potential therapeutics. Traditional methods for detection of protein-RNA binding include gel mobility shift, filter-binding and yeast-three-hybrid (Y3H) assays [19
]. While these assays are useful in low throughput applications, they lack adaptability to high throughput screening (HTS) since they require either an electrophoresis apparatus or multiple wash steps. In addition, yeast-three-hybrid assay has added disadvantages due to the indirect nature of the assay. Two types of homogeneous assays, scintillation proximity and AlphaScreen technology, have been used to assay the bindings of HIV RNAs and host proteins in a high throughput manner [24
]. A down side of scintillation proximity assays is the requirement of radio-labeled reagents that generate large amounts of radioactive waste. In addition, the advantages of a homogenous assay format are the simplification of assay workflow and reduction of assay noise due to plate wash steps. However, elimination of wash steps may lead to increased number of false positives. With sizes of many compound libraries currently expanding to over a million, there is increasing need of additional HTS-ready assays for orthogonal screening to eliminate detection related artifacts and allow fast hit triaging. Thus, the strategy of using one homogenous protein-RNA binding assay for HTS and the other as an orthogonal screen would facilitate hit prioritization efforts by rapidly eliminating detection-based false positives.
We have developed and compared two homogeneous assays to measure the binding of MBNL1 to CUG repeat RNA. One uses the AlphaScreen technology and the other uses homogeneous time-resolved fluorescence energy transfer (HTRF). Since these two assays apply different detection methods, they can be used as an orthogonal pair to eliminate false positive compounds. Both assays were optimized, miniaturized to 1536-well format and validated using a DMSO control plate. Our results indicate that while both assays are suitable for HTS, the HTRF assay is preferred for increased throughput and easier implementation, as no light shielding is needed. The HTRF assay was further validated in a pilot screen of 1280 pharmacologically active compounds (LOPAC1280, Sigma-Aldrich) and showed performance and hit rate that are acceptable for HTS. The hits were confirmed with the HTRF assay and assay artifacts were eliminated with the AlphaScreen assay as an orthogonal screen. The results indicate that the two assays can be paired for HTS and orthogonal screening use.