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BACKGROUND: Hypertension worsens outcomes in SARS-CoV-2 patients. Sartans, a type of antihypertensive angiotensin receptor blocker-(ARB), reduce COVID-19 morbidity and mortality by targeting angiotensin-converting enzyme-2 (ACE2). This study aimed to evaluate the antiviral and antihypertensive effects of nirmatrelvir, commercial sartans (candesartan, losartan, and losartan carboxylic (Exp3174)), and newly synthesized sartans (benzimidazole-N-biphenyl carboxyl (ACC519C) and benzimidazole-N-biphenyl tetrazole (ACC519T)), compared to nirmatrelvir, the antiviral component of Paxlovid. RESEARCH DESIGN AND METHODS: Surface plasmon resonance (SPR) and enzymatic studies assessed drug effects on ACE2. Antiviral abilities were tested with SARS-CoV-2-infected Vero E6 cells, and antihypertensive effects were evaluated using angiotensin II-contracted rabbit iliac arteries. RESULTS: Benzimidazole-based candesartan and ACC519C showed antiviral activity comparable to nirmatrelvir (95% inhibition). Imidazole-based losartan, Exp3174, and ACC519T were less potent (75%-80% and 50%, respectively), with Exp3174 being the least effective. SPR analysis indicated high sartans-ACE2 binding affinity. Candesartan and nirmatrelvir combined had greater inhibitory and cytopathic effects (3.96%) than individually (6.10% and 5.08%). ACE2 enzymatic assays showed varying effects of novel sartans on ACE2. ACC519T significantly reduced angiotensin II-mediated contraction, unlike nirmatrelvir and ACC519T(2). CONCLUSION: This study reports the discovery of a new class of benzimidazole-based sartans that significantly inhibit SARS-CoV-2, likely due to their interaction with ACE2.
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Enzima Convertidora de Angiotensina 2 , Antivirales , Bencimidazoles , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Bencimidazoles/farmacología , Animales , Antivirales/farmacología , Humanos , Chlorocebus aethiops , Enzima Convertidora de Angiotensina 2/metabolismo , SARS-CoV-2/efectos de los fármacos , Células Vero , Conejos , Antagonistas de Receptores de Angiotensina/farmacología , Compuestos de Bifenilo/farmacología , Antihipertensivos/farmacología , Tetrazoles/farmacología , Masculino , Hipertensión/tratamiento farmacológico , COVID-19 , Losartán/farmacología , Resonancia por Plasmón de SuperficieRESUMEN
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.
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Sitio Alostérico , Inhibidores Enzimáticos , Insulina , Sulfonamidas , Humanos , Dominio Catalítico/efectos de los fármacos , Cistinil Aminopeptidasa/antagonistas & inhibidores , Cistinil Aminopeptidasa/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Insulina/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacología , Cristalografía por Rayos X , Regulación Alostérica , Sitio Alostérico/efectos de los fármacos , Células HEK293 , Células CHO , Animales , CricetulusRESUMEN
[This corrects the article DOI: 10.3389/fphar.2019.00474.].
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The ß3 subunit of nicotinic acetylcholine receptors (nAChRs) participates in heteropentameric assemblies with some α and other ß neuronal subunits forming a plethora of various subtypes, differing in their electrophysiological and pharmacological properties. While ß3 has for several years been considered an accessory subunit without direct participation in the formation of functional binding sites, recent electrophysiology data have disputed this notion and indicated the presence of a functional (+) side on the extracellular domain (ECD) of ß3. In this study, we present the 2.4 Å resolution crystal structure of the monomeric ß3 ECD, which revealed rather distinctive loop C features as compared to those of α nAChR subunits, leading to intramolecular stereochemical hindrance of the binding site cavity. Vigorous molecular dynamics simulations in the context of full length pentameric ß3-containing nAChRs, while not excluding the possibility of a ß3 (+) binding site, demonstrate that this site cannot efficiently accommodate the agonist nicotine. From the structural perspective, our results endorse the accessory rather than functional role of the ß3 nAChR subunit, in accordance with earlier functional studies on ß3-containing nAChRs.
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Receptores Nicotínicos , Sitios de Unión , Nicotina/farmacología , Receptores Nicotínicos/metabolismoRESUMEN
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.
