Characterization of Apo‐Form Selective Inhibition of Indoleamine 2,3‐Dioxygenase**

Abstract

Indoleamine‐2,3‐dioxygenase 1 (IDO1) is a heme‐containing enzyme that catalyzes the rate‐limiting step in the kynurenine pathway of tryptophan (TRP) metabolism. As an inflammation‐induced immunoregulatory enzyme, pharmacological inhibition of IDO1 activity is currently being pursued as a potential therapeutic tool for the treatment of cancer and other disease states. As such, a detailed understanding of the mechanism of action of established and novel IDO1 inhibitors remains of great interest. Comparison of a newly‐developed IDO1 inhibitor (GSK5628) to the existing best‐in‐class compound, epacadostat (Incyte), allows us to report on a unique inhibition mechanism for IDO1. Here, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme‐free conformation of the enzyme (apo‐IDO1) while epacadostat coordinates its binding with the iron atom of the IDO1 heme cofactor. Comparison of these two compounds in cellular systems reveals a long‐lasting inhibitory effect of GSK5628, undescribed for other known IDO1 inhibitors. Detailed characterization of this apo‐binding mechanism for IDO1 inhibition may help design superior inhibitors or may confer a unique competitive advantage over other IDO1 inhibitors vis‐á‐vis specificity and pharmacokinetic parameters.