Abstract C060: Discovery of a novel IL4I1 inhibitor by high-throughput screening of the SymeGold library

Abstract Immune checkpoint inhibitors, such as anti-PD1, anti-CTLA4 and anti-LAG3 antibodies, have significantly improved outcomes in patients with various solid tumors. However, their effectiveness is influenced by local amino acid levels, in particular tryptophan, and derived metabolites, which are regulated by enzymes in the tumor microenvironment. IDO1 is a key immunosuppressive enzyme that depletes tryptophan and produces metabolites that activate the aryl hydrocarbon receptor (AhR), thereby promoting immune tolerance. Although IDO1 inhibitors were tested in clinical trials to enhance immune checkpoint inhibitor efficacy, the combination of the IDO1 inhibitor epacadostat and the anti-PD1 antibody pembrolizumab failed in a phase III trial, showing no added benefit of the combination in comparison to pembrolizumab monotherapy. The enzyme interleukin 4 induced 1 (IL4I1) metabolizes aromatic amino acids, i.e., phenylalanine, tryptophan and tyrosine, into their α-keto acids. Its role in immune modulation is not fully understood, but it is found that tryptophan metabolites generated by IL4I1 can act as ligands of the AhR. To investigate the role of IL4I1 in cancer immunotherapy, we developed a fluorescent biochemical assay for high-throughput screening of small-molecule inhibitors. Recombinant human IL4I1 was expressed in CHO cells. Kinetic studies showed that IL4I1 has similar affinity (K M) for its three main substrates, but highest activity with phenylalanine, and the assay was optimized accordingly. The assay was validated using reference inhibitors of IL4I1, and was subsequently used to screen a proprietary collection of low molecular weight synthetic chemicals (the SymeGold library). This screen revealed a hit with a novel molecular structure, which was subsequently confirmed via newly developed cellular assays in the endogenously IL4I1 expressing cell line L-428 and in an IL4I1 transfected HEK293 cell line. The hit showed no inhibition in IDO1, IDO2 and TDO biochemical assays. Analogs are being synthesized for further testing in cancer models to evaluate the therapeutic potential of IL4I1 inhibition. Citation Format: Guido Zaman, Jay Beerden, Sjoerd van der Leeuw, Jacob Ytsma, Yorik Bruseker, Leon van Berkum, Koen Hekking, Yvonne Grobben. Discovery of a novel IL4I1 inhibitor by high-throughput screening of the SymeGold library [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C060.