In vitro activity: ML414 (also known NGI-1) is a novel and cell-permeable inhibitor of oligosaccharyltransferase (OST), which is a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. ML414 was identified from a cell-based high-throughput screen and lead-compound-optimization campaign. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.

Kinase Assay: The HTS approach using the bioluminescent N-linked glycosylation reporter in D54-ERLucT and D54-LucT cells has been previously described. Briefly, the primary cell-based screen detects N-linked glycan site occupancy using a modified and ER translated luciferase protein with three N-linked glycosylation consensus sequons. Inhibition of glycosylation in D54-ERLucT restores and increases luciferase activity over controls whereas it does not increase activity in the non-ER translated D54-LucT cell line. The methodology for the primary (D54-ERlucT), secondary false positive (D54-LucT), and tertiary (luciferase inhibition) screens as well as toxicity assays with CellTitre Glo are deposited in Pubchem (AID 588693). Genedata Screener software with the Smartfit algorithm was used for to generate AC40 values for comparative analysis of analogs.

Cell Assay: In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.