Insulin-regulated Aminopeptidase Inhibitors Research Articles

Stabilization of the open conformation οf insulin-regulated aminopeptidase by a novel substrate-selective small-molecule inhibitor.

Insulin-regulated aminopeptidase (IRAP) is an enzyme with important biological functions and the target of drug-discovery efforts. We combined in silico screening with a medicinal chemistry optimization campaign to discover a nanomolar inhibitor of IRAP based on a pyrazolylpyrimidine scaffold. This compound displays an excellent selectivity profile versus homologous aminopeptidases, and kinetic analysis suggests it utilizes an uncompetitive mechanism of action when inhibiting the cleavage of a typical dipeptidic substrate. Surprisingly, the compound is a poor inhibitor of the processing of the physiological cyclic peptide substrate oxytocin and a 10mer antigenic epitope precursor but displays a biphasic inhibition profile for the trimming of a 9mer antigenic peptide. While the compound reduces IRAP-dependent cross-presentation of an 8mer epitope in a cellular assay, it fails to block in vitro trimming of select epitope precursors. To gain insight into the mechanism and basis of this unusual selectivity for this inhibitor, we solved the crystal structure of its complex with IRAP. The structure indicated direct zinc(II) engagement by the pyrazolylpyrimidine scaffold and revealed that the compound binds to an open conformation of the enzyme in a pose that should block the conformational transition to the enzymatically active closed conformation previously observed for other low-molecular-weight inhibitors. This compound constitutes the first IRAP inhibitor targeting the active site that utilizes a conformation-specific mechanism of action, provides insight into the intricacies of the IRAP catalytic cycle, and highlights a novel approach to regulating IRAP activity by blocking its conformational rearrangements.

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

With the ambition to identify novel chemical starting points that can be further optimized into small drug-like inhibitors of insulin-regulated aminopeptidase (IRAP) and serve as potential future cognitive enhancers in the clinic, we conducted an ultra-high-throughput screening campaign of a chemically diverse compound library of approximately 400,000 drug-like small molecules. Three biochemical and one biophysical assays were developed to enable large-scale screening and hit triaging. The screening funnel, designed to be compatible with high-density microplates, was established with two enzyme inhibition assays employing either fluorescent or absorbance readouts. As IRAP is a zinc-dependent enzyme, the remaining active compounds were further evaluated in the primary assay, albeit with the addition of zinc ions. Rescreening with zinc confirmed the inhibitory activity for most compounds, emphasizing a zinc-independent mechanism of action. Additionally, target engagement was confirmed using a complementary biophysical thermal shift assay where compounds causing positive/negative thermal shifts were considered genuine binders. Triaging based on biochemical activity, target engagement, and drug-likeness resulted in the selection of 50 qualified hits, of which the IC50 of 32 compounds was below 3.5 µM. Despite hydroxamic acid dominance, diverse chemotypes with biochemical activity and target engagement were discovered, including non-hydroxamic acid compounds. The most potent compound (QHL1) was resynthesized with a confirmed inhibitory IC50 of 320 nM. Amongst these compounds, 20 new compound structure classes were identified, providing many new starting points for the development of unique IRAP inhibitors. Detailed characterization and optimization of lead compounds, considering both hydroxamic acids and other diverse structures, are in progress for further exploration.

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

Inhibition of insulin-regulated aminopeptidase (IRAP) has been shown to improve cognitive functions in several animal models. Recently, we performed a screening campaign of approximately 10,000 compounds, identifying novel small-molecule-based compounds acting as inhibitors of the enzymatic activity of IRAP. Here we report on the chemical synthesis, structure-activity relationships (SAR) and initial characterization of physicochemical properties of a series of 48 imidazo [1,5-α]pyridine-based inhibitors, including delineation of their mode of action as non-competitive inhibitors with a small L-leucine-based IRAP substrate. The best compound displays an IC50 value of 1.0 µM. We elucidate the importance of two chiral sites in these molecules and find they have little impact on the compound's metabolic stability or physicochemical properties. The carbonyl group of a central urea moiety was initially believed to mimic substrate binding to a catalytically important Zn2+ ion in the active site, although the plausibility of this binding hypothesis is challenged by observation of excellent selectivity versus the closely related aminopeptidase N (APN). Taken together with the non-competitive inhibition pattern, we also consider an alternative model of allosteric binding.

