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1.
J Mol Biol ; 436(6): 168449, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38244767

RESUMO

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.


Assuntos
Sítio Alostérico , Inibidores Enzimáticos , Insulina , Sulfonamidas , Humanos , Domínio Catalítico/efeitos dos fármacos , Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Insulina/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacologia , Cristalografia por Raios X , Regulação Alostérica , Sítio Alostérico/efeitos dos fármacos , Células HEK293 , Células CHO , Animais , Cricetulus
2.
Angew Chem Int Ed Engl ; 61(39): e202203560, 2022 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-35904863

RESUMO

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.


Assuntos
Aminopeptidases , Apresentação de Antígeno , Aminopeptidases/metabolismo , Antígenos de Histocompatibilidade Classe I , Peptídeos/metabolismo
3.
J Med Chem ; 65(14): 10098-10117, 2022 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-35833347

RESUMO

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.


Assuntos
Aminopeptidases , Insulina , Aminoácidos/farmacologia , Aminopeptidases/química , Cistinil Aminopeptidase , Leucina/análogos & derivados
4.
Front Immunol ; 13: 863529, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35514997

RESUMO

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.


Assuntos
Aminopeptidases , Peptídeos , Aminopeptidases/genética , Aminopeptidases/metabolismo , Antígenos , Antígenos de Histocompatibilidade Menor/genética , Domínios Proteicos
5.
ACS Med Chem Lett ; 13(2): 218-224, 2022 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-35178178

RESUMO

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is an intracellular enzyme involved in the processing of antigenic peptides intended for presentation by major histocompatibility complex class I (MHCI) molecules. Because of its role in regulating immune responses, ERAP2 is an emerging pharmacological target. Phosphinic pseudopeptides are potent transition-state analogue inhibitors of ERAP2. Previous structure-activity studies have revealed a complex but ambiguous relationship between the occupation of putative specificity pockets and the inhibitor efficacy. To address these problems, we solved crystal structures of ERAP2 in complex with two phosphinic pseudotripeptide inhibitors. Both compounds are found in the catalytic site in a canonical orientation for transition-state analogues and utilize the S1 and S2' pockets in a similar fashion. Strikingly, their P1' side chains exhibit different orientations and make interactions with distinct shallow pockets near the ERAP2 active site. These structures suggest that S1' pocket usage in ERAP2 may be inhibitor-dependent and constitute useful starting templates for the further optimization of this class of compounds.

6.
Sci Rep ; 11(1): 16475, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389743

RESUMO

Processing of N-terminally elongated antigenic peptide precursors by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) is a key step in antigen presentation and the adaptive immune response. Although ERAP1 can efficiently process long peptides in solution, it has been proposed that it can also process peptides bound onto Major Histocompatibility Complex I molecules (MHCI). In a previous study, we suggested that the occasionally observed "ontο MHCI" trimming by ERAP1 is likely due to fast peptide dissociation followed by solution trimming, rather than direct action of ERAP1 onto the MHCI complex. However, other groups have proposed that ERAP1 can trim peptides covalently bound onto MHCI, which would preclude peptide dissociation. To explore this interaction, we constructed disulfide-linked MHCI-peptide complexes using HLA-B*08 and a 12mer kinetically labile peptide, or a 16mer carrying a phosphinic transition-state analogue N-terminus with high-affinity for ERAP1. Kinetic and biochemical analyses suggested that while both peptides could access the ERAP1 active site when free in solution, they were unable to do so when tethered in the MHCI binding groove. Our results suggest that MHCI binding protects, rather than promotes, antigenic peptide precursor trimming by ERAP1 and thus solution trimming is the more likely model of antigenic peptide processing.


Assuntos
Aminopeptidases/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Domínio Catalítico , Antígeno HLA-B8/metabolismo , Estrutura Terciária de Proteína
7.
Org Lett ; 23(5): 1726-1730, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33617265

RESUMO

The first example of a carbodiimide-mediated P-C bond-forming reaction is described. The reaction involves activation of ß-carboxyethylphosphinic acids and subsequent reaction with Boc-aminals using acid-catalysis. Mechanistic experiments using 31P NMR spectroscopy and DFT calculations support the contribution of unusually reactive cyclic phosphinic/carboxylic mixed anhydrides in a reaction pathway involving ion-pair "swapping". The utility of this protocol is highlighted by the direct synthesis of Boc-protected phosphinic dipeptides, as precursors to potent Zn-aminopeptidase inhibitors.


Assuntos
Carbodi-Imidas/química , Dipeptídeos/química , Compostos Organofosforados/química , Alquilação , Aminoácidos/química , Anidridos/química , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular
8.
Cancers (Basel) ; 13(1)2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33406696

RESUMO

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.

9.
J Proteome Res ; 19(11): 4398-4406, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-32931291

RESUMO

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.


