RESUMEN
BACKGROUND AND PURPOSE: The endocannabinoid (eCB) system plays an important homeostatic role in the regulation of stress circuits and has emerged as a therapeutic target to treat stress disorders and alcohol use disorder (AUD). Extensive research has elucidated a role for the eCB anandamide (AEA), but less is known about 2-arachidonoylglycerol (2-AG) mediated signalling. EXPERIMENTAL APPROACH: We pharmacologically enhanced eCB signalling by inhibiting the 2-AG metabolizing enzyme, monoacylglycerol lipase (MAGL), in male and female Marchigian Sardinian alcohol-preferring (msP) rats, a model of innate alcohol preference and stress hypersensitivity, and in control Wistar rats. We tested the acute effect of the selective MAGL inhibitor MJN110 in alleviating symptoms of alcohol drinking, anxiety, irritability and fear. KEY RESULTS: A single systemic administration of MJN110 increased 2-AG levels in the central amygdala, prelimbic and infralimbic cortex but did not acutely alter alcohol drinking. MAGL inhibition reduced aggressive behaviours in female msPs, and increased defensive behaviours in male msPs, during the irritability test. Moreover, in the novelty-induced hypophagia test, MJN110 selectively enhanced palatable food consumption in females, mitigating stress-induced food suppression. Lastly, msP rats showed increased conditioned fear behaviour compared with Wistar rats, and MJN110 reduced context-associated conditioned fear responses, but not cue-probed fear expression, in male msPs. CONCLUSIONS AND IMPLICATIONS: Acute inhibition of MAGL attenuated some stress-related responses in msP rats but not voluntary alcohol drinking. Our results provide new insights into the sex dimorphism documented in stress-induced responses. Sex-specific eCB-based approaches should be considered in the clinical development of therapeutics.
Asunto(s)
Monoacilglicerol Lipasas , Monoglicéridos , Ratas , Masculino , Femenino , Animales , Ratas Wistar , Etanol/farmacología , Endocannabinoides/metabolismoRESUMEN
The nicotinamide adenine dinucleotide hydrolase (NADase) sterile alpha toll/interleukin receptor motif containing-1 (SARM1) acts as a central executioner of programmed axon death and is a possible therapeutic target for neurodegenerative disorders. While orthosteric inhibitors of SARM1 have been described, this multidomain enzyme is also subject to intricate forms of autoregulation, suggesting the potential for allosteric modes of inhibition. Previous studies have identified multiple cysteine residues that support SARM1 activation and catalysis, but which of these cysteines, if any, might be selectively targetable by electrophilic small molecules remains unknown. Here, we describe the chemical proteomic discovery of a series of tryptoline acrylamides that site-specifically and stereoselectively modify cysteine-311 (C311) in the noncatalytic, autoregulatory armadillo repeat (ARM) domain of SARM1. These covalent compounds inhibit the NADase activity of WT-SARM1, but not C311A or C311S SARM1 mutants, show a high degree of proteome-wide selectivity for SARM1_C311 and stereoselectively block vincristine- and vacor-induced neurite degeneration in primary rodent dorsal root ganglion neurons. Our findings describe selective, covalent inhibitors of SARM1 targeting an allosteric cysteine, pointing to a potentially attractive therapeutic strategy for axon degeneration-dependent forms of neurological disease.
Asunto(s)
Proteínas del Dominio Armadillo , Cisteína , Proteínas del Citoesqueleto , Proteínas del Dominio Armadillo/antagonistas & inhibidores , Proteínas del Dominio Armadillo/química , Proteínas del Dominio Armadillo/genética , Axones , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Homeostasis , NAD+ Nucleosidasa , ProteómicaRESUMEN
The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing ß-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory 'sweet spot', achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector.
Asunto(s)
Adenosina Trifosfato/farmacología , Endorribonucleasas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Adenosina Trifosfato/química , Endorribonucleasas/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Desplegamiento Proteico/efectos de los fármacosRESUMEN
The dual kinase endoribonuclease IRE1 is a master regulator of cell fate decisions in cells experiencing endoplasmic reticulum (ER) stress. In mammalian cells, there are two paralogs of IRE1: IRE1α and IRE1ß. While IRE1α has been extensively studied, much less is understood about IRE1ß and its role in signaling. In addition, whether the regulation of IRE1ß's enzymatic activities varies compared to IRE1α is not known. Here, we show that the RNase domain of IRE1ß is enzymatically active and capable of cleaving an XBP1 RNA mini-substrate in vitro. Using ATP-competitive inhibitors, we find that, like IRE1α, there is an allosteric relationship between the kinase and RNase domains of IRE1ß. This allowed us to develop a novel toolset of both paralog specific and dual-IRE1α/ß kinase inhibitors that attenuate RNase activity (KIRAs). Using sequence alignments of IRE1α and IRE1ß, we propose a model for paralog-selective inhibition through interactions with nonconserved residues that differentiate the ATP-binding pockets of IRE1α and IRE1ß.