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

Angiotensin IV (Ang IV), a metabolite of Angiotensin II, is a bioactive hexapeptide that inhibits the insulin-regulated aminopeptidase (IRAP). This transmembrane zinc metallopeptidase with many biological functions has in recent years emerged as a new pharmacological target. IRAP is expressed in a variety of tissues and can be found in high density in the hippocampus and neocortex, brain regions associated with cognition. Ang IV is known to improve memory tasks in experimental animals. One of the most potent IRAP inhibitors known today is the macrocyclic compound HA08 that is significantly more stable than the endogenous Ang IV. HA08 combines structural elements from Ang IV and the physiological substrates oxytocin and vasopressin, and binds to the catalytic site of IRAP. In the present study we evaluate whether HA08 can restore cell viability in rat primary cells submitted to hydrogen peroxide damage. After damaging the cells with hydrogen peroxide and subsequently treating them with HA08, the conceivable restoring effects of the IRAP inhibitor were assessed. The cellular viability was determined by measuring mitochondrial activity and lactate dehydrogenase (LDH) release. The mitochondrial activity was significantly higher in primary hippocampal cells, whereas the amount of LDH was unaffected. We conclude that the cell viability can be restored in this cell type by blocking IRAP with the potent macrocyclic inhibitor HA08, although the mechanism by which HA08 exerts its effects remains unclear.

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

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.

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

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.

Enhanced recombinant expression and purification of human IRAP for biochemical and crystallography studies

Insulin-regulated aminopeptidase (IRAP) in humans is a membrane bound enzyme that has multiple functions. It was first described as a companion protein of the insulin-responsive glucose transporter, Glut4, in specialized vesicles. The protein has subsequently been shown to be identical to the oxytocinase/aminopeptidase or the angiotensin IV (Ang IV) receptor (AT4 receptor). Some AT4 ligand peptides, such as Ang IV and LVV-hemorphin-7, have been shown to act as IRAP inhibitors that exert memory-enhancing properties. As such IRAP has been a target for developing cognitive enhancers. To facilitate detailed mechanistic studies of IRAP catalysis and inhibition, and to pave the way for biophysical and structural studies of IRAP in complex with peptide inhibitors, we report here an optimized expression and purification system using High Five insect cells. We also report biochemical characterizations of the purified recombinant IRAP with a standard aminopeptidase substrate and an optimized IRAP peptide inhibitor with a Ki of 98 nM.

Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Benzopyran-Based Inhibitors.

Inhibition of the insulin-regulated aminopeptidase (IRAP) improves memory and cognition in animal models. The enzyme has recently been crystallized and several series of inhibitors reported. We herein focused on one series of benzopyran-based inhibitors of IRAP known as the HFI series, with unresolved binding mode to IRAP, and developed a robust computational model to explain the structure-activity relationship (SAR) and potentially guide their further optimization. The binding model here proposed places the benzopyran ring in the catalytic binding site, coordinating the Zn2+ ion through the oxygen in position 3, in contrast to previous hypothesis. The whole series of HFI compounds was then systematically simulated, starting from this binding mode, using molecular dynamics and binding affinity estimated with the linear interaction energy (LIE) method. The agreement with experimental affinities supports the binding mode proposed, which was further challenged by rigorous free energy perturbation (FEP) calculations. Here, we found excellent correlation between experimental and calculated binding affinity differences, both between selected compound pairs and also for recently reported experimental data concerning the site directed mutagenesis of residue Phe544. The computationally derived structure-activity relationship of the HFI series and the understanding of the involvement of Phe544 in the binding of this scaffold provide valuable information for further lead optimization of novel IRAP inhibitors.