Assuntos
Aminopeptidases/metabolismo , Antígenos Virais , Epitopos , Glicoproteína da Espícula de Coronavírus , Antígenos Virais/química , Antígenos Virais/metabolismo , Cromatografia Líquida , Epitopos/química , Epitopos/metabolismo , Células HEK293 , Antígenos HLA/química , Antígenos HLA/metabolismo , Humanos , Peptídeos/análise , Peptídeos/química , Peptídeos/metabolismo , Proteômica/métodos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Espectrometria de Massas em Tandem
10.
ACS Med Chem Lett ; 11(7): 1429-1434, 2020 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-32676150

RESUMO

Insulin-regulated aminopeptidase (IRAP) is a transmembrane zinc metallopeptidase with many important biological functions and an emerging pharmacological target. Although previous structural studies have given insight on how IRAP recognizes linear peptides, how it recognizes its physiological cyclic ligands remains elusive. Here, we report the first crystal structure of IRAP with the macrocyclic peptide inhibitor HA08 that combines structural elements from angiotensin IV and the physiological substrates oxytocin and vasopressin. The compound is found in the catalytic site in a near canonical substrate-like configuration and inhibits by a competitive mechanism. Comparison with previously solved structures of IRAP along with small-angle X-ray scattering experiments suggests that IRAP is in an open conformation in solution but undergoes a closing conformational change upon inhibitor binding. Stabilization of the closed conformation in combination with catalytic water exclusion by the tightly juxtaposed GAMEN loop is proposed as a mechanism of inhibition.

11.
J Biol Chem ; 295(21): 7193-7210, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32184355

RESUMO

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mature antigenic peptides for presentation by major histocompatibility complex class I (MHCI) molecules and regulates adaptive immune responses. ERAP1 has been proposed to trim peptide precursors both in solution and in preformed MHCI-peptide complexes, but which mode is more relevant to its biological function remains controversial. Here, we compared ERAP1-mediated trimming of antigenic peptide precursors in solution or when bound to three MHCI alleles, HLA-B*58, HLA-B*08, and HLA-A*02. For all MHCI-peptide combinations, peptide binding onto MHCI protected against ERAP1-mediated trimming. In only a single MHCI-peptide combination, trimming of an HLA-B*08-bound 12-mer progressed at a considerable rate, albeit still slower than in solution. Results from thermodynamic, kinetic, and computational analyses suggested that this 12-mer is highly labile and that apparent on-MHC trimming rates are always slower than that of MHCI-peptide dissociation. Both ERAP2 and leucine aminopeptidase, an enzyme unrelated to antigen processing, could trim this labile peptide from preformed MHCI complexes as efficiently as ERAP1. A pseudopeptide analogue with high affinity for both HLA-B*08 and the ERAP1 active site could not promote the formation of a ternary ERAP1/MHCI/peptide complex. Similarly, no interactions between ERAP1 and purified peptide-loading complex were detected in the absence or presence of a pseudopeptide trap. We conclude that MHCI binding protects peptides from ERAP1 degradation and that trimming in solution along with the dynamic nature of peptide binding to MHCI are sufficient to explain ERAP1 processing of antigenic peptide precursors.


Assuntos
Aminopeptidases/química , Antígeno HLA-A2/química , Antígenos HLA-B/química , Antígenos de Histocompatibilidade Menor/química , Oligopeptídeos/química , Aminopeptidases/genética , Domínio Catalítico , Antígeno HLA-A2/genética , Antígenos HLA-B/genética , Humanos , Antígenos de Histocompatibilidade Menor/genética
12.
Proc Natl Acad Sci U S A ; 116(52): 26709-26716, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31843903

RESUMO

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that optimizes the peptide cargo of major histocompatibility class I (MHC-I) molecules and regulates adaptive immunity. It has unusual substrate selectivity for length and sequence, resulting in poorly understood effects on the cellular immunopeptidome. To understand substrate selection by ERAP1, we solved 2 crystal structures of the enzyme with bound transition-state pseudopeptide analogs at 1.68 Å and 1.72 Å. Both peptides have their N terminus bound at the active site and extend away along a large internal cavity, interacting with shallow pockets that can influence selectivity. The longer peptide is disordered through the central region of the cavity and has its C terminus bound in an allosteric pocket of domain IV that features a carboxypeptidase-like structural motif. These structures, along with enzymatic and computational analyses, explain how ERAP1 can select peptides based on length while retaining the broad sequence-specificity necessary for its biological function.

13.
Cancer Immunol Immunother ; 68(8): 1245-1261, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31222486

RESUMO

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.


Assuntos
Aminopeptidases/metabolismo , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/metabolismo , Imunoterapia/métodos , Melanoma/tratamento farmacológico , Antígenos de Histocompatibilidade Menor/metabolismo , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Linfócitos T Citotóxicos/imunologia , Aminopeptidases/antagonistas & inibidores , Apresentação de Antígeno , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunogenicidade da Vacina , Ativação Linfocitária , Terapia de Alvo Molecular , Peptídeos/genética , Peptídeos/imunologia , Ligação Proteica
14.
Mol Immunol ; 113: 50-57, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-29678301

RESUMO

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.


