RESUMEN
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.
RESUMEN
Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.
Asunto(s)
Antiprotozoarios/administración & dosificación , Leishmania donovani/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Leishmaniasis Visceral/diagnóstico por imagen , Inhibidores de Proteasoma/administración & dosificación , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Antiprotozoarios/química , Sitios de Unión , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Leishmania donovani/química , Leishmania donovani/enzimología , Leishmania infantum/química , Leishmania infantum/enzimología , Leishmaniasis Visceral/parasitología , Masculino , Ratones , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Secuencia de Aminoácidos , Animales , Biocatálisis/efectos de los fármacos , Dominio Catalítico , Células Cultivadas , Inhibidores Enzimáticos/metabolismo , Evolución Molecular , Histonas/química , Histonas/metabolismo , Humanos , Concentración 50 Inhibidora , Histona Demetilasas con Dominio de Jumonji/química , Histona Demetilasas con Dominio de Jumonji/clasificación , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Macrófagos/enzimología , Macrófagos/metabolismo , Metilación/efectos de los fármacos , Ratones , Modelos Moleculares , Especificidad por Sustrato , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
A series of potent, competitive and highly selective kynurenine monooxygenase inhibitors have been discovered via a substrate-based approach for the treatment of acute pancreatitis. The lead compound demonstrated good cellular potency and clear pharmacodynamic activity in vivo.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Quinurenina 3-Monooxigenasa/antagonistas & inhibidores , Pancreatitis/tratamiento farmacológico , Animales , Benzoxazoles/química , Benzoxazoles/farmacocinética , Benzoxazoles/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacocinética , Células HEK293 , Humanos , Indazoles/química , Indazoles/farmacocinética , Indazoles/farmacología , Quinurenina/metabolismo , Quinurenina 3-Monooxigenasa/metabolismo , Pancreatitis/enzimología , Pancreatitis/metabolismo , Ratas , Triptófano/metabolismoRESUMEN
Malaria is a preventable and treatable disease; yet half of the world's population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of Plasmodium falciparum (Pf) DHODH inhibitors via in vitro resistance selections. We found 11 point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.
Asunto(s)
Antimaláricos/química , Inhibidores Enzimáticos/química , Malaria Falciparum/parasitología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Antimaláricos/farmacología , Sitios de Unión , Cristalografía por Rayos X , Dihidroorotato Deshidrogenasa , Evaluación Preclínica de Medicamentos , Resistencia a Medicamentos , Inhibidores Enzimáticos/farmacología , Humanos , Malaria Falciparum/tratamiento farmacológico , Modelos Moleculares , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Mutación Puntual , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
This study describes the first long-term (14-year) evaluation of the efficacy of an established (>100 years) natural wetland to remediate highly acidic mine drainage (AMD). Although natural wetlands are highly valued for their biodiversity, this study demonstrates that they also provide important ecosystem service functions through their ability to consistently and reliably improve water quality by mitigating AMD. The Afon Goch river flows from Parys Mountain copper mine via a natural wetland, and was the major source of Zn and Cu contamination to the Irish Sea. Prior to 2003 the wetland received severe acidic metal contamination and retained a large proportion of the contamination (55, 64, and 37% in dissolved Fe, Zn, and Cu) leading to a greatly reduced metal flow to the Irish Sea. Reduced wetland loadings midway through the sampling period led to a reduction of metals by 83-94% and a pH increase from 2.7 to 5.5, resulting in long-term improvements in the downstream benthic invertebrate community. High root metal accumulation by the dominant wetland plant species and the association of acidophilic bacteria in the wetland rhizosphere indicate that multiple interacting processes provide an efficient and self-sustaining system to remediate AMD.
Asunto(s)
Restauración y Remediación Ambiental/métodos , Metales , Minería , Calidad del Agua , Humedales , Ecosistema , Agua Dulce , Metales/análisis , Plantas , ARN Ribosómico 16S , Ríos , Reino Unido , Microbiología del Agua , Contaminantes Químicos del Agua/análisis , Contaminación del AguaRESUMEN
There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the Leishmania proteasome. A related analogue, active against Trypanosoma cruzi, showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the T. cruzi proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.
Asunto(s)
Enfermedad de Chagas , Leishmaniasis Visceral , Tripanocidas , Trypanosoma cruzi , Ratones , Animales , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Complejo de la Endopetidasa Proteasomal , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Leishmaniasis Visceral/tratamiento farmacológico , Tripanocidas/farmacología , Tripanocidas/uso terapéutico , Tripanocidas/químicaRESUMEN
The Janus family of tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) play an essential role in the receptor signaling of cytokines that have been implicated in the pathogenesis of severe asthma, and there is emerging interest in the development of small-molecule-inhaled JAK inhibitors as treatments. Here, we describe the optimization of a quinazoline series of JAK inhibitors and the results of mouse lung pharmacokinetic (PK) studies where only low concentrations of parent compound were observed. Subsequent investigations revealed that the low exposure was due to metabolism by aldehyde oxidase (AO), so we sought to identify quinazolines that were not metabolized by AO. We found that specific substituents at the quinazoline 2-position prevented AO metabolism and this was rationalized through computational docking studies in the AO binding site, but they compromised kinome selectivity. Results presented here highlight that AO metabolism is a potential issue in the lung.