IRAP Endosomes Control Phagosomal Maturation in Dendritic Cells.

Dendritic cells (DCs) contribute to the immune surveillance by sampling their environment through phagocytosis and endocytosis. We have previously reported that, rapidly following uptake of extracellular antigen into phagosomes or endosomes in DCs, a specialized population of storage endosomes marked by Rab14 and insulin-regulated aminopeptidase (IRAP) is recruited to the nascent antigen-containing compartment, thereby regulating its maturation and ultimately antigen cross-presentation to CD8+ T lymphocytes. Here, using IRAP–/– DCs, we explored how IRAP modulates phagosome maturation dynamics and cross-presentation. We find that in the absence of IRAP, phagosomes acquire more rapidly late endosomal markers, are more degradative, and show increased microbicidal activity. We also report evidence for a role of vesicle trafficking from the endoplasmic reticulum (ER)–Golgi intermediate compartment to endosomes for the formation or stability of the IRAP compartment. Moreover, we dissect the dual role of IRAP as a trimming peptidase and a critical constituent of endosome stability. Experiments using a protease-dead IRAP mutant and pharmacological IRAP inhibition suggest that IRAP expression but not proteolytic activity is required for the formation of storage endosomes and for DC-typical phagosome maturation, whereas proteolysis is required for fully efficient cross-presentation. These findings identify IRAP as a key factor in cross-presentation, trimming peptides to fit the major histocompatibility complex class-I binding site while preventing their destruction through premature phagosome maturation.

From Angiotensin IV to Small Peptidemimetics Inhibiting Insulin-Regulated Aminopeptidase.

It was reported three decades ago that intracerebroventricular injection of angiotensin IV (Ang IV, Val-Tyr-Ile-His-Pro-Phe) improved memory and learning in the rat. There are several explanations for these positive effects of the hexapeptide and related analogues on cognition available in the literature. In 2001, it was proposed that the insulin-regulated aminopeptidase (IRAP) is a main target for Ang IV and that Ang IV serves as an inhibitor of the enzyme. The focus of this review is the efforts to stepwise transform the hexapeptide into more drug-like Ang IV peptidemimetics serving as IRAP inhibitors. Moreover, the discovery of IRAP inhibitors by virtual and substance library screening and direct design applying knowledge of the structure of IRAP and of related enzymes is briefly presented.

Is There an Interplay Between the Functional Domains of IRAP?

As a member of the M1 family of aminopeptidases, insulin regulated aminopeptidase (IRAP) is characterized by distinct binding motifs at the active site in the C-terminal domain that mediate the catalysis of peptide substrates. However, what makes IRAP unique in this family of enzymes is that it also possesses trafficking motifs at the N-terminal domain which regulate the movement of IRAP within different intracellular compartments. Research on the role of IRAP has focused predominantly on the C-terminus catalytic domain in different physiological and pathophysiological states ranging from pregnancy to memory loss. Many of these studies have utilized IRAP inhibitors, that bind competitively to the active site of IRAP, to explore the functional significance of its catalytic activity. However, it is unknown whether these inhibitors are able to access intracellular sites where IRAP is predominantly located in a basal state as the enzyme may need to be at the cell surface for the inhibitors to mediate their effects. This property of IRAP has often been overlooked. Interestingly, in some pathophysiological states, the distribution of IRAP is altered. This, together with the fact that IRAP possesses trafficking motifs, suggest the localization of IRAP may play an important role in defining its physiological or pathological functions and provide insights into the interplay between the two functional domains of the protein.

IRAP Inhibitors: M1-Aminopeptidase Family Inspiration.

The insulin regulated aminopeptidase (IRAP) has been proposed as an important therapeutic target for indications including Alzheimer’s disease and immune disorders. To date, a number of IRAP inhibitor designs have been investigated but the total number of molecules investigated remains quite small. As a member the M1 aminopeptidase family, IRAP shares numerous structural features with the other M1 aminopeptidases. The study of those enzymes and the development of inhibitors provide key learnings and new approaches and are potential sources of inspiration for future IRAP inhibitors.