Assuntos
Aminopeptidases/imunologia , Apresentação de Antígeno/imunologia , Antígenos/imunologia , Peptídeos/imunologia , Transdução de Sinais/imunologia , Animais , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Ligantes
15.
Curr Med Chem ; 26(15): 2715-2729, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29446724

RESUMO

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.


Assuntos
Aminopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Aminopeptidases/química , Aminopeptidases/genética , Aminopeptidases/fisiologia , Animais , Autoimunidade/fisiologia , Domínio Catalítico , Linhagem Celular Tumoral , Desenho de Fármacos , Inibidores Enzimáticos/química , Antígenos de Histocompatibilidade/imunologia , Antígenos de Histocompatibilidade/metabolismo , Humanos , Imunidade Inata/fisiologia , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/fisiologia , Neoplasias/enzimologia , Neoplasias/imunologia , Polimorfismo de Nucleotídeo Único
16.
ACS Med Chem Lett ; 8(3): 333-337, 2017 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-28337326

RESUMO

Endoplasmic reticulum aminopeptidase 2 assists with the generation of antigenic peptides for presentation onto Major Histocompatibility Class I molecules in humans. Recent evidence has suggested that the activity of ERAP2 may contribute to the generation of autoimmunity, thus making ERAP2 a possible pharmacological target for the regulation of adaptive immune responses. To better understand the structural elements of inhibitors that govern their binding affinity to the ERAP2 active site, we cocrystallized ERAP2 with a medium activity 3,4-diaminobenzoic acid inhibitor and a poorly active hydroxamic acid derivative. Comparison of these two crystal structures with a previously solved structure of ERAP2 in complex with a potent phosphinic pseudopeptide inhibitor suggests that engaging the substrate N-terminus recognition properties of the active site is crucial for inhibitor binding even in the absence of a potent zinc-binding group. Proper utilization of all five major pharmacophores is necessary, however, to optimize inhibitor potency.

17.
J Med Chem ; 60(7): 2963-2972, 2017 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-28328206

RESUMO

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.


Assuntos
Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/metabolismo , Inibidores Enzimáticos/farmacologia , Conformação Proteica/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Cistinil Aminopeptidase/química , Inibidores Enzimáticos/química , Humanos , Ligantes , Modelos Moleculares , Peptídeos/metabolismo , Especificidade por Substrato
18.
Biochemistry ; 56(10): 1546-1558, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28218509

RESUMO

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that is important for the generation of antigenic epitopes and major histocompatibility class I-restricted adaptive immune responses. ERAP1 processes a vast variety of different peptides but still shows length and sequence selectivity, although the mechanism behind these properties is poorly understood. X-ray crystallographic analysis has revealed that ERAP1 can assume at least two distinct conformations in which C-terminal domain IV is either proximal or distal to active site domain II. To improve our understanding of the role of this conformational change in the catalytic mechanism of ERAP1, we used site-directed mutagenesis to perturb key salt bridges between domains II and IV. Enzymatic analysis revealed that these mutations, although located away from the catalytic site, greatly reduce the catalytic efficiency and change the allosteric kinetic behavior. The variants were more efficiently activated by small peptides and bound a competitive inhibitor with weaker affinity and faster dissociation kinetics. Molecular dynamics analysis suggested that the mutations affect the conformational distribution of ERAP1, reducing the population of closed states. Small-angle X-ray scattering indicated that both the wild type and the ERAP1 variants are predominantly in an open conformational state in solution. Overall, our findings suggest that electrostatic interactions between domains II and IV in ERAP1 are crucial for driving a conformational change that regulates the structural integrity of the catalytic site. The extent of domain opening in ERAP1 probably underlies its specialization for antigenic peptide precursors and should be taken into account in inhibitor development efforts.


Assuntos
Aminopeptidases/química , Retículo Endoplasmático/enzimologia , Antígenos de Histocompatibilidade Menor/química , Mutação , Sequência de Aminoácidos , Aminopeptidases/genética , Aminopeptidases/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Biocatálise , Domínio Catalítico , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Humanos , Cinética , Lepidópteros/citologia , Lepidópteros/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Simulação de Dinâmica Molecular , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sais/química , Eletricidade Estática , Termodinâmica
19.
J Med Chem ; 59(19): 9107-9123, 2016 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-27606717

RESUMO

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.


Assuntos
Aminopeptidases/antagonistas & inibidores , Cistinil Aminopeptidase/antagonistas & inibidores , Fosfinas/química , Fosfinas/farmacologia , Aminopeptidases/imunologia , Animais , Linhagem Celular , Cistinil Aminopeptidase/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Desenho de Fármacos , Humanos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Peptídeos/imunologia , Relação Estrutura-Atividade
20.
J Biol Chem ; 290(43): 26021-32, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26381406

RESUMO

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.


Assuntos
Aminopeptidases/metabolismo , Antígenos/metabolismo , Retículo Endoplasmático/enzimologia , Peptídeos/metabolismo , Aminopeptidases/química , Animais , Linhagem Celular , Cristalografia , Modelos Moleculares , Conformação Proteica
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