Mechanisms of Allosteric Inhibition of Insulin-Regulated Aminopeptidase.

Inhibition of Insulin-Regulated Aminopeptidase is being actively explored for the treatment of several human diseases and several classes of inhibitors have been developed although no clinical applications have been reported yet. Here, we combine enzymological analysis with x-ray crystallography to investigate the mechanism employed by two of the most studied inhibitors of IRAP, an aryl sulfonamide and a 2-amino-4H-benzopyran named HFI-419. Although both compounds have been hypothesized to target the enzyme's active site by competitive mechanisms, we discovered that they instead target previously unidentified proximal allosteric sites and utilize non-competitive inhibition mechanisms. X-ray crystallographic analysis demonstrated that the aryl sulfonamide stabilizes the closed, more active, conformation of the enzyme whereas HFI-419 locks the enzyme in a semi-open, and likely less active, conformation. HFI-419 potency is substrate-dependent and fails to effectively block the degradation of the physiological substrate cyclic peptide oxytocin. Our findings demonstrate alternative mechanisms for inhibiting IRAP through allosteric sites and conformational restricting and suggest that the pharmacology of HFI-419 may be more complicated than initially considered. Such conformation-specific interactions between IRAP and small molecules can be exploited for the design of more effective second-generation allosteric inhibitors.

Discovery of the First Selective Nanomolar Inhibitors of ERAP2 by Kinetic Target-Guided Synthesis.

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a key enzyme involved in the trimming of antigenic peptides presented by Major Histocompatibility Complex class I. It is a target of growing interest for the treatment of autoimmune diseases and in cancer immunotherapy. However, the discovery of potent and selective ERAP2 inhibitors is highly challenging. Herein, we have used kinetic target-guided synthesis (KTGS) to identify such inhibitors. Co-crystallization experiments revealed the binding mode of three different inhibitors with increasing potency and selectivity over related enzymes. Selected analogues engage ERAP2 in cells and inhibit antigen presentation in a cellular context. 4 d (BDM88951) displays favorable in vitro ADME properties and in vivo exposure. In summary, KTGS allowed the discovery of the first nanomolar and selective highly promising ERAP2 inhibitors that pave the way of the exploration of the biological roles of this enzyme and provide lead compounds for drug discovery efforts.

Discovery of Selective Nanomolar Inhibitors for Insulin-Regulated Aminopeptidase Based on α-Hydroxy-ß-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-ß-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-ß-amino acid derivatives may constitute useful chemical tools and drug leads for this group of aminopeptidases.

Can ERAP1 and ERAP2 Form Functional Heterodimers? A Structural Dynamics Investigation.

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) play important roles in the generation of antigenic peptides presented by Major Histocompatibility Class I (MHCI) molecules and indirectly regulate adaptive immune responses. Although the discrete function of these enzymes has been extensively characterized, recent reports have suggested that they can also form heterodimers with functional consequences. However, lack of structural characterization of a putative ERAP1/ERAP2 dimer has limited our understanding of its biological role and significance. To address this, we employed computational molecular dynamics calculations to explore the topology of interactions between these two, based on experimentally determined homo-dimerization interfaces observed in crystal structures of ERAP2 or homologous enzymes. Our analysis of 8 possible dimerization models, suggested that the most likely ERAP1/ERAP2 heterodimerization topology involves the exon 10 loop, a non-conserved loop previously implicated in interactions between ERAP1 and the disulfide-bond shuffling chaperone ERp44. This dimerization topology allows access to the active site of both enzymes and is consistent with a previously reported construct in which ERAP1 and ERAP2 were linked by Fos/Jun zipper tags. The proposed model constitutes a tentative structural template to help understand the physiological role and significance of ERAP1/ERAP2 molecular interactions.

The Role of Antigen Processing and Presentation in Cancer and the Efficacy of Immune Checkpoint Inhibitor Immunotherapy.

Recent clinical successes of cancer immunotherapy using immune checkpoint inhibitors (ICIs) are rapidly changing the landscape of cancer treatment. Regardless of initial impressive clinical results though, the therapeutic benefit of ICIs appears to be limited to a subset of patients and tumor types. Recent analyses have revealed that the potency of ICI therapies depends on the efficient presentation of tumor-specific antigens by cancer cells and professional antigen presenting cells. Here, we review current knowledge on the role of antigen presentation in cancer. We focus on intracellular antigen processing and presentation by Major Histocompatibility class I (MHCI) molecules and how it can affect cancer immune evasion. Finally, we discuss the pharmacological tractability of manipulating intracellular antigen processing as a complementary approach to enhance tumor immunogenicity and the effectiveness of ICI immunotherapy.

Generation of SARS-CoV-2 S1 Spike Glycoprotein Putative Antigenic Epitopes in Vitro by Intracellular Aminopeptidases.

Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2, and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.

Editing the immunopeptidome of melanoma cells using a potent inhibitor of endoplasmic reticulum aminopeptidase 1 (ERAP1).

