Discovery of Selective Inhibitor Leads by Targeting an Allosteric Site in Insulin-Regulated Aminopeptidase

Abstract

Insulin-Regulated aminopeptidase (IRAP) is a zinc-dependent aminopeptidase with several important biological functions and is an emerging pharmaceutical target for cognitive enhancement and immune system regulation. Aiming to discover lead-like IRAP inhibitors with enhanced selectivity versus homologous enzymes, we targeted an allosteric site at the C-terminal domain pocket of IRAP. We compiled a library of 2.5 million commercially available compounds from the ZINC database, and performed molecular docking at the target pocket of IRAP and the corresponding pocket of the homologous endoplasmic reticulum aminopeptidase 1 (ERAP1). Of the top compounds that showed high selectivity, 305 were further analyzed by molecular dynamics simulations and free energy calculations, leading to the selection of 33 compounds for in vitro evaluation. Two orthogonal functional assays were employed: one using a small fluorogenic substrate and one following the degradation of oxytocin, a natural peptidic substrate of IRAP. In vitro evaluation suggested that several of the compounds tested can inhibit IRAP, but the inhibition profile was dependent on substrate size, consistent with the allosteric nature of the targeted site. Overall, our results describe several novel leads as IRAP inhibitors and suggest that the C-terminal domain pocket of IRAP is a promising target for developing highly selective IRAP inhibitors.

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

“…It is, therefore, important to emphasize that we have previously demonstrated that 6a binds reversibly to IRAP through jump-dilution experiments [ 49 ]. Although there are literature examples of non-competitive binding also for active site binders [ 54 ], these data, together with the observed selectivity, required us to consider an allosteric binding site alongside the active site model [ 55 ]. To enable structure-guided compound optimization and to firmly establish the inhibitor binding site, we have initiated co-crystallization efforts with the recombinant human protein [ 10 ].…”

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 ].…”

The Discovery of New Inhibitors of Insulin-Regulated Aminopeptidase by a High-Throughput Screening of 400,000 Drug-like Compounds

“…More recently, our group screened a library of 10,500 small molecules and identified various lead compounds [44], including arylsulfonamides [45], that enhance dendritic spine density in primary hippocampal neuron cultures [46], and un-competitive IRAP inhibitors encompassing a spiro-oxindole dihydroquinazolinone scaffold [47] and an imidazo [1,5α]pyridine scaffold [48]. Additional selective inhibitor leads targeting an allosteric site in IRAP were identified after applying a virtual docking approach [49], and it was reported that modulators of the structurally related hERAP2 have also been identified from a highthroughput screen (HTS) [50].…”