Characterization of a Novel Small Molecule Inhibitor of Asparagine Synthetase

Abstract

Asparagine synthetase (ASNS) is a protein in human cells that is responsible for synthesizing asparagine (ASN), a nonessential amino acid, from glutamine and aspartate. ASNS is frequently overexpressed in cancers, leading to increased growth and resistance to nutrient deprivation. There has been a demand for an effective pharmacological inhibitor of ASNS to starve the tumors by preventing ASN synthesis. In this study, the ASNS specific inhibitor ASX-173 was utilized to determine how human embryonic kidney 293A (HEK293A) cells would respond to inhibition of ASNS in ASN-depleted conditions. Given that amino acid (AA) depletion is a well-documented inducer of GCN2 eIF2 kinase and the integrated stress response (ISR), we utilized multiple cell-based techniques to determine how the cells were responding to the stress of ASNS inhibition. This study showed that biomarkers of ISR, including phosphorylated GCN2 and its substrate eIF2α, ATF4, GADD34, and TRIB3 were all increased in the HEK293A cells treated with ASX-173. The ISR induction by the ASNS inhibitory drug was also supported by the increased expression of ATF4, GADD34, and TRIB3 mRNAs, along with luciferase reporter assays showing that ASX-173 induced ATF4 transcriptional activity. ASX-173 induction of the ISR was dependent on GCN2 activity and was reversed by the addition of exogenous L-Asparagine. It is noteworthy that the levels of ASNS protein and mRNA were not affected by ASX-173. Cell growth was greatly diminished during ASX-173 exposure in ASN-depleted conditions. Emphasizing the importance of GCN2 and the ISR for resistance to amino acid limitation, the growth reduction of HEK293A cells with the combined treatment of ASX-173 was accentuated with the combined addition of GCN2iB, a potent inhibitor of GCN2. These results support the idea that the pharmacological ASNS inhibition could be utilized to treat and eliminate certain types of cancers and other diseases where nutrient starvation is a potent strategy.