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 orient 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 orient 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 was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the Ki 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.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This research was supported by the Robert J. Kleberg Jr. and Helen C. Kleberg Foundation; the Alzheimer's Drug Discovery Foundation; a Neurosciences Victoria/Victorian State Government Science, Technology, and Innovation Grant; the National Health and Medical Research Council (NHMRC) [Development Grants 454714 and 520695]; an NHMRC Peter Doherty fellowship (to V.P.); an Australian Research Council Federation Fellowship (to M.W.P.); an NHMRC Honorary Fellowship (to M.W.P.); and an NHMRC Senior Research Fellowship (to S.Y.C.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.065458.
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ABBREVIATIONS:
- IRAP
- insulin-regulated aminopeptidase
- APN
- aminopeptidase N
- Ang IV
- angiotensin IV
- AVP
- arginine8 vasopressin
- HEK
- human embryonic kidney
- HFI
- Howard Florey Institute
- HFI-142
- ethyl 2-amino-7-hydroxy-4-pyridin-3-yl-4H-chromene-3-carboxylate
- HFI-419
- ethyl 2-acetylamino-7-hydroxy-4-pyridin-3-yl-4H-chromene-3-carboxylate
- HFI-435
- ethyl 2-amino-7-hydroxy-4-quinolin-3-yl-4H-chromene-3-carboxylate
- HFI-437
- ethyl 2-acetylamino-7-hydroxy-4-quinolin-3-yl-4H-chromene-3-carboxylate
- Leu-MCA
- l-leucine-4-methyl-7-coumarinylamide
- Leu-Enk
- leu-enkephalin
- LTA4H
- leukotriene A4 hydrolase
- LVV-H7
- Leu-Val-Val-hemorphin-7
- MCA
- 7-amino-4-methylcoumarin
- TFA
- trifluoroacetic acid.
- Received April 7, 2010.
- Accepted July 2, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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