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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, Mg\n                  <jats:sup>2+<\/jats:sup>\n                  , and pyrophosphate in the C-terminal synthetase domain of ASNS, thereby enabling multivalent, high-affinity binding. Based on\n                  <jats:italic>in vitro<\/jats:italic>\n                  kinetic and thermal shift assays, we find that ASX-173 binds to the ASNS\/Mg\n                  <jats:sup>2+<\/jats:sup>\n                  \/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.\n                <\/jats:p>","DOI":"10.1101\/2025.10.16.682859","type":"posted-content","created":{"date-parts":[[2025,10,17]],"date-time":"2025-10-17T04:40:14Z","timestamp":1760676014000},"source":"Crossref","is-referenced-by-count":1,"title":["Cryo-EM reveals how ASX-173 inhibits human asparagine synthetase to activate the integrated stress response"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8722-9585","authenticated-orcid":false,"given":"Kirk A.","family":"Staschke","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2375-6599","authenticated-orcid":false,"given":"Nicholas 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