The signaling function of IDO1 incites the malignant progression of mouse B16 melanoma.

Indoleamine 2,3 dioxygenase 1 (IDO1), a leader tryptophan-degrading enzyme, represents a recognized immune checkpoint molecule. In neoplasia, IDO1 is often highly expressed in dendritic cells infiltrating the tumor and/or in tumor cells themselves, particularly in human melanoma. In dendritic cells, IDO1 does not merely metabolize tryptophan into kynurenine but, after phosphorylation of critical tyrosine residues in the non-catalytic small domain, it triggers a signaling pathway prolonging its immunoregulatory effects by a feed-forward mechanism. We here investigated whether the non-enzymatic function of IDO1 could also play a role in tumor cells by using B16-F10 mouse melanoma cells transfected with either the wild-type Ido1 gene (Ido1WT ) or a mutated variant lacking the catalytic, but not signaling activity (Ido1H350A ). As compared to the Ido1WT -transfected counterpart (B16WT), B16-F10 cells expressing Ido1H350A (B16H350A) were characterized by an in vitro accelerated growth mediated by increased Ras and Erk activities. Faster growth and malignant progression of B16H350A cells, also detectable in vivo, were found to be accompanied by a reduction in tumor-infiltrating CD8+ T cells and an increase in Foxp3+ regulatory T cells. Our data, therefore, suggest that the IDO1 signaling function can also occur in tumor cells and that alternative therapeutic approach strategies should be undertaken to effectively tackle this important immune checkpoint molecule.

Identification of a 2-propanol analogue modulating the non-enzymatic function of indoleamine 2,3-dioxygenase 1.

Indoleamine 2,3 dioxygenase 1 (IDO1) is a metabolic enzyme that catalyzes the conversion of the essential amino acid tryptophan (Trp) into a series of immunoactive catabolites, collectively known as kynurenines. Through the depletion of Trp and the generation of kynurenines, IDO1 represents a key regulator of the immune responses involved in physiologic homeostasis as well as in neoplastic and autoimmune pathologies. The IDO1 enzyme has been described as an important immune checkpoint to be targeted by catalytic inhibitors in the treatment of cancer. In contrast, a defective expression/activity of the enzyme has been demonstrated in autoimmune diseases. Beside its catalytic activity, the IDO1 protein is endowed with an additional function associated with the presence of two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which, once phosphorylated, bind SHP phosphatases and mediate a long-term immunoregulatory activity of IDO1. Herein, we report the screening of a focused library of molecules bearing a propanol core by a protocol combining microscale thermophoresis (MST) analysis and a cellular assay. As a result, the combined screening identified a 2-propanolol analogue, VIS351, as the first potent activator of the ITIM-mediated function of the IDO1 enzyme. VIS351 displayed a good dissociation constant (Kd = 1.90 µM) for IDO1 and a moderate cellular inhibitor activity (IC50 = 11.463 µM), although it did not show any catalytic inhibition of the recombinant IDO1 enzyme. Because we previously demonstrated that the enzymatic and non-enzymatic (i.e., ITIM-mediated) functions of IDO1 reside in different conformations of the protein, we hypothesized that in the cellular system VIS351 may shift the dynamic conformational balance towards the ITIM-favoring folding of IDO1, resulting in the activation of the signaling rather than catalytic activity of IDO1. We demonstrated that VIS351 activated the ITIM-mediated signaling of IDO1 also in mouse plasmacytoid dendritic cells, conferring those cells an immunosuppressive phenotype detectable in vivo. Thus the manuscript describes for the first time a small molecule as a positive modulator of IDO1 signaling function, paving the basis for an innovative approach to develop first-in-class drugs acting on the IDO1 target.