Project 4.  Development of assay systems for small molecule high throughput screens for muscular dystrophy. 

Carrie Miceli, PhD.   University of California, Los Angeles.

            An important experimental approach to the development of new therapies and biological probes involves high throughput drug and small molecule screening.  In the most common iteration of this approach, a cell culture-based assay system is developed upon which tens of thousands of potential therapeutic agents are screened.  Potential therapeutic agents are applied to the assays, with some resulting “read out” indicating the rare agent that has achieved the desired therapeutic effect.  High throughput screens have not been systematically applied to the muscular dystrophies.  The two high throughput screens performed to date are attempts to up-regulate endogenous utrophin in cells to compensate for dystrophin deficiency, and a screen of agents able to effect read-through of stop codons [1]. 

Here, we propose a systematic approach to high throughput drug screening relevant to the muscular dystrophies, where we target five different processes, the modulation of which, are known or likely to show therapeutic benefit.  The specific aims focus on developing sensitive and specific assays appropriate for high throughput testing of the effects of individual molecules via screening of complex libraries in 384 well culture plates. 

The assays developed can then be deployed in subsequent small molecule screens to allow the detection of molecular probes which affect:

1) exon skipping (both endogenous and artificial/exogenous);

2) muscle membrane stability;

3) surface glycosylation and stability of the dystroglycan complex;

4) myocyte cell calcineurin/NFAT signal transduction; and

5) fibrosis.

These assays are highly portable and thus can be applied broadly in various high throughput screening facilities which have different sets of small compounds for testing.   Additionally, we propose to pilot the most promising and well developed assay system on an existing library of 500 known biologically active compounds in the UCLA Molecular Screening Facility. The development of cellullar reporter assays which model processes relevant to muscular dystrophies will enable high throughput screens of small molecule libraries for the identification of lead compounds which can purturb these processes.  Such compounds have tremendous potential as probes for understanding normal and pathologic muscle cell biology and in the development of novel therapeutics for DMD.