Drug discovery for dystroglycanopathies via LARGE promoter activation screening

The project aims to establish and validate a system of screening of compounds able to induce an increase of the protein levels of the N-acetylglucosaminyltransferase LARGE in myoblastic cells. LARGE is an enzyme involved in the glycosylation of α-dystroglycan (α-DG), a transmembrane protein expressed in myoblastic and neural cells. A-DG, once appropriately glycosylated, binds to extracellular matrix ligands. Loss of this binding causes muscle membrane and cytoskeletal instability and may trigger secondary damage events which ultimately lead to the death of the muscle fibers. Dystroglycanopathies are congenital muscular dystrophies due to mutations in eight glycosyltransferases or accessory proteins and are associated to α-DG hypoglycosylation. The clinical phenotype can be extremely severe with central nervous system, eyes abnormalities and early infantile death. The mildest end of the clinical spectrum may present in adult life with just a limb girdle muscular dystrophy phenotype. Several previous descriptions in literature have shown that up-regulation of LARGE protein levels can functionally bypass α-DG hypoglycosylation caused by defects in other glycosyltransferases, thus improving the muscle diseases. However, no drug or compound able to stimulate LARGE has been identified yet. Toward this objective, we propose to utilize a high-throughput screening of small compounds or existing drugs able to activate LARGE promoter and hence LARGE transcription. This methodology has the advantage to investigate in a short time a large collection of chemicals. However, the screens require a robust and reproducible signal which has to be designed accurately in a first phase of the project. In this exploratory grant, we plan to develop and test all the optimal conditions required for the establishment of an accurate assay for LARGE promoter activation in myoblastic cells. The actual screening, hit confirmation, and validation will be the object of our future application.