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bioRxiv · 10.1101/2025.10.16.682859

Cryo-EM reveals how ASX-173 inhibits human asparagine synthetase to activate the integrated stress response

Staschke, K. A.·Walda, N. T.·Pearce, L. A.·Wek, R. C.·Zhu, W.·Takagi, Y.
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Abstract

Targeting asparagine metabolism is a promising strategy for treating asparaginase-resistant acute lymphoblastic leukemia (ALL), sarcoma, and potentially other solid tumors. Here, we characterize the molecular mechanism by which a cell-penetrable small molecule, ASX-173, inhibits human asparagine synthetase (ASNS), the enzyme that catalyzes intracellular asparagine biosynthesis. ASX-173 reduces cellular asparagine levels, induces the integrated stress response (ISR), and reduces cell growth in HEK-293A cells. A cryo-EM structure reveals that ASX-173 engages a unique, hydrophobic pocket formed by AMP, Mg2+, and pyrophosphate in the C-terminal synthetase domain of ASNS, thereby enabling multivalent, high-affinity binding. Based on in vitro kinetic and thermal shift assays, we find that ASX-173 binds to the ASNS/Mg2+/ATP complex and is therefore a rare example of an uncompetitive enzyme inhibitor with potential therapeutic use. These findings provide a structural and mechanistic basis for targeting ASNS with small molecules, which have application in treating cancer and other human diseases.

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Staschke, K. A., Walda, N. T., Pearce, L. A., Wek, R. C., Zhu, W., Takagi, Y.. 2025-10-16. Cryo-EM reveals how ASX-173 inhibits human asparagine synthetase to activate the integrated stress response. https://doi.org/10.1101/2025.10.16.682859

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