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 site-directed 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 orientate with the benzopyran oxygen interacting with the Zn²⁺ ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast the two 4-quinolinyl derivatives orientate in a different manner interacting with the Zn²⁺ 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 is substrate dependent. Phe544 is also important for inhibitor binding as 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.