Asunto(s)
Aldehído Oxidasa/metabolismo , Inhibidores de las Cinasas Janus/farmacocinética , Pulmón/metabolismo , Administración Intranasal , Administración Intravenosa , Animales , Sitios de Unión , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Inhibidores de las Cinasas Janus/administración & dosificación , Inhibidores de las Cinasas Janus/síntesis química , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Simulación del Acoplamiento Molecular , Quinazolinas/síntesis química , Quinazolinas/farmacocinética , Quinazolinas/farmacología , Relación Estructura-ActividadRESUMEN
The predominant assay detection methodologies used for enzyme inhibitor identification during early-stage drug discovery are fluorescence-based. Each fluorophore has a characteristic fluorescence decay, known as the fluorescence lifetime, that occurs throughout a nanosecond-to-millisecond timescale. The measurement of fluorescence lifetime as a reporter for biological activity is less common than fluorescence intensity, even though the latter has numerous issues that can lead to false-positive readouts. The confirmation of hit compounds as true inhibitors requires additional assays, cost, and time to progress from hit identification to lead drug-candidate optimization. To explore whether the use of fluorescence lifetime technology (FLT) can offer comparable benefits to label-free-based approaches such as RapidFire mass spectroscopy (RF-MS) and a superior readout compared to time-resolved fluorescence resonance energy transfer (TR-FRET), three equivalent assays were developed against the clinically validated tyrosine kinase 2 (TYK2) and screened against annotated compound sets. FLT provided a marked decrease in the number of false-positive hits when compared to TR-FRET. Further cellular screening confirmed that a number of potential inhibitors directly interacted with TYK2 and inhibited the downstream phosphorylation of the signal transducer and activator of transcription 4 protein (STAT4).
Asunto(s)
Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/normas , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/normas , Colorantes Fluorescentes , TYK2 Quinasa/antagonistas & inhibidores , TYK2 Quinasa/química , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Optimization of a previously reported lead series of PI3Kδ inhibitors with a novel binding mode led to the identification of a clinical candidate compound 31 (GSK251). Removal of an embedded Ames-positive heteroaromatic amine by reversing a sulfonamide followed by locating an interaction with Trp760 led to a highly selective compound 9. Further optimization to avoid glutathione trapping, to enhance potency and selectivity, and to optimize an oral pharmacokinetic profile led to the discovery of compound 31 (GSK215) that had a low predicted daily dose (45 mg, b.i.d) and a rat toxicity profile suitable for further development.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Sulfonamidas/farmacología , Animales , Cristalografía por Rayos X , Femenino , Masculino , Ratones Endogámicos BALB C , Estructura Molecular , Inhibidores de las Quinasa Fosfoinosítidos-3/síntesis química , Inhibidores de las Quinasa Fosfoinosítidos-3/metabolismo , Unión Proteica , Ratas Wistar , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/metabolismoRESUMEN
Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. Herein, is described a series of potent inhibitors based on an hydroxyethylamine (HEA) transition state mimetic template. These inhibitors interact with the non prime side of the enzyme using a novel edge-to-face interaction with Arg-296.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Arginina/química , Etilaminas/química , Inhibidores de Proteasas/química , Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Etilaminas/síntesis química , Etilaminas/uso terapéutico , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/uso terapéutico , Ratas , Relación Estructura-ActividadRESUMEN
We have designed and synthesized a novel series of alpha-amino cyclic boronates and incorporated them successfully in several acyclic templates at the P1 position. These compounds are inhibitors of the HCV NS3 serine protease, and structural studies show that they inhibit the NS3 protease by trapping the Ser-139 hydroxyl group in the active site. Synthetic methodologies and SARs of this series of compounds are described.
Asunto(s)
Ácidos Borónicos/síntesis química , Hepacivirus/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Ácidos Borónicos/farmacología , Ácidos Borónicos/uso terapéutico , Dominio Catalítico , Diseño de Fármacos , Hepacivirus/enzimología , Estructura Molecular , Serina/química , Relación Estructura-ActividadRESUMEN
A macrocyclization approach has been explored on a series of benzoxazine phosphoinositide 3-kinase δ inhibitors, resulting in compounds with improved potency, permeability, and in vivo clearance while maintaining good solubility. The thermodynamics of binding was explored via surface plasmon resonance, and the binding of lead macrocycle 19 was found to be almost exclusively entropically driven compared with progenitor 18, which demonstrated both enthalpic and entropic contributions. The pharmacokinetics of macrocycle 19 was also explored in vivo, where it showed reduced clearance when compared with the progenitor 18. This work adds to the growing body of evidence that macrocyclization could provide an alternative and complementary approach to the design of small-molecule inhibitors, with the potential to deliver differentiated properties.
