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N-Glycosylation is one of the most important posttranslational modifications, and the disturbance of N-glycosylation can lead to serious diseases. Identification of potent N-glycosylation inhibitors is a promising strategy for novel drug design to treat these diseases. However, the current N-glycosylation assays with high throughput screening (HTS) still depend on many traditional methods, which is a time-consuming process and lack automation. Herein, we developed an HTS assay of N-glycosylation by cell-based HTS (cell-HTA) using Saccharomyces cerevisiae mutant strain deficient in N-glycan processing to measure the inhibition of N-glycosylation. The assay uses a self-made microplate reader to automatically screen for yeast mutants that are defective in N-glycan processing by plate imaging. Finally, the inhibitory effects of nine molecules (candidate N-glycosylation inhibitors) on the N-glycosylation of cell surface-exposed α-1,6-mannose and/or β-1,2-xylose residues in living yeast cells were examined. The microplate reader-based HTS assay for the cell-HTA platform was then validated for specificity and reproducibility. Our results show that this cell-HTA platform has the potential for identification of N-glycosylation inhibitors and is a promising alternative method for the rapid screening of N-glycosylation-inhibitory agents.//
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