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. 2024 Apr 25;187(9):2209-2223.e16.
doi: 10.1016/j.cell.2024.03.022.

Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB

Benjamin L Lampson  1 , Ana S Ramίrez  2 , Marta Baro  3 , Lixia He  1 , Mudra Hegde  4 , Vidyasagar Koduri  5 , Jamie L Pfaff  1 , Ruth E Hanna  4 , Julia Kowal  2 , Nitin H Shirole  1 , Yanfeng He  1 , John G Doench  4 , Joseph N Contessa  3 , Kaspar P Locher  6 , William G Kaelin Jr  7
Affiliations

Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB

Benjamin L Lampson et al. Cell. .

Abstract

Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.

Keywords: CRISPR screening; N-glycosylation; NF-κB; Toll-like receptors; inhibition mechanism; oligosaccharyltransferase.

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Conflict of interest statement

Declaration of interests B.L.L. is currently a paid employee of Blueprint Medicines. M.H. is currently a paid employee of Thermo Fisher Scientific. W.G.K. is a paid advisor to Casdin Capital, Circle Pharma, FibroGen, Nextech Invest, and Tango Therapeutics. W.G.K. receives compensation for serving as a board director for Eli Lilly and Company, IconOVir Bio, and LifeMine Therapeutics.

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