一种激活整合应激反应的新型天冬酰胺合成酶抑制剂的表征

Asparagine synthetase (ASNS) catalyzes the biosynthesis of L-asparagine (L-Asn) from L-aspartate, L-glutamine, and ATP, and plays a central role in anticancer therapies such as L-asparaginase treatment of acute lymphoblastic leukemia. In response to L-Asn depletion, ASNS induction is a key effector of the GCN2-ATF4 arm of the integrated stress response (ISR), enabling tumor cells to maintain amino acid homeostasis under limiting nutrient conditions. However, direct ASNS targeting has long been hindered by the poor cellular potency of existing inhibitors. Here, we delineate the molecular and cellular mechanism of ASX-173, a cell-penetrant ASNS inhibitor with potent biochemical and cellular activity with the potential to starve tumors of asparagine and enhance the impact of combination therapies targeting tumor metabolism. Using HEK293A cells cultured without exogenous L-Asn, ASX-173 markedly reduced intracellular L-Asn levels, activated the ISR, and stimulated ATF4 transcriptional activity. ISR induction was reversed by physiological levels of L-Asn, confirming on-target activity. Deletion of GCN2 lowered basal ASNS expression, impaired ATF4 induction, and sensitized cells to ASX-173. Similarly, the GCN2 inhibitor GCN2iB suppressed ASX-173-induced ATF4 transcriptional activity and synergized to inhibit the growth of multiple cancer lines, including renal (RENCA) and prostate (Myc-CaP) models. Cell-based thermal protein profiling experiments confirmed direct ASNS binding, shifting its melting temperature from 45 °C to 54 °C. Using recombinant ASNS, ASX-173 inhibited L-Asn production without affecting L-glutamate and showed reduced activity at the Km for Mg²⁺-ATP. Differential scanning fluorimetry experiments demonstrated that binding of ASX-173 to ASNS requires Mg²⁺-ATP. Moreover, a 2.58 Å cryo-EM structure revealed ASX-173 bound in the C-terminal synthetase subdomain alongside AMP, PPi, and two Mg²⁺ ions, stabilized by hydrophobic, π-interactions, and hydrogen bonding with AMP to form a composite pocket. The structure indicates that ASX-173 promotes ATP hydrolysis and blocks ammonia transfer, supporting an uncompetitive mechanism. These findings establish ASX-173 as a potent ASNS inhibitor with strong cellular target engagement and ISR-inducing activity. The heightened sensitivity of GCN2-deficient cells and the synergy with GCN2 inhibition highlight the therapeutic potential of dual ISR targeting and support ASNS inhibition-alone or in combination with ISR-modulating strategies-as a promising approach for cancers dependent on asparagine metabolism.