Assay Development to Identify Small Molecule Drugs for DM1

Criticality of Small Molecule Assays

Small molecule drug development relies upon unbiased cellular, biochemical, or molecular level assays (screens) that read out the ability of test compounds to alter pathogenic mechanisms operative in the disease under study. Structure-activity relationships (SAR) of hits from such screens, along with established knowledge of how to improve the drug-like properties of such hits, can identify lead compounds for further non-clinical and clinical development.

Best practices in small molecule assay development have been codified by the National Center for Advancing Translational Sciences (NCATS) in their on-line Assay Guidance Manual. At the core of NCATS guidance is pilot development of assays that can easily transition into high throughput screening formats (HTS; >100K compounds per screen) and thus are valuable for iterative SAR efforts and collaborations with major pharmaceutical firm operations and their multi-million compound libraries. 

Given the variety of hurdles presented by the multi-system involvement in DM1, small molecule drugs are likely to play a significant role in reducing burden of disease. Thus, development and optimization of HTS assays utilizing expertise residing in academia will attract expertise and capacity of industry to the benefit of DM patients.

A Screen for Compounds Targeting Expanded CTG Repeats

 A paper describing a new drug screening assay for DM1 has been published in Proceedings of the National Academy of Sciences by Dr. Andy Berglund (University of Florida College of Medicine and University of Albany) and colleagues, including Dr. Kaalak Reddy, recipient of a 2017 MDF Postdoctoral Research Fellowship (Reddy et al., 2019). The team’s new assay is CTG repeat-selective, targeting the production and/or stability of construct containing a 480 CUG repeat tract in the context of a human DMPK segment, in a HeLa cell model. The model also contains a control, non-expanded CUG and uses a ratio-metric qRT-PCR readout of expanded repeat to non-expanded repeat levels. Cell lines generated by the team were evaluated for formation of nuclear foci and MBNL-dependent splicing events consistent with a DM1 phenotype.

After generating and selecting for optimal properties among several DM1 HeLa cell lines, properties of the selected line were evaluated using a pilot screen of the publicly available LOPAC1280 small molecule library; actinomycin D was included as a positive control (Siboni et al., 2015). Top hits from the screen included three microtubule inhibitors, a DNA intercalator, and a purine nucleoside analog. Based upon further characterization of hits in an MBNL-dependent splicing assay, one of the microtubule inhibitors, colchicine, was carried forward into testing and subsequently validated in both DM1 mice (HSALR) and a DM1 patient cell line. Studies of the mechanism of action for colchicine support selectively reduced transcription of the CTG expansion. While known toxicity of colchicine might preclude its further development for DM1, this work has identified a novel potential therapeutic target (microtubule dynamics as a means of reducing DM1 expanded repeat transcription) and provides proof of principle for the new screening assay.

Going Forward

The research team has established a cell-based small molecule drug screening assay that reads out the selective reduction of the expanded trinucleotide repeat that is pathogenic in DM1. Moreover, their pilot screen establishes the feasibility of the assay to identify putative small molecule drug hits and to identify potentially new mechanisms of action for therapeutic development in DM1. The next steps are translation of the assay into a concerted drug discovery and development effort.

At a minimum, this new assay has importance as a secondary screening tool for validating HTS screening assay hits and for conducting SAR using analogs of primary screen hits. Key properties that are essential for HTS assays (see NCATS’ Assay Guidance Manual) are not reported out by the research team in this particular publication, thus its value for use in the large format (e.g., 1536-well) screens needed with very large (> 1M) pharmaceutical company compound libraries is not yet known.


A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels.
Reddy K, Jenquin JR, McConnell OL, Cleary JD, Richardson JI, Pinto BS, Haerle MC, Delgado E, Planco L, Nakamori M, Wang ET, Berglund JA.
Proc Natl Acad Sci U S A. 2019 Sep 30. pii: 201901893. doi: 10.1073/pnas.1901893116. [Epub ahead of print]

Actinomycin D Specifically Reduces Expanded CUG Repeat RNA in Myotonic Dystrophy Models.
Siboni RB, Nakamori M, Wagner SD, Struck AJ, Coonrod LA, Harriott SA, Cass DM, Tanner MK, Berglund JA.
Cell Rep. 2015 Dec 22;13(11):2386-2394. doi: 10.1016/j.celrep.2015.11.028. Epub 2015 Dec 10.

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