Structure-based virtual screening identifies small-molecule inhibitors of O-fucosyltransferase SPINDLY in Arabidopsis
Yalikunjiang Aizezi, Hongming Zhao, Zhenzhen Zhang, Yang Bi, Qiuhua Yang, G. Guo, Hongliang Zhang, Hongwei Guo, Kai Jiang, Zhiyong Wang
Abstract
Protein O-glycosylation is a nutrient signaling mechanism that plays an essential role in maintaining cellular homeostasis across different species. In plants, SPINDLY (SPY) and SECRET AGENT (SEC) posttranslationally modify hundreds of intracellular proteins with O-fucose and O-linked N-acetylglucosamine, respectively. SPY and SEC play overlapping roles in cellular regulation, and loss of both SPY and SEC causes embryo lethality in Arabidopsis (Arabidopsis thaliana). Using structure-based virtual screening of chemical libraries followed by in vitro and in planta assays, we identified a SPY O-fucosyltransferase inhibitor (SOFTI). Computational analyses predicted that SOFTI binds to the GDP-fucose-binding pocket of SPY and competitively inhibits GDP-fucose binding. In vitro assays confirmed that SOFTI interacts with SPY and inhibits its O-fucosyltransferase activity. Docking analysis identified additional SOFTI analogs that showed stronger inhibitory activities. SOFTI treatment of Arabidopsis seedlings decreased protein O-fucosylation and elicited phenotypes similar to the spy mutants, including early seed germination, increased root hair density, and defective sugar-dependent growth. In contrast, SOFTI did not visibly affect the spy mutant. Similarly, SOFTI inhibited the sugar-dependent growth of tomato (Solanum lycopersicum) seedlings. These results demonstrate that SOFTI is a specific SPY O-fucosyltransferase inhibitor that can be used as a chemical tool for functional studies of O-fucosylation and potentially for agricultural management.