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Introduction: Indoleamine 2,3-dioxygenase (IDO1) is a heme-containing oxidoreductase enzyme that converts L-tryptophan (Trp) into N’-formylkynurenine (NFK). IDO1 is broadly expressed by tumor cells as well as immune cells in the tumor microenvironment of many cancers, which is often correlated with poor prognosis. Depletion of Trp and increased L-kynurenine (Kyn) levels induce immune tolerance by suppression of effector T-cell and natural killer cell functions, and activation of regulatory T-cells and myeloid-derived suppressor cells. Four small molecule inhibitors are currently investigated in phase III (linrodostat/BMS-986205), phase II (epacadostat/INCB024360) or phase I (MK7162 and LY3381916) clinical trials. Linrodostat, epacadostat and LY3381916 are reported as being selective for IDO1 over TDO, while details on MK7162 have not yet been disclosed. The compounds inhibit IDO1 with different mechanisms, with epacadostat binding to the heme-group in the catalytic center of IDO1, while linrodostat and LY3381916 bind to apo-IDO1 and compete with heme for the active site [1-3]. Experimental procedures Clinical and reference IDO1 inhibitors were characterized in biochemical assays for IDO1 and tryptophan 2,3-dioxygenase (TDO), a structurally different enzyme, which also catalyzes the conversion of Trp to NFK. Furthermore, the compounds were profiled in a panel of functional cell-based assays, including human cancer cell lines and assays based on IDO1- or TDO2-overexpressing HEK293 cells. A IDO1-expressing subline of the mouse B16F10 melanoma cell line was generated and used to develop a syngeneic mouse model at Charles River Laboratories (USA) to determine target modulation in vivo [4]. Stable gene expression was confirmed by qPCR and target modulation was examined by measurement of Trp and Kyn levels using LC-MS/MS. Results: Linrodostat has sub-nanomolar cellular potency, despite the absence of any biochemical activity on the timescale of our assays, which is consistent with its reported heme-competing mechanism of inhibition [1]. Linrodostat also inhibits different Cytochrome P450 enzymes with micromolar activity. In our biochemical assays, epacadostat was not selective for IDO1 over TDO, whereas in the IDO1- and TDO2-overexpressing HEK293 cell lines it was 2000 times selective for IDO1. High level expression of IDO1 in B16F10 cells did not result in enhanced tumor growth after grafting in syngeneic mice, which contrasts published data with a similar model [4]. Nonetheless, we observed strong modulation of Trp and Kyn levels in plasma and in tumors of the IDO1-overexpressing mouse model, compared to non-tumor bearing mice. Treatment of the B16F10-IDO1 model with epacadostat did not result in a reduction of tumor growth, though epacadostat did induce clear changes in Trp and Kyn in both plasma and tumor tissue. Conclusion: Our comparative study of the potencies and selectivities of IDO1 inhibitors, as well as our model for measuring in vivo target modulation, helps to identify strengths and weaknesses of current IDO1 inhibitors, and supports the development of new inhibitors. [1] Nelp et al. (2018) Proc. Natl.Acad. Sci. U.S.A. 115, 3249-3254; [2] Yue et al. (2017) ACS Med. Chem. Lett. 8, 486-491; [3] Dorsey et al. (2018) Proceedings: AACR Annual Meeting 2018, Abstract nr. 5245; [4] Holmgaard et al. (2015) Cell Rep. 13, 412-424. Citation Format: Yvonne Grobben, Joost C.M. Uitdehaag, Antoon M. van Doornmalen, Nicole Willemsen-Seegers, Diep Vu-Pham, Jos de Man, Rogier C. Buijsman, Guido J.R. Zaman. Side-by-side comparison of small molecule IDO1 inhibitors in biochemical and cell-based assays and development of a IDO1-expressing mouse model to evaluate target modulation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B060. doi:10.1158/1535-7163.TARG-19-B060