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Aminopeptidasas , Presentación de Antígeno , Aminopeptidasas/metabolismo , Antígenos de Histocompatibilidad Clase I , Péptidos/metabolismoRESUMEN
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.
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Aminopeptidasas , Insulina , Aminoácidos/farmacología , Aminopeptidasas/química , Cistinil Aminopeptidasa , Leucina/análogos & derivadosRESUMEN
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.
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The pharmacological profile of medicinally relevant Ru(III) coordination compounds has been ascribed to their interactions with proteins, as several studies have provided evidence that DNA is not the primary target. In this regard, numerous spectroscopic and crystallographic studies have indicated that the Ru(III) ligands play an important role in determining the metal binding site, acting as the recognition element in the early stages of the protein-complex formation. Herein, we present a series of near-atomic-resolution X-ray crystal structures of the adducts formed between the antimetastatic metallodrug imidazolium trans-[tetrachlorido(S-dimethyl sufoxide)(1H-imidazole)ruthenate(III)] (NAMI-A) and hen egg-white lysozyme (HEWL). These structures elucidate a series of binding events starting from the noncovalent interaction of intact NAMI-A ions with HEWL (1.5 h), followed by the stepwise exchange of all Ru ligands except for 1H-imidazole (26 h) to the final "ruthenated" protein comprising one aquated Ru ion coordinated to histidine-15 of HEWL (98 h). Our structural data clearly support a two-step mechanism of protein ruthenation, illustrating the ligand-mediated recognition step of the process.
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Antineoplásicos/química , Antineoplásicos/farmacología , Estructuras Metalorgánicas/química , Estructuras Metalorgánicas/farmacología , Muramidasa/química , Rutenio/química , Línea Celular Tumoral , Cristalografía por Rayos X , Humanos , Imidazoles/química , Modelos Moleculares , Metástasis de la Neoplasia , Conformación ProteicaRESUMEN
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.
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The high-resolution X-ray crystal structures of the adducts formed between the "half sandwich"-type Ru(II) coordination compound [RuII(1,4,7-trithiacyclononane)(ethane-1,2-diamine)Cl]+ and two proteins, namely hen egg-white lysozyme and proteinase K, are presented. The structures unveil that upon reaction with both enzymes the Ru(II) compound is coordinated by solvent-exposed aspartate residues after releasing the chloride ligand (Asp101 in lysozyme, Asp200 and Asp260 in proteinase K), while retaining the two chelating ligands. The adduct with Asp101 residue at the catalytic cleft of lysozyme is accompanied by residue-specific conformational changes to accommodate the Ru(II) fragment, whereas the complexes bound at the two calcium-binding sites of proteinase K revealed minimal structural perturbation of the enzyme. To the best of our knowledge, proteinase K is used here for the first time as a model system of protein metalation and these are the first X-ray crystal structures of protein adducts of a Ru(II) coordination compound that maintains its coordination sphere almost intact upon binding. Our data demonstrate the role of ligands in stabilizing the protein adducts via hydrophobic/aromatic or hydrogen-bonding interactions, as well as their underlying role in the selection of specific sites on the electrostatic potential surface of the enzymes.
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Complejos de Coordinación/química , Endopeptidasa K/química , Muramidasa/química , Rutenio/química , Complejos de Coordinación/metabolismo , Cristalografía por Rayos X , Endopeptidasa K/metabolismo , Modelos Moleculares , Conformación Molecular , Muramidasa/metabolismo , Rutenio/metabolismoRESUMEN
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.
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Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that helps generate peptides presented by Major Histocompatibility Complex Class I (MHC class I) molecules and is an emerging target for immunotherapy applications. Despite almost two decades of research on ERAP1, lack of high-resolution crystal structures has hampered drug-development efforts. By optimizing the protein construct, we obtained a high-resolution (1.60 Å) crystal structure of the closed-conformation of ERAP1 with a potent phosphinic pseudopeptide inhibitor bound in its active site. The structure provides key insight on the mechanism of inhibition as well as selectivity toward homologous enzymes and allows detailed mapping of the internal cavity of the enzyme that accommodates peptide-substrates. Bis-tris propane and malic acid molecules, found bound in pockets in the internal cavity, reveal potential druggable secondary binding sites. The ability to obtain high-resolution crystal structures of ERAP1 removes a major bottleneck in the development of compounds that regulate its activity and will greatly accelerate drug-discovery efforts.
