Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells

Abstract

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, NGI-1. NGI-1 targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. 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.

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Figure 1: HTS for inhibitors of N-linked glycosylation.
Figure 2: NGI-1 blocks LLO transfer and hydrolysis.
Figure 3: NGI-1 blocks OST function.
Figure 4: NGI-1 disrupts EGFR glycosylation and cell-surface expression.
Figure 5: NGI-1 blocks RTK-driven proliferation.
Figure 6: NGI-1 induces G1 arrest and senescence in EGFR addicted tumor cells.

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Acknowledgements

This work was funded by US National Institutes of Health (NIH) R03DA033178 and R01CA172391, and in part by a Research Scholar Grant from the American Cancer Society (J.N.C.) and by NIH RO1GM43768 (R.G.) and RO1GM038545 (M.A.L.). Additional funding was provided by the NIH-MLPCN program U54HG005031 (Kansas University) and U54HG005032 (Broad Institute). The mass spectrometry work was supported by NIH grant P41GM103490.

Author information

C.L.-S. designed and performed the cell biology experiments in NSCLC cells and contributed to analysis and interpretation of all presented data. N.R. performed and analyzed the CTSA assays; J.C.C. performed experiments in CHO and Lec cells. J.N.C. designed the study and was involved in all experimental designs, data analysis, and data interpretation. S.S. and R.G. designed and performed in vitro glycosylation studies. C.K. and T.A.L. performed and triaged the HTS; D.P.F. and J.E.G. performed analysis of SAR and synthesis of chemical analogs; N.G. and M.A.L. performed the LLO analysis; P.Z. and L.W. collected and analyzed the MS data. All authors contributed to writing the manuscript.

Correspondence to Joseph N Contessa.

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Competing interests

J.N.C. and J.E.G. are listed as inventors on a provisional patent application for the analogs reported in this manuscript.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–9 and Supplementary Tables 1–6. (PDF 7267 kb)

Supplementary Note

Synthetic Procedures. (PDF 7757 kb)

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Lopez-Sambrooks, C., Shrimal, S., Khodier, C. et al. Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells. Nat Chem Biol 12, 1023–1030 (2016). https://doi.org/10.1038/nchembio.2194

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