Discovery of Selective Nanomolar Inhibitors for Insulin-Regulated Aminopeptidase Based on α-Hydroxy-β-amino Acid Derivatives of Bestatin

Abstract

The oxytocinase subfamily of M1 zinc aminopeptidases comprises emerging drug targets, including the ER-resident aminopeptidases 1 and 2 (ERAP1 and ERAP2) and insulin-regulated aminopeptidase (IRAP); however, reports on clinically relevant inhibitors are limited. Here we report a new synthetic approach of high diastereo- and regioselectivity for functionalization of the α-hydroxy-β-amino acid scaffold of bestatin. Stereochemistry and mechanism of inhibition were investigated by a high-resolution X-ray crystal structure of ERAP1 in complex with a micromolar inhibitor. By exploring the P1 side-chain functionalities, we achieve significant potency and selectivity, and we report a cell-active, low-nanomolar inhibitor of IRAP with >120-fold selectivity over homologous enzymes. X-ray crystallographic analysis of IRAP in complex with this inhibitor suggest that interactions with the GAMEN loop is an unappreciated key determinant for potency and selectivity. Overall, our results suggest that α-hydroxy-β-amino acid derivatives may constitute useful chemical tools and drug leads for this group of aminopeptidases.

Stabilization of the open conformation of Insulin-Regulated Aminopeptidase by a novel substrate-selective small molecule inhibitor

“…The overall conformation of the enzyme is distinct from all previously published structures determined in complex with active site binding small-molecule inhibitors and corresponds to the open conformation in which domain IV moves away from domains I and II using domain III as a hinge (Figure 5). The observed conformation is consistent with the original template used for the virtual screening which corresponds to the open conformation of IRAP but comes in sharp contrast with all other known structures of IRAP with small inhibitors occupying the active site (PDB IDs: 6YDX,7ZYF, 5MJ6) 19,20,31 , suggesting that the previously stipulated hypothesis that active-site binding induces conformational closing of the enzyme may not hold true for all ligands 19,20 . To help visualize the differences in relative domain orientation, lines between Glu661-Asp341 and Glu661-Lys911 are drawn and the angle between them is indicated.…”

Hydrogen Peroxide Induced Toxicity Is Reversed by the Macrocyclic IRAP-Inhibitor HA08 in Primary Hippocampal Cell Cultures

“…To conclude, inhibitors of IRAP, such as Ang IV, improve memory and cognition in animal models, and the aminopeptidase IRAP is recognized as a new potential target for drugs aimed at treatment of cognitive disorders [ 2 , 9 , 16 , 17 , 18 , 19 , 37 , 38 ]. As a consequence, in recent years a large number of IRAP inhibitors have been reported from different laboratories [ 9 , 22 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ] and HA08, a macrocyclic disulfide analogue of the unstable endogenous Ang IV, has been identified as one of the most potent IRAP inhibitors known to date [ 12 , 34 ]. HA08 enhances dendritic spine density in rat hippocampal primary cultures [ 30 ], and as reported herein, restores cell viability by increasing the mitochondrial activity in primary hippocampal cultures after hydrogen-peroxide-induced damage.…”

“…These observations stimulated an interest in developing improved IRAP inhibitors, more potent than the short-lived endogenous Ang IV, as a new class of cognitive enhancers and as potential therapeutics for treating a variety of cognitive disorders such as Alzheimer’s disease [ 22 , 23 , 24 ]. A large series of IRAP inhibitors have been reported as well as support for the hypothesis that these types of compounds could serve as suitable starting points for drug discovery projects aimed at developing cognitive enhancers useful in clinic [ 9 , 22 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ]. A characteristic feature of IRAP is the ability to bind to cyclic substrates [ 33 ].…”

Inhibition of Insulin-Regulated Aminopeptidase by Imidazo [1,5-α]pyridines—Synthesis and Evaluation

“…These include compounds incorporating β -homo amino acids and constrained amino acid analogs of Ang IV, or peptide-like transition state mimicking compounds. Notably, these inhibitors have consistently demonstrated excellent potency and a relatively good selectivity profile toward other aminopeptidases [ 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. Our group has used the structures of Ang IV and the IRAP substrate oxytocin to design both linear and macrocyclic inhibitors with high potency and stability [ 38 , 39 , 40 ].…”