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The α9 subunit of nicotinic acetylcholine receptors (nAChRs) exists mainly in heteropentameric assemblies with α10. Accumulating data indicate the presence of three different binding sites in α9α10 nAChRs: the α9(+)/α9(-), the α9(+)/α10(-), and the α10(+)/α9(-). The major role of the principal (+) side of the extracellular domain (ECD) of α9 subunit in binding of the antagonists methyllylcaconitine and α-bungarotoxin was shown previously by the crystal structures of the monomeric α9-ECD with these molecules. Here we present the 2.26-Å resolution crystal structure of α9-ECD in complex with α-conotoxin (α-Ctx) RgIA, a potential drug for chronic pain, the first structure reported for a complex between an nAChR domain and an α-Ctx. Superposition of this structure with those of other α-Ctxs bound to the homologous pentameric acetylcholine binding proteins revealed significant similarities in the orientation of bound conotoxins, despite the monomeric state of the α9-ECD. In addition, ligand-binding studies calculated a binding affinity of RgIA to the α9-ECD at the low micromolar range. Given the high identity between α9 and α10 ECDs, particularly at their (+) sides, the presented structure was used as template for molecular dynamics simulations of the ECDs of the human α9α10 nAChR in pentameric assemblies. Our results support a favorable binding of RgIA at α9(+)/α9(-) or α10(+)/α9(-) rather than the α9(+)/α10(-) interface, in accordance with previous mutational and functional data.
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Ba0331 is a putative polysaccharide deacetylase from Bacillus anthracis, the etiological agent of the disease anthrax, that contributes to adaptation of the bacterium under extreme conditions and to maintenance of the cell shape. In the present study, the crystal structure of Ba0331 was determined at 2.6â Å resolution. The structure consists of two domains: a fibronectin type 3-like (Fn3-like) domain and a NodB catalytic domain. The latter is present in all carbohydrate esterase family 4 enzymes, while a comparative analysis of the Fn3-like domain revealed structural plasticity despite the retention of the conserved Fn3-like domain characteristics.
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Amidohidrolasas/química , Amidohidrolasas/genética , Bacillus anthracis/enzimología , Expresión Génica , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Dominios Proteicos , Electricidad Estática , Zinc/metabolismoRESUMEN
Functional and folding constraints impose interdependence between interacting sites along the protein chain that are envisaged through protein sequence evolution. Studying the influence of structure in phylogenetic models requires detailed and reliable structural models. Polysaccharide deacetylases (PDAs), members of the carbohydrate esterase family 4, perform mainly metal-dependent deacetylation of O- or N-acetylated polysaccharides such as peptidoglycan, chitin and acetylxylan through a conserved catalytic core termed the NodB homology domain. Genomes of Bacillus anthracis and its relative Bacillus cereus contain multiple genes of putative or known PDAs. A comparison of the functional domains of the recently determined PDAs from B. anthracis and B. cereus and multiple amino acid and nucleotide sequence alignments and phylogenetic analysis performed on these closely related species showed that there were distinct differences in binding site formation, despite the high conservation on the protein sequence, the folding level and the active site assembly. This may indicate that, subject to biochemical verification, the binding site-forming sequence fragments are under functionally driven evolutionary pressure to accommodate and recognize distinct polysaccharide residues according to cell location, use, or environment. Finally, we discuss the suggestion of the paralogous nature of at least two genes of B. anthracis, ba0330 and ba0331, via specific differences in gene sequence, protein structure, selection pressure and available localization patterns. This study may contribute to understanding the mechanisms under which sequences evolve in their structures and how evolutionary processes enable structural variations.
