Novel Quinic Acid Glycerates from Tussilago farfara Inhibit Polypeptide GalNAc-Transferase.

The discovery of a bioactive inhibitor tool for human polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts), the initiating enzyme for mucin-type O-glycosylation, remains challenging. In the present study, we identified an array of quinic acid derivatives, including four new glycerates (1-4) from Tussilago farfara, a traditional Chinese medicinal plant, as active inhibitors of GalNAc-T2 using a combined screening approach with a cell-based T2-specific sensor and purified enzyme assay. These inhibitors dose-dependently inhibited human GalNAc-T2 but did not affect O-linked N-acetylglucosamine transferase (OGT), the other type of glycosyltransferase. Importantly, they are not cytotoxic and retain inhibitory activity in cells lacking elongated O-glycans, which are eliminated by the CRISPR/Cas9 gene editing tool. A structure-activity relationship study unveiled a novel quinic acid-caffeic acid conjugate pharmacophore that directs inhibition. Overall, these new natural product inhibitors could serve as a basis for developing an inhibitor tool for GalNAc-T2.

Inhibitor of ppGalNAc-T3-mediated O-glycosylation blocks cancer cell invasiveness and lowers FGF23 levels.

Small molecule inhibitors of site-specific O-glycosylation by the polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) family are currently unavailable but hold promise as therapeutics, especially if selective against individual ppGalNAc-T isozymes. To identify a compound targeting the ppGalNAc-T3 isozyme, we screened libraries to find compounds that act on a cell-based fluorescence sensor of ppGalNAc-T3 but not on a sensor of ppGalNAc-T2. This identified a hit that subsequent in vitro analysis showed directly binds and inhibits purified ppGalNAc-T3 with no detectable activity against either ppGalNAc-T2 or ppGalNAc-T6. Remarkably, the inhibitor was active in two medically relevant contexts. In cell culture, it opposed increased cancer cell invasiveness driven by upregulated ppGalNAc-T3 suggesting the inhibitor might be anti-metastatic. In cells and mice, it blocked ppGalNAc-T3-mediated glycan-masking of FGF23 thereby increasing its cleavage, a possible treatment of chronic kidney disease. These findings establish a pharmacological approach for the ppGalNAc-transferase family and suggest that targeting specific ppGalNAc-transferases will yield new therapeutics.

Small molecule inhibitors of mucin-type O-linked glycosylation from a uridine-based library.

The polypeptide N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs, also abbreviated ppGaNTases) initiate mucin-type O-linked glycosylation and therefore play pivotal roles in cell-cell communication and protection of tissues. In order to develop new tools for studying mucin-type O-linked glycosylation, we screened a 1338 member uridine-based library to identify small molecule inhibitors of ppGalNAcTs. Using a high-throughput enzyme-linked lectin assay (ELLA), two inhibitors of murine ppGalNAcT-1 (K(I) approximately 8 microM) were identified that also inhibit several other members of the family. The compounds did not inhibit other mammalian glycosyltransferases or nucleotide sugar utilizing enzymes, suggesting selectivity for the ppGalNAcTs. Treatment of cells with the compounds abrogated mucin-type O-linked glycosylation but not N-linked glycosylation and also induced apoptosis. These uridine analogs represent the first generation of chemical tools to study the functions of mucin-type O-linked glycosylation.