Phenylalanine-544 Plays a Key Role in Substrate and Inhibitor Binding by Providing a Hydrophobic Packing Point at the Active Site of Insulin-Regulated Aminopeptidase

Abstract

Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and sitedirected mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orient with the benzopyran oxygen interacting with the Zn 2ϩ ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast, the two 4-quinolinyl derivatives orient in a different manner, interacting with the Zn 2ϩ ion via the quinoline nitrogen, and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine, or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the K i in some cases, and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis.

Inhibition of Insulin‐Regulated Aminopeptidase (IRAP) by Arylsulfonamides

“…Furthermore, the compound is stacked between Phe 472 (IRAP: Phe 544) and Phe 896 (IRAP: Tyr 961) in the active site. The stacking interaction with Phe 544 in IRAP has previously been reported as a key interaction for ligand and substrate binding . Two of the amino acids in contact with compound 3 differ between APN and IRAP.…”

Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides

“…Furthermore, all three enzymes fail to process substrates with side chains that carry beta-carbon or oxygen branching (such as valine, isoleucine or threonine) due to the limited space in the S1 pocket and the stringent stereochemical requirements for the recognition of the N-terminus of the substrate by the conserved GAMEN motif. Phenylalanine 544 plays a key role in this phenomenon and is critical for enzyme activity [40]. These findings however, raise a crucial question.…”

Discovery of Inhibitors of Insulin-Regulated Aminopeptidase as Cognitive Enhancers

“…The compounds bind with low affinity to structurally related enzymes, such as AP-N and leukotriene A 4 hydrolase itself, despite the latter having been used in the homology modelling [186]. Computational docking suggests that the S -isomer is the preferred binding mode in all examples, and two alternate binding conformations for these structurally analogous inhibitors have been proposed [187]. …”

“…It has been predicted that the quinoline compounds are more active than the pyridinyl compounds, partly due to a more favourable coordination to the zinc ion in IRAP. In addition, computational docking experiments have suggested that Phe 544 of IRAP provides an important hydrophobic packing point at one side of the active site [187]. No comparative modelling has been carried out, but it is tempting to suggest that the N-terminal of Ang IV, the macrocyclic compound 13 and the quinoline HIF-437 bind to IRAP as shown in Figure 15.…”