Drug-like modulators of the large enzyme family that initiates site-specific O-glycosylation in the Golgi complex (UDP-N-acetyl-?-D-galactosamine polypeptide N-acetyl-galactosaminyltransferases or GalNAc-Ts) hold promise as entirely new therapeutics for major diseases such as osteoporosis, dyslipidemia, heart disease, chronic obstructive lung disease, cancer, and viral outbreaks. Significantly, there are currently no known inhibitors or activators of these initiating enzymes. To address this shortcoming we developed cell-based fluorescent sensors to be used for high-throughput screening to identify isoform-specific, small molecule modulators of GalNAc-T2 and GalNAcT3 mediated O-glycosylation. We will carryout simultaneous screening of compounds with these sensors, which will greatly minimize off-target effects allowing identification of candidates that directly target either enzyme. Preliminary work including a pilot screen has resulted in a few promising candidates arguing that further screening will be successful. Hits will be validated using, among other tests, biochemical assays with purified enzyme preparations. Beyond the scope of this proposal, we plan structural characterization and optimization of any lead compounds as well as initial tests of therapeutic value. Successful identification of isoform-specific modulators promises to be transformative to glycobiology research, and potentially, the clinic.
Site-specific O-glycosylation is a modification of proteins that controls their activities in crucial ways. Some diseases mentioned in the proposal are caused by misregulation of this process and could be treated by drug- like modulators of site-specific O-glycosylation. Other diseases, viral outbreaks being an example, depend on this process to carryout their destruction. So again, drugs targeting site-specific O-glycosylation could provide an effective therapy. Interestingly, a family of closely related isozymes initiates site-specific O-glycosylation with individual isozymes acting on distinct sets of proteins. This avails a therapeutic opportunity in the sense that a single disease-relevant isozyme may be targeted leaving the others to carryout their myriad of functions critical to normal health. But, crucially, there are no known drugs targeting these isozymes. We are developing a new tool that ?reads out? the activity in living cells of the individual isozymes initiating site-specific O- glycosylation. It is easy to use, inexpensive, and adaptable to pretty much any cellular system. Using the tool, we will initiate a search for compounds that modify the activity of individual isozymes, which could then be developed into effective therapeutics.
| Simon, Emily J; Linstedt, Adam D (2018) Site-specific glycosylation of Ebola virus glycoprotein by human polypeptide GalNAc-transferase 1 induces cell adhesion defects. J Biol Chem 293:19866-19873 |
| Song, Lina; Linstedt, Adam D (2017) Inhibitor of ppGalNAc-T3-mediated O-glycosylation blocks cancer cell invasiveness and lowers FGF23 levels. Elife 6: |
| Song, Lina; Bachert, Collin; Linstedt, Adam D (2016) Activity Detection of GalNAc Transferases by Protein-Based Fluorescence Sensors In Vivo. Methods Mol Biol 1496:123-31 |
