Effect of Molecular Crowding Agents on the Activity and Stability of Immunosuppressive Enzyme Indoleamine 2,3‐Dioxygenase 1

Indoleamine 2,3‐dioxygenase 1 (IDO1) plays pivotal role in regulating the metabolism of L‐tryptophan (L‐Trp) through kynurenine pathway, which is a well‐established therapeutic target for the treatment of diseases associated with immunosuppression. Disproportionate expression of the enzyme and poor prediction of various types of malignancies make the development and clinical trials of IDO1 inhibitors extremely relevant. However, IDO1‐based drug development has been hindered due to the use of idealized dilute buffer solution media during the screening and optimization of inhibitors, which do not reflect the highly crowded intracellular environment. In this report, IDO1 enzyme activity and its inhibitory activity against few potent compounds of N′ ‐hydroxyamidine and aryl‐substituted pyran scaffolds were investigated under macromolecularly crowded environment. The synthetic crowding agents were used to generate a cellular mimetic condition where IDO1 enzyme was found to retain its activity. A non‐linear relationship between the size and volume of the crowding agent with enzyme kinetic parameters was observed. The results suggest that judicious use of size and volume of the crowding agent could provide cellular mimetic environment. A very similar catalytic efficiency, inhibitory activity along with preservation of secondary structural content of IDO1 enzyme under the selected crowded media makes these crowding agents appropriate for further therapeutic application.