RESUMEN
ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that generates antigenic peptides and is an emerging target for cancer immunotherapy and the control of autoimmunity. ERAP1 inhibitors described previously target the active site and are limited in selectivity, minimizing their clinical potential. To address this, we targeted the regulatory site of ERAP1 using a high-throughput screen and discovered a small molecule hit that is highly selective for ERAP1. (4aR,5S,6R,8S,8aR)-5-(2-(Furan-3-yl)ethyl)-8-hydroxy-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid is a natural product found in Dodonaea viscosa that constitutes a submicromolar, highly selective, and cell-active modulator of ERAP1. Although the compound activates hydrolysis of small model substrates, it is a competitive inhibitor for physiologically relevant longer peptides. Crystallographic analysis confirmed that the compound targets the regulatory site of the enzyme that normally binds the C-terminus of the peptide substrate. Our findings constitute a novel starting point for the development of selective ERAP1 modulators that have potential for further clinical development.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Presentación de Antígeno/efectos de los fármacos , Diterpenos de Tipo Clerodano/farmacología , Epítopos/metabolismo , Péptidos/metabolismo , Inhibidores de Proteasas/farmacología , Sitio Alostérico , Aminopeptidasas/química , Aminopeptidasas/metabolismo , Animales , Dominio Catalítico , Cristalografía por Rayos X , Diterpenos de Tipo Clerodano/química , Diterpenos de Tipo Clerodano/metabolismo , Activadores de Enzimas/química , Activadores de Enzimas/metabolismo , Activadores de Enzimas/farmacología , Epítopos/química , Células HeLa , Humanos , Ratones , Antígenos de Histocompatibilidad Menor/química , Antígenos de Histocompatibilidad Menor/metabolismo , Péptidos/química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Unión Proteica , Proteolisis/efectos de los fármacosRESUMEN
Optimization of a lead series of PI3Kδ inhibitors based on a dihydroisobenzofuran core led to the identification of potent, orally bioavailable compound 19. Selectivity profiling of compound 19 showed similar potency for class III PI3K, Vps34, and PI3Kδ, and compound 19 was not well-tolerated in a 7-day rat toxicity study. Structure-based design led to an improvement in selectivity for PI3Kδ over Vps34 and, a focus on oral phramacokinetics properties resulted in the discovery of compound 41, which showed improved toxicological outcomes at similar exposure levels to compound 19.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas Clase III/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacocinética , Animales , Unión Competitiva , Disponibilidad Biológica , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Isoenzimas , Modelos Moleculares , Simulación del Acoplamiento Molecular , Inhibidores de las Quinasa Fosfoinosítidos-3/toxicidad , Ratas , Relación Estructura-ActividadRESUMEN
Our first generation of hydroxyethylamine BACE-1 inhibitors proved unlikely to provide molecules that would lower amyloid in an animal model at low oral doses. This observation led us to the discovery of a second generation of inhibitors having nanomolar activity in a cell-based assay and with the potential for improved pharmacokinetic profiles. In this Letter, we describe our successful strategy for the optimization of oral bioavailability and also give insights into the design of compounds with the potential for improved brain penetration.
Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Etilaminas/química , Inhibidores de Proteasas/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Disponibilidad Biológica , Perros , Etilaminas/síntesis química , Etilaminas/farmacocinética , Ratones , Ratones Noqueados , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
Our first generation of hydroxyethylamine transition-state mimetic BACE-1 inhibitors allowed us to validate BACE-1 as a key target for Alzheimer's disease by demonstrating amyloid lowering in an animal model, albeit at rather high doses. Finding a molecule from this series which was active at lower oral doses proved elusive and demonstrated the need to find a novel series of inhibitors with improved pharmacokinetics. This Letter describes the discovery of such inhibitors.
Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Etilaminas/química , Inhibidores de Proteasas/química , Administración Oral , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Etilaminas/síntesis química , Etilaminas/farmacología , Humanos , Ratones , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Ratas , Relación Estructura-Actividad , Tiazinas/química , Tiazinas/farmacologíaRESUMEN
Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. We have recently disclosed a series of transition-state mimetic BACE-1 inhibitors showing nanomolar potency in cell-based assays. Amongst them, GSK188909 (compound 2) had favorable pharmacokinetics and was the first orally bioavailable inhibitor reported to demonstrate brain amyloid lowering in an animal model. In this Letter, we describe the reasons that led us to favor a second generation of inhibitors for further in vivo studies.
Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Tiazinas/química , Administración Oral , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Simulación por Computador , Etilaminas/síntesis química , Etilaminas/química , Etilaminas/farmacología , Humanos , Ratones , Inhibidores de Proteasas/farmacocinética , Ratas , Relación Estructura-Actividad , Tiazinas/síntesis química , Tiazinas/farmacocinéticaRESUMEN
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.