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 it is 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 IDO1 inhibitors with various mechanisms of inhibition is of great interest. Comparison of an apo‐form‐binding IDO1 inhibitor (GSK5628) to the heme‐coordinating compound, epacadostat (Incyte), allows us to explore the details of the apo‐binding inhibition of IDO1. Herein, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme‐free conformation of the enzyme (apo‐IDO1), whereas 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, previously undescribed for other known IDO1 inhibitors. Detailed characterization of this apo‐binding mechanism for IDO1 inhibition might help design superior inhibitors or could confer a unique competitive advantage over other IDO1 inhibitors vis‐à‐vis specificity and pharmacokinetic parameters.

The [1,2,4]Triazolo[4,3‐a]pyridine as a New Player in the Field of IDO1 Catalytic Holo‐Inhibitors

“…While apo inhibitors show a lack of inhibition in the enzyme‐based assay in face of a high cellular activity, [18,19,45] VS9 provided an rhIDO1 inhibition of 54.6±3.1 % (see also Supporting Information), comparable to the result from the cellular‐based assay. To further confirm the absence of binding to the apo‐form, the interference of the compound with the amount of free heme produced after incubation with IDO1 was evaluated [19] . Similarly to epacadostat, VS9 did not increase the levels of free heme (see Supporting Information), confirming the molecule as a catalytic holo‐inhibitor.…”

“…rhIDO1 was treated with 10 μM of VS9 and the conversion of L-Trp to L-Kyn was determined spectrophotometrically using p-dimethylaminobenzaldehyde. While apo inhibitors show a lack of inhibition in the enzymebased assay in face of a high cellular activity, [18,19,45] VS9 provided an rhIDO1 inhibition of 54.6 � 3.1 % (see also Supporting Information), comparable to the result from the cellularbased assay. To further confirm the absence of binding to the apo-form, the interference of the compound with the amount of free heme produced after incubation with IDO1 was evaluated.…”

“…Absorbance at 570 nm was measured on a Tecan microplate reader. Values are reported relative to the untreated sample and are the mean of n=3±SD [19] …”

Structural Basis of Selective Human Indoleamine‐2,3‐dioxygenase 1 (hIDO1) Inhibition

“…Röhrig et al., [67] Ortiz‐Moez et al., [68] and others [69] described Linrodostat as an apo‐form IDO1 inhibitor standing for the kinetic study results and spectroscopic determinations [67–68] . Moreover, Ortiz‐Moez's study [68] raised the concern of the physiological conditions that support the existence of IDO1 in apo‐form more than the holo‐form, to justify obtaining results agree with Nelp et al. study [35] and disagrees with the Pham et al.…”

A Complex Metabolic Network Confers Immunosuppressive Functions to Myeloid-Derived Suppressor Cells (MDSCs) within the Tumour Microenvironment

“…However, a number of clinical trials were terminated due to the lack of reported benefits [150], thus suggesting the need to establish a novel strategy to block L-Trp catabolism. Interestingly, a novel inhibitor of IDO1 that blocks the binding of the haem-free conformation of the enzyme (apo-IDO1) has been identified (GSK5628) and was recently synthesised [151]. In comparison to traditional drugs that bind to the iron atom of the IDO1 heme cofactor (holo-IDO1), this compound guarantees a durable inhibitory effect.…”