The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, often is limited by ineffective presentation of antigenic peptides that elicit T-cell-mediated anti-tumor cytotoxic responses. Manipulation of antigen presentation pathways is an emerging approach for enhancing the immunogenicity of tumors in immunotherapy settings. ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides as part of the system that generates peptides for binding to MHC class I molecules (MHC-I). We hypothesized that pharmacological inhibition of ERAP1 in cells could regulate the cellular immunopeptidome. To test this hypothesis, we treated A375 melanoma cells with a recently developed potent ERAP1 inhibitor and analyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting bound peptides, and identifying them using capillary chromatography and tandem mass spectrometry (LC-MS/MS). Although the inhibitor did not reduce cell-surface MHC-I expression, it induced qualitative and quantitative changes in the presented peptidomes. Specifically, inhibitor treatment altered presentation of about half of the total 3204 identified peptides, including about one third of the peptides predicted to bind tightly to MHC-I. Inhibitor treatment altered the length distribution of eluted peptides without change in the basic binding motifs. Surprisingly, inhibitor treatment enhanced the average predicted MHC-I binding affinity, by reducing presentation of sub-optimal long peptides and increasing presentation of many high-affinity 9-12mers, suggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes. Our results suggest that chemical inhibition of ERAP1 may be a viable approach for manipulating the immunopeptidome of cancer.

Inhibitors of ER Aminopeptidase 1 and 2: From Design to Clinical Application.

Endoplasmic Reticulum aminopeptidase 1 and 2 are two homologous enzymes that help generate peptide ligands for presentation by Major Histocompatibility Class I molecules. Their enzymatic activity influences the antigenic peptide repertoire and indirectly controls adaptive immune responses. Accumulating evidence suggests that these two enzymes are tractable targets for the regulation of immune responses with possible applications ranging from cancer immunotherapy to treating inflammatory autoimmune diseases. Here, we review the state-of-the-art in the development of inhibitors of ERAP1 and ERAP2 as well as their potential and limitations for clinical applications.

Molecular pathways for antigenic peptide generation by ER aminopeptidase 1.

Endoplasmic Reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that can generate or destroy potential peptide ligands for MHC class I molecules. ERAP1 activity influences the cell-surface immunopeptidome and epitope immunodominance patterns but in complex and poorly understood manners. Two main distinct pathways have been proposed to account for ERAP1's effects on the nature and quantity of MHCI-bound peptides: i) ERAP1 trims peptides in solution, generating the correct length for binding to MHCI or overtrimming peptides so that they are too short to bind, and ii) ERAP1 trims peptides while they are partially bound onto MHCI in manner that leaves the peptide amino terminus accessible. For both pathways, once an appropriate length peptide is generated it could bind conventionally to MHCI, competing with further trimming by ERAP1. The two pathways, although not necessarily mutually exclusive, provide distinct vantage points for understanding of the rules behind the generation of the immunopeptidome. Resolution of the mechanistic details of ERAP1-mediated antigenic peptide generation can have important consequences for pharmacological efforts to regulate the immunopeptidome for therapeutic applications, and for understanding association of ERAP1 alleles with susceptibility to autoimmune disease and cancer. We review current evidence in support of these two pathways and discuss their relative importance and potential complementarity.

Ligand-Induced Conformational Change of Insulin-Regulated Aminopeptidase: Insights on Catalytic Mechanism and Active Site Plasticity.

Insulin-regulated aminopeptidase (IRAP) is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates, although the mechanism behind this wide specificity is not clearly understood. We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at 2.53 Å resolution. In the presence of this inhibitor, the enzyme adopts a novel conformation in which domains II and IV are juxtaposed, forming a hollow structure that excludes external solvent access to the catalytic center. A loop adjacent to the enzyme's GAMEN motif undergoes structural reconfiguration, allowing the accommodation of bulky inhibitor side chains. Atomic interactions between the inhibitor and IRAP that are unique to this conformation can explain the strong selectivity compared to homologous aminopeptidases ERAP1 and ERAP2. This conformation provides insight on IRAP's catalytic cycle and reveals significant active-site plasticity that may underlie its substrate permissiveness.

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.

Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2.

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.

Crystal Structure of Insulin-Regulated Aminopeptidase with Bound Substrate Analogue Provides Insight on Antigenic Epitope Precursor Recognition and Processing.

Aminopeptidases that generate antigenic peptides influence immunodominance and adaptive cytotoxic immune responses. The mechanisms that allow these enzymes to efficiently process a vast number of different long peptide substrates are poorly understood. In this work, we report the structure of insulin-regulated aminopeptidase, an enzyme that prepares antigenic epitopes for cross-presentation in dendritic cells, in complex with an antigenic peptide precursor analog. Insulin-regulated aminopeptidase is found in a semiclosed conformation with an extended internal cavity with limited access to the solvent. The N-terminal moiety of the peptide is located at the active site, positioned optimally for catalysis, whereas the C-terminal moiety of the peptide is stabilized along the extended internal cavity lodged between domains II and IV. Hydrophobic interactions and shape complementarity enhance peptide affinity beyond the catalytic site and support a limited selectivity model for antigenic peptide selection that may underlie the generation of complex immunopeptidomes.