Biology subjects

Stratikos, E.

Publications and source records attributed to Stratikos, E..

9 recordsLinked to original sources

bioRxiv2025

Discovery of an orally available potent ER aminopeptidase 1 (ERAP1) inhibitor that enhances anti-tumor responses and limits inflammatory autoimmunity in vivo.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that can regulate immune responses primarily by proteolytically processing peptides before loading and presentation on the cell surface by major histocompatibility class I molecules (MHC-I). ERAP1 activity can either reduce the immunogenicity of cancer cells by over-trimming cancer-associated antigenic peptides or contribute to autoimmunity by generating self-antigenic peptides. As a result, ERAP1 inhibition has emerged as a tractable approach for cancer immunotherapy and specific classes of autoimmunity. Here, we describe the discovery, after hit-to-lead optimization, of a potent and selective ERAP1 inhibitor based on the pyrrolidine 3-carboxylic acid scaffold that targets the regulatory allosteric site. The compound has favourable in vivo pharmacokinetics, including oral bioavailability, and can regulate the immunopeptidome of cancer cells and enhance cancer cell antigenicity in vivo in a dose-dependent manner, controlling tumor growth. In addition, when administered in the murine collagen-induced arthritis model, it does not induce any exacerbation of autoimmune responses but rather results in a dose-dependent therapeutic benefit. Our results demonstrate that ERAP1 inhibition can constitute a tractable approach to modulating immune responses for therapeutic applications, providing mechanistic insight and a valuable lead and in vivo tool for further drug development efforts and for interrogating ERAP1 biology.

immunology↗

bioRxiv2025

ERAP1 allotypes 2 and 10 differentially regulate the immunopeptidome of melanocytes

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a polymorphic enzyme that shapes the peptide repertoire presented by MHC class I molecules and can regulate adaptive immune responses in cancer and autoimmunity. Common missense polymorphisms in ERAP1 modulate its activity and are found in specific allotypes in humans. ERAP1 allotypes are linked to predisposition to HLA-associated inflammatory diseases. ERAP1 allotypic variation has been correlated with the development of psoriasis and Bechets disease, through the generation of specific CD8+ T cell populations targeting disease-specific HLAs. Given the established broad effects of ERAP1 activity on the cellular immunopeptidome, we hypothesized that ERAP1 allotypic variation may lead to broad immunopeptidome shifts that underlie antigenic responses. To test this hypothesis, we generated two A375 melanoma cell lines, each one expressing one of the most common, disease-associated, ERAP1 allotypes, namely allotypes 2 or 10. Comparison of the immunopeptidome of these two cell lines showed only minor differences in peptide sequences presented but extensive changes in abundance that included alterations in length distribution, binding affinity and sequence motifs. Our results suggest that enzymatic differences between ERAP1 allotypes are reflected primarily on the quantitative composition of the cellular immunopeptidome. These quantitative changes may constitute a mechanism that underlies ERAP1-allotypic associations with HLA-associated autoimmunity and variable anti-tumor responses.

immunology↗

bioRxiv2024

ERAP1 activity modulates the immunopeptidome but also affects the proteome, metabolism and stress responses in cancer cells

Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) metabolizes peptides inside the ER and shapes the peptide repertoire available for binding to Major Histocompatibility Complex Class I molecules (MHC-I). Moreover, it may have additional effects on cellular homeostasis, which have not been explored. To address these questions, we used both genetic silencing of ERAP1 expression as well as treatment with a selective allosteric ERAP1 inhibitor to probe changes in the immunopeptidome and proteome of the A375 melanoma cancer cell line. We observed significant immunopeptidome shifts with both methods of functional ERAP1 disruption, which were distinct for each method. Both methods of inhibition led to an enhancement, albeit slight, in tumor cell killing by stimulated human PBMCs and in significant proteomic alterations in pathways related to metabolism and cellular stress. Similar proteomic changes were also observed in the leukemia cell line THP-1. Biochemical analyses suggested that ERAP1 inhibition affected sensitivity to ER stress, reactive oxygen species production and mitochondrial metabolism. Although the proteomics shifts were significant, their potential in shaping immunopeptidome shifts was limited since only 15.8% of differentially presented peptides belonged to proteins with altered expression and only 5.0% of proteins with altered expression were represented in the immunopeptidome shifts. Taken together, our findings suggest that modulation of ERAP1 activity can generate unique immunopeptidomes, mainly due to altered peptide processing in the ER, but also induce changes in the cellular proteome and metabolic state which may have further effects on tumor cells.

biochemistry↗

bioRxiv2024

Stabilization of the open conformation of 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 several drug-discovery efforts although no clinically useful inhibitors have been reported yet. 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 and is active in blocking IRAP-dependent cross-presentation of an 8mer epitope. To better understand the mechanism of action and the basis for the unusual substrate selectivity of this inhibitor, we solved the crystal structure of the compound in 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 closed conformation previously observed with other low molecular weight inhibitors and hypothesized to be important for catalysis. 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.

biochemistry↗

bioRxiv2024

Polymorphic positions 349 and 725 of the autoimmunity-protective allotype 10 of ER aminopeptidase 1 are key in determining its unique enzymatic properties

