Insulin-Regulated Aminopeptidase (IRAP, EC 3.4.11.3) is a transmembrane zinc metalloprotease that belongs to the M1 family of metalloproteases and in particular in the oxytocinase subfamily.1 IRAP has been reported to have several important biological functions that include roles in glucose metabolism through the regulation of trafficking of glucose transporter 4,2 the degradation of placental oxytocin levels,3 the regulation of immune responses through the generation of antigenic peptides or effects on T-cell receptor signalling,4, 5 as well as roles in cognition through the regulation of brain oxytocin and vasopressin.6, 7 IRAP is a transmembrane protein consisting of a small cytoplasmic domain that plays roles in intracellular trafficking and a large extra-cellular domain that carries the aminopeptidase activity. Several potential physiological substrates have been identified to mediate IRAP biological functions. For example, IRAP's role in adaptive immunity is driven by the trimming of precursor antigenic peptides in specialized compartments of antigen-presenting cells performing cross-presentation.8 Other biological functions of IRAP are likely mediated by the degradation of peptidic hormones. Notably, IRAP can degrade macrocyclic peptides such as oxytocin and vasopressin in the placenta or the brain.3, 9 The latter likely mediates the role of IRAP in cognitive functions.10 Other possible substrates include angiotensin III, metenkephalin, dynorphin A, neurokinin A, neuromedin B, and somatostatin.11 Regulation of IRAP’s enzymatic activity for pharmaceutical applications using small molecular weight inhibitors has attracted significant experimental efforts, in particular in the area of cognition and treatment of ischemic stroke.10, 12, 13, 14, 15, 16 In addition, circulating, soluble IRAP is being actively explored as a biomarker for diabetes, cardio-metabolic diseases and ischemic stroke.17, 18

Over the last decade, several inhibitors of IRAP have been developed and tested in a variety of cellular and in vivo systems. These include pseudophosphinic peptide transition-state-analogues, aryl sulfonamides, benzopyran derivatives, diaminobenzoic acid derivatives and cyclic peptide analogues.10, 13, 19, 20, 21, 22, 23, 24, 25 IRAP inhibitors have been discovered either after rational substrate-inspired design or after random chemical or virtual library screening. In both cases, the proposed mechanism of action is direct competition with the substrate for the catalytic site of the enzyme. Two main classes of IRAP inhibitors discovered by library screening are aryl sulfonamides and benzopyran derivatives.24, 25 Compounds from both chemical scaffolds have been shown to be active in cellular systems relating to IRAP biological activities. In particular, aryl sulfonamide inhibitors as well as the benzopyran derivative HFI-419 have been shown to enhance spatial working memory by promoting the formation of functional dendritic spines in primary hippocampal neuron cultures, and this effect was proposed to rely on enhanced glucose uptake.14, 26 Most notable, HFI-419 has been utilized in numerous in vivo studies and its activity has been associated with biological functions of IRAP such as acetylcholine-mediated vasoconstriction,27 glucose tolerance in insulin-resistant Zucker fatty rats,28 glucose handling in normal and diabetic rats,29in vivo Leucine Enkephalin Hydrolysis30 and ischemic stroke.16 Despite these results, however, no clinical application of an IRAP inhibitor has been reported yet.

Of the IRAP inhibitors reported, aryl sulfonamides and benzopyran derivatives, such as compound HFI-419, are of particular interest pharmacologically because they carry a good selectivity profile, and they are not of peptidic nature and thus can avoid common pharmacokinetic pitfalls of peptidic inhibitors. The mechanism of action of these compounds has been investigated by biochemical and computational studies and the most commonly proposed mode of action is a competitive mechanism through the occupation of the catalytic site and chelation of the active-site zinc(II) atom by the sulfonamide group and either an oxygen atom in position 3 or the nitrogen atom of the pyridine ring of the benzopyran derivative.24, 29, 31, 32, 33 In one study, alternative non-competitive mechanisms of inhibition were suggested for specific analogues but experiments were carried out using membrane preparations of the enzyme complicating interpretation.14 For benzopyran derivatives, site-directed mutagenesis suggested that Phenylalanine-544, a residue lining the S1 specificity pocket of the enzyme, is important for inhibitor binding by forming p-stacking interactions with the main aromatic rings of the compound.33 These mechanistic insights are guiding the further development of these two chemical scaffolds.

The extra-cellular aminopeptidase component of IRAP has been studied by X-ray crystallography revealing four structural domains that organize into a concave structure that hosts the catalytic site adjacent to an internal large cavity.34, 35 One of those structures contained a peptidic substrate analogue that extended from the catalytic site, throughout the length of the cavity towards the external solvent.34 Structures solved more recently, in complex with a phosphinic pseudopeptide inhibitor, a macrocyclic peptidic inhibitor and a bestatin analogue, revealed that IRAP can change conformation and assume a more closed configuration in which the active site and the adjacent large cavity are isolated in the interior of the protein with no direct access to the external solvent.36, 37, 38 Significant structural reconfigurations in the active site, including the reorientation of a catalytic Tyrosine residue and the GAMEN motif, were linked to this conformational change and proposed to differentiate the two conformations in terms of enzymatic activity, with the closed conformation considered to be more active due to optimized configuration of its catalytic site elements. Analysis by small-angle X-ray scattering suggested that IRAP is primarily in the open conformation in solution and undergoes conformational closing upon inhibitor binding.37 The role and importance of this conformational flexibility to the mechanism of inhibition by non-peptidic classes of inhibitors have not been investigated.

In this study, we explored the mechanism of action of an aryl-sulfonamide inhibitor of IRAP as well as the benzopyran derivative HFI-419, which is widely used in in vivo experiments as a tool compound. By a combination of enzymatic kinetic analyses and x-ray crystallography, we show that, in contrast to what has been proposed before, both compounds do not operate by binding in the active site and engaging the catalytic zinc(II) atom, but rather bind to nearby allosteric sites. In particular, HFI-419 appears to lock the enzyme to the open and less active conformation by bridging domains I and IV and sterically blocking further closing. Surprisingly, although HFI-419 is a potent inhibitor of a small dipeptidic substrate commonly used in previous studies, it was found to be unable to block the degradation of the physiological IRAP peptide substrate, oxytocin, an observation consistent with its binding site and preferred conformation. Our data, taken together, allow us to propose a structural and kinetic framework for IRAP function and inhibition and suggest that in vivo and in cellulo results obtained using HFI-419 may need to be re-evaluated in the context of targeted physiological IRAP substrates.