Molecular Mechanism of the Immunomodulatory Drug Halofuginone

Cellular sensing of amino acid availability has recently emerged as an important mechanism of immunomodulation. Small molecules that manipulate amino acid metabolism serve as powerful tools for studying these poorly characterized signaling pathways. The compound halofuginone, derived from an herb in traditional Chinese medicine, induces a cellular state of proline starvation by inhibiting the prolyl-tRNA synthetase domain of EPRS. The resulting accumulation of uncharged prolyl-tRNA correlated with inhibition of Th17 differentiation and protection against autoimmune disease. Halofuginone invoked the canonical amino acid starvation response (AAR) by increasing phosphorylation of GCN2 and eIF2α to adapt to nutrient stress. However, knockout of GCN2 failed to fully account for the anti-inflammatory properties of halofuginone and did not rescue Th17 differentiation, suggesting immunomodulation through non-canonical AAR pathways. Since GCN2-independent AAR pathways are better characterized in yeast than in mammals, we identified and investigated their orthologs. Co-immunoprecipitation studies validated the formation of an orthologous human GCN1/RWDD1/DRG2 complex. Knockdown of the adaptor protein RWDD1 in fibroblast-like synoviocytes reduced the ability of halofuginone to suppress CXCL10 and MMP-13 expression during TNF-α-induced inflammation. A non-canonical AAR pathway involving GCN1, RWDD1, and DRG2 could expand our understanding of how amino acid metabolism regulates immunity and may reveal new drug targets for immunotherapy.