L-Trp is an essential amino acid necessary for protein synthesis in mammalian cells, and the L-Trp to kynurenine (Kyn) pathway is firmly established as a key regulator of innate and adaptive immunity. Catabolism of L-Trp to Kyn maintains an immunosuppressive microenvironment by starving immune cells of L-Trp and releasing degradation products of L-Trp that have immunosuppressive functions. Indoleamine 2,3- dioxygenases (IDO1 & IDO2), two of the rate limiting enzymes in this pathway, are upregulated in many tumors, providing cancer cells with an avenue for immune evasion.

Inhibitors of IDO1 can bind either irreversibly (covalent binding to the protein) or reversibly (non-covalent association) to the enzyme. The IDO1 Inhibitor Mechanism of Action Assay Kit allows researchers to determine the mechanism of IDO1 inhibitor binding. At high concentrations, e.g., 10x the IC50 concentration of the inhibitor, both reversible and irreversible inhibitors will interfere with IDO1 activity. However, after sufficient dilution, e.g. to 0.3x the IC50 concentration, reversible inhibitors will dissociate, relieving inhibition of the IDO1 enzyme. Irreversible inhibitors will not dissociate, and will continue to inhibit the IDO1 enzyme at the same level. Therefore, by monitoring enzymatic activity after inhibitor dilution, the mechanism of binding to the IDO1 protein can be determined.