ER aminopeptidase 1 (ERAP1) is a polymorphic intracellular aminopeptidase with key roles in antigen presentation and adaptive immune responses. ERAP1 allotype 10 is highly protective towards developing some forms of autoimmunity and displays unusual functional properties, including very low activity versus some substrates. To understand the molecular mechanisms that underlie the biology of allotype 10 we studied its enzymatic and biophysical properties focusing on its unique polymorphisms V349M and Q725R. Compared to ancestral allotype 1, allotype 10 is much less effective in trimming small substrates but presents allosteric kinetics that ameliorate activity differences at high substrate concentrations. Furthermore, it is inhibited by a transition-state analogue via a non-competitive mechanism and is much less responsive to an allosteric small-molecule modulator. It also presents opposite enthalpy, entropy and heat capacity of activation compared to allotype 1 and its catalytic rate is highly dependent on viscosity. Polymorphisms V349M and Q725R significantly contribute to the lower enzymatic activity of allotype 10 for small substrates, especially at high substrate concentrations, influence the cooperation between the regulatory and active sites and regulate viscosity dependence, likely by limiting product release. Overall, our results suggest that allotype 10 is not just an inactive variant of ERAP1 but rather carries distinct enzymatic properties that largely stem from changes at positions 349 and 725. These changes affect kinetic and thermodynamic parameters that likely control rate-limiting steps in the catalytic cycle, resulting in an enzyme optimized for sparing small substrates and contributing to the homeostasis of antigenic epitopes in the ER.

biochemistry↗

bioRxiv2021

Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 Spike Glycoprotein

Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both SARS-CoV-2 infection severity and long-term immunity, either after recovery or vaccination. A hallmark of COVID-19 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides by 10 common allotypes of ER aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for MHC class I molecules, including known SARS-CoV-2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses to COVID-19.

biochemistry↗

bioRxiv2020

Common allotypes of ER aminopeptidase 1 have substrate- dependent and highly variable enzymatic properties

ObjectivePolymorphic variation of immune system proteins can drive variability of individual immune responses. ER aminopeptidase 1 (ERAP1) generates antigenic peptides for presentation by MHC class I molecules. Coding single nucleotide polymorphisms (SNPs) in ERAP1 have been associated with predisposition to inflammatory rheumatic disease and shown to affect functional properties of the enzyme, but the interplay between combinations of these SNPs as they exist in allotypes, has not been thoroughly explored. MethodsWe used phased genotype data to estimate ERAP1 allotype frequency in 2,504 individuals across five major human populations, generated highly pure recombinant enzymes corresponding to the 10 most common ERAP1 allotypes and systematically characterized their in vitro enzymatic properties. ResultsWe find that ERAP1 allotypes possess a wide range of enzymatic activities, up to 60-fold, whose ranking is substrate-dependent. Strikingly, allotype 10, previously associated with Behcets disease, is consistently a low-activity outlier, suggesting that a significant percentage of individuals carry a sub-active ERAP1 gene. Enzymatic analysis revealed that ERAP1 allotypes can differ in both catalytic efficiency and substrate affinity, differences that can change intermediate accumulation in multi-step trimming reactions. Alterations in efficacy of an allosteric inhibitor that targets the regulatory site suggest that allotypic variation influences the communication between the regulatory and the active site. ConclusionOur work defines the wide landscape of ERAP1 activity in human populations and demonstrates how common allotypes can induce substrate-dependent variability in antigen processing, thus contributing, in synergy with MHC haplotypes, to immune response variability and to predisposition to chronic inflammatory conditions.

biochemistry↗

bioRxiv2020

ERAP2 facilitates a subpeptidome of Birdshot Uveitis-associated HLA-A29

Birdshot Uveitis (BU) is a blinding inflammatory eye condition that only affects HLA-A29-positive individuals. Genetic association studies linked ERAP2 with BU, an aminopeptidase which trims peptides before their presentation by HLA class I at the cell surface, which suggests that ERAP2-dependent peptide presentation by HLA-A29 drives the pathogenesis of BU. However, it remains poorly understood whether the effects of ERAP2 on the HLA-A29 peptidome are distinct from its effect on other HLA allotypes. To address this, we focused on the effects of ERAP2 on the immunopeptidome in patient-derived antigen presenting cells. Using complementary HLA-A29-based and pan-class I immunopurifications, isotope-labelled naturally processed and presented HLA-bound peptides were sequenced by mass spectrometry. We show that the effects of ERAP2 on the N-terminus of ligands of HLA-A29 are shared across endogenous HLA allotypes, but discover and replicate that one peptide motif generated in the presence of ERAP2 is specifically bound by HLA-A29. This motif can be found in the amino acid sequence of putative autoantigens. We further show evidence for internal sequence specificity for ERAP2 imprinted in the immunopeptidome. These results reveal that ERAP2 can generate an HLA-A29-specific antigen repertoire, which supports that antigen presentation is a key disease pathway in BU.

immunology↗

bioRxiv2020

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.

biochemistry↗