Artemether and Euphorbia factor L9 suppress kynurenine production through distinct effects on tryptophan metabolism.

l-Tryptophan (Trp) is an essential amino acid catabolised through the kynurenine pathway, which is mediated by the enzymes indoleamine-2,3-dioxygenase 1 (IDO1), IDO2, or Trp-2,3-deoxygenase. In cancer, IDO1 acts as an immune checkpoint, suppressing effector T cell function. Yet, direct inhibition of IDO1 has had limited success in clinical trials. Therefore, alternative approaches to Trp metabolism therapeutic targeting are needed. We screened a library of 597 natural products (NPs) or NP derivatives for their effect on kynurenine production in triple-negative breast cancer cells. This revealed 24 candidate inhibitors of kynurenine production. Among them, artemether, a member of the artemisinin family of anti-malarial drugs, suppressed kynurenine production, likely via an endoperoxide bridge-dependent mechanism. The Euphorbia factor L9 (EFL9) inhibited kynurenine production, likely via a C7-benzoylation-dependent mechanism. Neither artemether nor EFL9 affected JAK/STAT signalling or IDO1 levels. Targeted metabolomics and molecular docking analyses demonstrated that artemether suppressed kynurenine production through heme sequestration and potential interactions with the IDO1 heme-binding pocket A. EFL9 affected Trp metabolism through heme-independent mechanisms and resulted in changes in purine and amino acid metabolism and the cellular redox balance. Notably, ouabain, a regulator of IDO1 levels, and linrodostat, a clinically approved IDO1 inhibitor, revealed distinct metabolic profiles, with ouabain and EFL9 showing the largest overlap. Importantly, the kynurenine-suppressing activities of artemether and EFL9 were observed in non-transformed primary mammary epithelial cells and also lung cancer cells. Overall, our findings set the foundation for future studies exploring the use of artemether or EFL9 as novel Trp metabolism-targeting therapeutics.