The long term goal of our research is to discover effective treatments for muscular dystrophies. A number of muscular dystrophies (MD) are associated with abnormal O-mannosylation of alpha dystroglycan (1-DG) that plays a central role in linking the extracellular matrix (ECM) to the actin-based cytoskeleton in muscle. Hypoglycosylation of 1-DG impairs its ability to bind to the ECM proteins such as laminins and results in pathogenesis in the muscle and nerve systems and often causes devastating diseases in humans. Currently, there is no specific treatment for any form of MD. Increasing evidence suggests that over-expression of glycosyltransferases enhancing the glycosylation of 1-DG can restore the functions of 1-DG in MD mouse models. We hypothesize that cells can enhance glycosylation of 1-DG in response to certain chemical stimuli. We recently developed a cell-based assay for determination of O-mannosylation of alpha-DG (ADOMA). With ADOMA, we are able to quantitatively monitor the levels of O-mannosylation of 1-DG and its functional binding to the ligands, laminins, on the cell surface with remarkable reliability and reproducibility. This assay has great potential to become a high throughput screening (HTS) platform. A pilot screening of 2000 bioactive compounds with ADOMA has been conducted to test our hypothesis and the performance of ADOMA under HTS conditions. The result has shown that the performance of the pilot screen is acceptable for HTS. Furthermore, we have identified several positive hits which are able to enhance the functional binding of laminins to the cell surface, thus proved our hypothesis. However, the current ADOMA screening procedure requires multiple washing steps and need to be reconfigured to HTS friendly format for large scale HTS campaign. In this project, thus, we will focus on the specific aim: Reconfiguration of ADOMA into a homogeneous, miniaturizable, and wash-free HTS assay. We plan to integrate homogeneous time-resolved fluorescence (HTRF) methodology into our HTS assay to develop HTRF based HTS assay.
The goal of this project is to discover effective treatments for muscular dystrophies. A number of muscular dystrophies (MD) are associated with deficiency of sugar chains synthesis. The abnormality of sugar chain on alpha dystroglycan, a key protein in muscle, results in pathogenesis in the muscle and nerve systems and often causes devastating diseases in humans. Currently, there is no specific treatment for any form of MD. This project aims to develop a drug discovery assay to identify compounds and develop drug for MD.
| Lv, Fengping; Li, Zhi-Fang; Hu, Wenhao et al. (2015) Small molecules enhance functional O-mannosylation of Alpha-dystroglycan. Bioorg Med Chem 23:7661-70 |