The Discovery of Insulin-Regulated Aminopeptidase (IRAP) Inhibitors: A Literature Review.

Insulin-Regulated Aminopeptidase (IRAP, EC 3.4.11.3) is a multi-tasking member of the M1 family of zinc aminopeptidases. Among its diverse biological functions, IRAP is a regulator of oxytocin levels during late stages of pregnancy, it affects cellular glucose uptake by trafficking of the glucose transporter type 4 and it mediates antigen cross-presentation by dendritic cells. Accumulating evidence show that pharmacological inhibition of IRAP may hold promise as a valid approach for the treatment of several pathological states such as memory disorders, neurodegenerative diseases, etc. Aiming to the investigation of physiological roles of IRAP and therapeutic potential of its regulation, intense research efforts have been dedicated to the discovery of small-molecule inhibitors. Moreover, reliable structure-activity relationships have been largely facilitated by recent crystal structures of IRAP and detailed computational studies. This review aims to summarize efforts of medicinal chemists toward the design and development of IRAP inhibitors, with special emphasis to factors affecting inhibitor selectivity.

Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro-Oxindole Dihydroquinazolinones as IRAP Inhibitors.

Insulin‐regulated aminopeptidase (IRAP) is a new potential macromolecular target for drugs aimed for treatment of cognitive disorders. Inhibition of IRAP by angiotensin IV (Ang IV) improves the memory and learning in rats. The majority of the known IRAP inhibitors are peptidic in character and suffer from poor pharmacokinetic properties. Herein, we present a series of small non‐peptide IRAP inhibitors derived from a spiro‐oxindole dihydroquinazolinone screening hit (pIC50 5.8). The compounds were synthesized either by a simple microwave (MW)‐promoted three‐component reaction, or by a two‐step one‐pot procedure. For decoration of the oxindole ring system, rapid MW‐assisted Suzuki‐Miyaura cross‐couplings (1 min) were performed. A small improvement of potency (pIC50 6.6 for the most potent compound) and an increased solubility could be achieved. As deduced from computational modelling and MD simulations it is proposed that the S‐configuration of the spiro‐oxindole dihydroquinazolinones accounts for the inhibition of IRAP.

Macrocyclic peptidomimetics as inhibitors of insulin-regulated aminopeptidase (IRAP).

Macrocyclic analogues of the linear hexapeptide, angiotensin IV (AngIV) have proved to be potent inhibitors of insulin-regulated aminopeptidase (IRAP, oxytocinase, EC 3.4.11.3). Along with higher affinity, macrocycles may also offer better metabolic stability, membrane permeability and selectivity, however predicting the outcome of particular cycle modifications is challenging. Here we describe the development of a series of macrocyclic IRAP inhibitors with either disulphide, olefin metathesis or lactam bridges and variations of ring size and other functionality. The binding mode of these compounds is proposed based on molecular dynamics analysis. Estimation of binding affinities (ΔG) and relative binding free energies (ΔΔG) with the linear interaction energy (LIE) method and free energy perturbation (FEP) method showed good general agreement with the observed inhibitory potency. Experimental and calculated data highlight the cumulative importance of an intact N-terminal peptide, the specific nature of the macrocycle, the phenolic oxygen and the C-terminal functionality.

Insulin-regulated aminopeptidase inhibitor-mediated increases in dendritic spine density are facilitated by glucose uptake.

