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 (TDO). 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. Amongst them, artemether, a member of the artemisinin family of anti-malarial drugs, suppressed kynurenine production, likely via an endoperoxide bridge-dependent mechanisms. The Euphorbia factor L9 (EFL9) inhibited kynurenine production likely via a C7-benzoylation-dependent mechanism. Neither artemether nor EFL9 affected JAK/STAT signaling 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.