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Nicotinic ACh receptors (nAChRs) are the best studied members of the superfamily of pentameric ligand-gated ion channels (pLGICs). Neuronal nAChRs regulate neuronal excitability and neurotransmitter release in the nervous system and form either homo- or hetero-pentameric complexes with various combinations of the 11 neuronal nAChR subunits (α2-7, α9, α10 and ß2-4) known to exist in humans. In addition to their wide distribution in the nervous system, neuronal nAChRs have been also found in immune cells and many peripheral tissues. These nAChRs are important drug targets for neurological and neuropsychiatric diseases (e.g. Alzheimer's, schizophrenia) and substance addiction (e.g. nicotine), as well as in a variety of diseases such as chronic pain, auditory disorders and some cancers. To decipher the functional mechanisms of human nAChRs and develop efficient and specific therapeutic drugs, elucidation of their high-resolution structures is needed. Recent studies, including the X-ray crystal structures of the near-intact α4ß2 nAChR and of the ligand-binding domains of the α9 and α2 subunits, have advanced our knowledge on the detailed structure of the ligand-binding sites formed between the same and different subunits and revealed many other functionally important interactions. The aim of this review is to highlight some of the structural and functional findings of these studies and to compare them with recent breakthrough findings on other pLGIC members and earlier data from their homologous ACh-binding proteins. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.
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Neuronas , Receptores Nicotínicos , Humanos , Modelos Moleculares , Neuronas/química , Neuronas/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Relación Estructura-ActividadRESUMEN
The cell wall peptidoglycan is recognized as a primary target of the innate immune system, and usually its disintegration results in bacterial lysis. Bacillus cereus, a close relative of the highly virulent Bacillus anthracis, contains 10 polysaccharide deacetylases. Among these, the peptidoglycan N-acetylglucosamine deacetylase Bc1974 is the highest homologue to the Bacillus anthracis Ba1977 that is required for full virulence and is involved in resistance to the host's lysozyme. These metalloenzymes belong to the carbohydrate esterase family 4 (CE4) and are attractive targets for the development of new anti-infective agents. Herein we report the first X-ray crystal structures of the NodB domain of Bc1974, the conserved catalytic core of CE4s, in the unliganded form and in complex with four known metalloenzyme inhibitors and two amino acid hydroxamates that target the active site metal. These structures revealed the presence of two conformational states of a catalytic loop known as motif-4 (MT4), which were not observed previously for peptidoglycan deacetylases, but were recently shown in the structure of a Vibrio clolerae chitin deacetylase. By employing molecular docking of a substrate model, we describe a catalytic mechanism that probably involves initial binding of the substrate in a receptive, more open state of MT4 and optimal catalytic activity in the closed state of MT4, consistent with the previous observations. The ligand-bound structures presented here, in addition to the five Bc1974 inhibitors identified, provide a valuable basis for the design of antibacterial agents that target the peptidoglycan deacetylase Ba1977.
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Amidohidrolasas/química , Bacillus cereus/enzimología , Proteínas Bacterianas/química , Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Catálisis , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Dominios Proteicos , Proteínas Recombinantes/química , Relación Estructura-Actividad , Zinc/químicaRESUMEN
The enantioselectivity of ß-cyclodextrin (ß-CD) towards L- and D-N-acetyltryptophan (NAcTrp) has been studied in aqueous solution and the crystalline state. NMR studies in solution show that ß-CD forms complexes of very similar but not identical geometry with both L- and D-NAcTrp and exhibits stronger binding with L-NAcTrp. In the crystalline state, only ß-CD-L-NAcTrp crystallizes readily from aqueous solutions as a dimeric complex (two hosts enclosing two guest molecules). In contrast, crystals of the complex ß-CD-D-NAcTrp were never obtained, although numerous conditions were tried. In aqueous solution, the orientation of the guest in both complexes is different than in the ß-CD-L-NAcTrp complex in the crystal. Overall, the study shows that subtle differences observed between the ß-CD-L,D-NAcTrp complexes in aqueous solution are magnified at the onset of crystallization, as a consequence of accumulation of many soft host-guest interactions and of the imposed crystallographic order, thus resulting in very dissimilar propensity of each enantiomer to produce crystals with ß-CD.
RESUMEN
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.
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Cistinil Aminopeptidasa/antagonistas & inhibidores , Cistinil Aminopeptidasa/metabolismo , Inhibidores Enzimáticos/farmacología , Conformación Proteica/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Cistinil Aminopeptidasa/química , Inhibidores Enzimáticos/química , Humanos , Ligandos , Modelos Moleculares , Péptidos/metabolismo , Especificidad por SustratoRESUMEN
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.