ABSTRACT The tryptophan degradation pathway is used to prevent unrestrained immune activation in healthy cells. However, tumors hijack this mechanism to escape immune surveillance. The key enzymes of this pathway, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), are established immune checkpoint proteins. Tumors enhance their expression to block T cell proliferation and induce T cell death, thereby avoiding immune system surveillance and increasing tumor cell migration capacity. Thus, inhibiting these checkpoint enzymes in cancer cells by small molecule-based therapies has emerged as a potential immunotherapeutic strategy. There is a critical need for new agents developed from an innovative approach with a solid understanding of their underlying chemistry and biology, to advance the overall scientific knowledge and to ultimately help the public live long healthy lives. From a joint basic science-clinical study we identified a non- catalytic L-tryptophan (L-Trp) binding site in human TDO, which binds L-Trp surprisingly much tighter than the catalytic heme site. The newly discovered L-Trp binding site is involved in regulating TDO activity and stability by suppressing ubiquitin-dependent degradation when loaded with L-Trp. This finding has inspired us to propose a central hypothesis that this newly discovered signaling site is an Achilles' heel of TDO for drug development. This application will fill the critical need to identify protein-degrading ligands for exploring their biomedical potential. Towards this end, we will design compounds with a novel mode of action that destabilize the signaling site of TDO or bind without enhancing the protein stability. These agents will not target the catalytic activity of TDO but instead will disrupt its degradation resistance signal. We will assess the effects of promising compounds on human TDO in cellular models to validate the innovative approach and target. In the end, this work will open the door for designing revolutionarily new inhibitors targeting the immune checkpoint protein human TDO.