Asunto(s)
Endorribonucleasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Regulación Alostérica , Animales , Estrés del Retículo Endoplásmico , Endorribonucleasas/metabolismo , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleasas/metabolismoRESUMEN
In cells experiencing unrelieved endoplasmic reticulum (ER) stress, the ER transmembrane kinase/endoribonuclease (RNase)-IRE1α-endonucleolytically degrades ER-localized mRNAs to promote apoptosis. Here we find that the ABL family of tyrosine kinases rheostatically enhances IRE1α's enzymatic activities, thereby potentiating ER stress-induced apoptosis. During ER stress, cytosolic ABL kinases localize to the ER membrane, where they bind, scaffold, and hyperactivate IRE1α's RNase. Imatinib-an anti-cancer tyrosine kinase inhibitor-antagonizes the ABL-IRE1α interaction, blunts IRE1α RNase hyperactivity, reduces pancreatic ß cell apoptosis, and reverses type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. A mono-selective kinase inhibitor that allosterically attenuates IRE1α's RNase-KIRA8-also efficaciously reverses established diabetes in NOD mice by sparing ß cells and preserving their physiological function. Our data support a model wherein ER-stressed ß cells contribute to their own demise during T1D pathogenesis and implicate the ABL-IRE1α axis as a drug target for the treatment of an autoimmune disease.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Estrés del Retículo Endoplásmico , Endorribonucleasas/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Transducción de Señal , Animales , Apoptosis/efectos de los fármacos , Diabetes Mellitus Tipo 1/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Femenino , Humanos , Mesilato de Imatinib/farmacología , Masculino , Ratones Endogámicos NOD , Modelos Biológicos , Unión Proteica/efectos de los fármacos , Pirimidinas/farmacología , Ratas , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
There is an allosteric relationship between the kinase and RNase domains of the ER stress sensor IRE1α. This relationship has been exploited to develop ATP-competitive inhibitors that are able to divergently modulate the RNase activity of IRE1α through its kinase domain. Here, we describe a series of biochemical methods for profiling the dual enzymatic activities of IRE1α. These methods can be used to ascertain how ATP-competitive inhibitors affect the kinase activity of IRE1α and for determining whether these ligands allosterically activate or inactivate RNase activity.
Asunto(s)
Adenosina Trifosfato/metabolismo , Endorribonucleasas/antagonistas & inhibidores , Pruebas de Enzimas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Dominio Catalítico , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Fluoresceínas/química , Colorantes Fluorescentes/química , Expresión Génica , Humanos , Concentración 50 Inhibidora , Ligandos , Proteína Básica de Mielina/metabolismo , Radioisótopos de Fósforo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , SpodopteraRESUMEN
The accumulation of unfolded proteins under endoplasmic reticulum (ER) stress leads to the activation of the multidomain protein sensor IRE1α as part of the unfolded protein response (UPR). Clustering of IRE1α lumenal domains in the presence of unfolded proteins promotes kinase trans-autophosphorylation in the cytosol and subsequent RNase domain activation. Interestingly, there is an allosteric relationship between the kinase and RNase domains of IRE1α, which allows ATP-competitive inhibitors to modulate the activity of the RNase domain. Here, we use kinase inhibitors to study how ATP-binding site conformation affects the activity of the RNase domain of IRE1α. We find that diverse ATP-competitive inhibitors of IRE1α promote dimerization and activation of RNase activity despite blocking kinase autophosphorylation. In contrast, a subset of ATP-competitive ligands, which we call KIRAs, allosterically inactivate the RNase domain through the kinase domain by stabilizing monomeric IRE1α. Further insight into how ATP-competitive inhibitors are able to divergently modulate the RNase domain through the kinase domain was gained by obtaining the first structure of apo human IRE1α in the RNase active back-to-back dimer conformation. Comparison of this structure with other existing structures of IRE1α and integration of our extensive structure activity relationship (SAR) data has led us to formulate a model to rationalize how ATP-binding site ligands are able to control the IRE1α oligomeric state and subsequent RNase domain activity.