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

Abstract

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.DOI: http://dx.doi.org/10.7554/eLife.24051.001

The Multiplicity of Polypeptide GalNAc‐Transferase: Assays, Inhibitors, and Structures

“…The isozyme‐specific sensor for GalNAc‐T is an invaluable tool for illustrating the cellular activity of GalNAc‐T and for evaluating the specificity and efficiency of GalNAc‐T inhibitors in cells. Indeed, the authors have applied this method to discover the first specific inhibitor for GalNAc‐T3 (for details, see the discussion on the inhibitor in Section 4.1. and Table ) …”

“…The limitation of detection of UDP in the luminescence‐based assay is in the sub‐micromolar range. Additionally, the assay kit has been successfully employed in academia to monitor the activities of GalNAc‐T2 and GalNAc‐T3 …”

“…Furthermore, a few direct inhibitors have been designed and developed for GalNAc‐Ts, and most of them are not potent or bioactive in cells. In the following section, we itemized the available inhibitors for GalNAc‐Ts (Table ).…”

“…In the following section, we itemized the available inhibitors for GalNAc‐Ts (Table ). These inhibitors include a synthetic inhibitor for GalNAc‐T3, natural product inhibitors, and UDP‐GalNAc‐analogue‐based pan‐inhibitors …”

“…. Upon screening 21 710 synthetic compounds, they identify one selective inhibitor for GalNAc‐T3, namely, T3Inh‐1, that can inhibit cellular GalNAc‐T3 activity determined by the GalNAc‐T3‐based fluorescence sensor with the half maximal inhibitory concentration (IC 50 ) value of 12 μ m . Moreover, T3Inh‐1 dose‐dependently inhibits GalNAc‐T3 activity with an IC 50 of 7 μ m without affecting the activity of GalNAc‐T2 or GalNAc‐T6 at a concentration up to 50 μ m in an in vitro purified enzyme coupling assay that has been commercially developed by the Promega Corporation (for details of the assay, see Section 3.1.).…”