Selective inhibition of indoleamine and tryptophan 2,3-dioxygenases: Comparative study on kynurenine pathway in cell lines via LC-MS/MS-based targeted metabolomics
Over the past decade, the kynurenine pathway, the main metabolic route for tryptophan (TRP) degradation, has gained significant attention in pharmaceutical sciences due to its involvement in immune regulation and cancer immunoediting. In this context, the development of cell-based assays offers a valuable tool to: (i) characterize the secretome of different cell types; (ii) deepen our understanding of the role of kynurenines in various disease conditions; and (iii) screen inhibitors of human indoleamine 2,3-dioxygenase (hIDO1) while assessing their impact on downstream TRP-metabolizing enzymes. This study presents a validated Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) method for the simultaneous quantification of TRP, L-kynurenine (KYN), xanthurenic acid (XA), 3-hydroxykynurenine (3OHKYN), kynurenic acid (KA), 3-hydroxyanthranilic acid (3OHAA), anthranilic acid (AA), 5-hydroxytryptamine (serotonin, 5HT), and tryptamine (TRYP) in Dulbecco’s Modified Eagle Medium (DMEM) and Eagle’s Minimum Essential Medium (EMEM). The method was validated following FDA, ICH, and EMA guidelines and was subsequently applied to: (i) evaluate the effects of selective inhibition of hIDO1 or human tryptophan 2,3-dioxygenase (hTDO) on the kynurenine pathway in A375 (melanoma), MDA-MB-231 (breast cancer), and U87 (glioblastoma) cell lines using multivariate analysis (MVA); and (ii) determine the IC50 values of well-known (epacadostat, linrodostat) and novel (BL5) hIDO1 inhibitors in these cell lines. The proposed LC-MS/MS method is robust, reliable, and highly adaptable, making it well-suited for preclinical drug research and in vitro assays.