Katarzyna B. Handing, Mu-Sen Liu, Douglas A. Whittington, Sining Sun, Rebecca Salerno, William D. Mallender, Jon Come, Scott Throner, Andrew Maynard, Patrick McCarren, John P. Maxwell, Serge Gueroussov, Kiera Vassallo, Yingnan Chen, Jannik N. Andersen, Wenhai Zhang

Molecular Cancer Therapeutics

DOI: 10.1158/1535-7163.targ-25-c104

Abstract

MGAT1 is an N-glycosyltransferase essential for the synthesis of N-glycans. Cell surface glycans serve as immune checkpoints, playing a key role in cancer immune evasion. Knockout (KO) of MGAT1 enhances immune recognition and promotes T-cell–mediated killing, with enzymatic activity being necessary for this phenotype. These findings position MGAT1’s catalytic function as an attractive target for cancer therapy. In this study, we report the discovery of novel MGAT1 binders and inhibitors with sub-micromolar potency. Compounds were identified through two independent screening approaches: a UDP-Glo^TM-based high-throughput screen (HTS) measuring inhibition of MGAT1 enzymatic activity, and a DNA-encoded library (DEL) screen selecting for molecules that reproducibly bind to MGAT1. High-resolution crystal structures reveal detailed interactions between MGAT1 and the compounds, clearly identifying a binding site distinct from the active site. Surface plasmon resonance (SPR) competition assays further demonstrate that these inhibitors bind noncompetitively with respect to the endogenous product, UDP. Together, these results validate allosteric inhibition of MGAT1 as a novel and tractable strategy for impeding MGAT1 activity. Additionally, our findings lay the foundation for future structure-based optimization of MGAT1 inhibitors with potential application in cancer immunotherapy.