Ethyl2-acetylamino-7-hydroxy-4-pyridin-3-yl-4H-chromene-3-carboxylate (HFI-419), the benzopyran-based inhibitor of insulin-regulated aminopeptidase (IRAP), has previously been shown to improve spatial working and recognition memory in rodents. However, the mechanism of its cognitive-enhancing effect remains unknown. There is a close correlation between dendritic spine density and learning in vivo and several studies suggest that increases in neuronal glucose uptake and/or alterations to the activity of matrix metalloproteinases (MMPs) may improve memory and increase dendritic spine density. We aimed to identify the potential mechanism by which HFI-419 enhances memory by utilizing rat primary cultures of hippocampal cells. Alterations to dendritic spine density were assessed in the presence of varying concentrations of HFI-419 at different stages of hippocampal cell development. In addition, glucose uptake and changes to spine density were assessed in the presence of indinavir, an inhibitor of the glucose transporter 4 (GLUT4 ), or the matrix metalloprotease inhibitor CAS 204140-01-2. We confirmed that inhibition of IRAP activity with HFI-419 enhanced spatial working memory in rats, and determined that this enhancement may be driven by GLUT4 -mediated changes to dendritic spine density. We observed that IRAP inhibition increased dendritic spine density prior to peak dendritic growth in hippocampal neurons, and that spine formation was inhibited when GLUT4 -mediated glucose uptake was blocked. In addition, during the peak phase of dendritic spine growth, the effect of IRAP inhibition on enhancement of dendritic spine density resulted specifically in an increase in the proportion of mushroom/stubby-like spines, a morphology associated with memory and learning. Moreover, these spines were deemed to be functional based on their expression of the pre-synaptic markers vesicular glutamate transporter 1 and synapsin. Overall, or findings suggest that IRAP inhibitors may facilitate memory by increasing hippocampal dendritic spine density via a GLUT4 -mediated mechanism. Cover Image for this issue: doi: 10.1111/jnc.14745.

Abstract 131: Insulin Regulated Aminopeptidase Inhibitors are More Effective Than the ACE Inhibitor, Perindopril in Preventing Unilateral Ureteral Obstruction-Induced Renal Fibrosis in Mice

New strategies are needed to combat pathological organ fibrosis that leads to end stage organ failure. We have identified the enzyme insulin regulated aminopeptidase (IRAP) as one such promising new target, with IRAP KO mice completely protected, and pharmacological inhibition of IRAP reversing, age-induced cardiac fibrosis. However, the effect of IRAP inhibition in renal fibrosis is unknown. Therefore the objective of this study was to: (1) compare anti-fibrotic efficacy of 2 chemically distinct IRAP inhibitors, HFI-419 and SJM4-164 to the ACE inhibitor, perindopril (PER) in a murine model of unilateral ureteral obstructive (UUO)-induced renal fibrosis; and (2) investigate role of IRAP substrates in mediating ant-fibrotic effect of IRAP inhibitors. Male C57Bl/6J mice (8 weeks old) were assigned to one of the following 7 day protocols (n=6/gp): sham, UUO+Veh, UUO+HFI (0.72mg/kg/d), UUO+SJM (0.72mg/kg/d), or UUO+PER (1mg/kg/d). IRAP substrate involvement was determined using specific receptor blockers for the oxytocin receptor (OTR; Atosiban; 0.6mg/kg/d) or the AT2 receptor (AT2R; PD123319; 10mg/kg/d). Interstitial fibrosis was significantly increased in UUO+Veh-injured kidneys compared to sham (% positive staining: UUO=5.9±0.7 vs Sham=1.2±0.3; p<0.05). HFI and SJM treatment were equally effective in preventing UUO-induced increase in interstitial renal fibrosis (% positive staining: HFI=2.5±0.3, SJM=2.8±0.3, all p<0.05 vs UUO). PER had no effect on UUO-induced increase in interstitial renal fibrosis (PER=6.6±0.7), however it was as effective as the IRAP inhibitors in reducing markers of inflammation, including NFκB activation and macrophage infiltration. The anti-fibrotic effects produced with IRAP inhibition were prevented by OTR or AT2R blockade (% positive staining: OTR=5.0±0.55, AT2R=6±0.8). Overall, this study demonstrated greater reno-protection with IRAP inhibition compared to that achieved using an ACE inhibitor, with IRAP inhibitors exhibiting potent anti-fibrotic and anti-inflammatory effects. Moreover, for the first time we show that the anti-fibrotic effects of IRAP inhibition involve activation of receptors associated with endogenous IRAP substrates.

Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Aryl Sulfonamides.

The insulin-regulated aminopeptidase (IRAP) is a membrane-bound zinc metallopeptidase with many important regulatory functions. It has been demonstrated that inhibition of IRAP by angiotensin IV (Ang IV) and other peptides, as well as more druglike inhibitors, improves cognition in several rodent models. We recently reported a series of aryl sulfonamides as small-molecule IRAP inhibitors and a promising scaffold for pharmacological intervention. We have now expanded with a number of derivatives, report their stability in liver microsomes, and characterize the activity of the whole series in a new assay performed on recombinant human IRAP. Several compounds, such as the new fluorinated derivative 29, present submicromolar affinity and high metabolic stability. Starting from the two binding modes previously proposed for the sulfonamide scaffold, we systematically performed molecular dynamics simulations and binding affinity estimation with the linear interaction energy method for the full compound series. The significant agreement with experimental affinities suggests one of the binding modes, which was further confirmed by the excellent correlation for binding affinity differences between the selected pair of compounds obtained by rigorous free energy perturbation calculations. The new experimental data and the computationally derived structure–activity relationship of the sulfonamide series provide valuable information for further lead optimization of novel IRAP inhibitors.

Insulin-regulated aminopeptidase inhibitors do not alter glucose handling in normal and diabetic rats.

Insulin-regulated aminopeptidase (IRAP) co-localizes with the glucose transporter 4 (GLUT4) in GLUT4 storage vesicles (GSV) in insulin-responsive cells. In response to insulin, IRAP is the only transmembrane enzyme known to translocate together with GLUT4 to the plasma membrane in adipocytes and muscle cells. Although the intracellular region of IRAP is associated with GLUT4 vesicle trafficking, the role of the aminopeptidase activity in insulin-responsive cells has not been elucidated. The aim of this study was to investigate whether the inhibition of the aminopeptidase activity of IRAP facilitates glucose uptake in insulin-responsive cells. In both in vitro and in vivo studies, inhibition of IRAP aminopeptidase activity with the specific inhibitor, HFI-419, did not modulate glucose uptake. IRAP inhibition in the L6GLUT4myc cell line did not alter glucose uptake in both basal and insulin-stimulated state. In keeping with these results, HFI419 did not affect peripheral, whole-body glucose handling after an oral glucose challenge, neither in normal rats nor in the streptozotocin (STZ)-induced experimental rat model of diabetes mellitus (DM). Therefore, acute inhibition of IRAP aminopeptidase activity does not affect glucose homeostasis.

Optimization and Structure-Activity Relationships of Phosphinic Pseudotripeptide Inhibitors of Aminopeptidases That Generate Antigenic Peptides.

The oxytocinase subfamily of M1 aminopeptidases, consisting of ER aminopeptidase 1 (ERAP1), ER aminopeptidase 2 (ERAP2), and insulin-regulated aminopeptidase (IRAP), plays critical roles in the generation of antigenic peptides and indirectly regulates human adaptive immune responses. We have previously demonstrated that phosphinic pseudotripeptides can constitute potent inhibitors of this group of enzymes. In this study, we used synthetic methodologies able to furnish a series of stereochemically defined phosphinic pseudotripeptides and demonstrate that side chains at P1' and P2' positions are critical determinants in driving potency and selectivity. We identified low nanomolar inhibitors of ERAP2 and IRAP that display selectivity of more than 2 and 3 orders of magnitude, respectively. Cellular analysis demonstrated that one of the compounds that is a selective IRAP inhibitor can reduce IRAP-dependent but not ERAP1-dependent cross-presentation by dendritic cells with nanomolar efficacy. Our results encourage further preclinical development of phosphinic pseudotripeptides as regulators of adaptive immune responses.