RESUMEN
Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic ß cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.
Asunto(s)
Estrés del Retículo Endoplásmico , Endorribonucleasas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Regulación Alostérica , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Retina/metabolismo , Ribonucleasas/antagonistas & inhibidoresRESUMEN
During endoplasmic reticulum (ER) stress, homeostatic signaling through the unfolded protein response (UPR) augments ER protein-folding capacity. If homeostasis is not restored, the UPR triggers apoptosis. We found that the ER transmembrane kinase/endoribonuclease (RNase) IRE1alpha is a key component of this apoptotic switch. ER stress induces IRE1alpha kinase autophosphorylation, activating the RNase to splice XBP1 mRNA and produce the homeostatic transcription factor XBP1s. Under ER stress--or forced autophosphorylation--IRE1alpha's RNase also causes endonucleolytic decay of many ER-localized mRNAs, including those encoding chaperones, as early events culminating in apoptosis. Using chemical genetics, we show that kinase inhibitors bypass autophosphorylation to activate the RNase by an alternate mode that enforces XBP1 splicing and averts mRNA decay and apoptosis. Alternate RNase activation by kinase-inhibited IRE1alpha can be reconstituted in vitro. We propose that divergent cell fates during ER stress hinge on a balance between IRE1alpha RNase outputs that can be tilted with kinase inhibitors to favor survival.
Asunto(s)
Endorribonucleasas/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Células/metabolismo , Retículo Endoplásmico/metabolismo , Insulina/genética , Complejos Multienzimáticos , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas , Estabilidad del ARN , Ratas , RibonucleasasRESUMEN
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
After lung injury, damage-associated transient progenitors (DATPs) emerge, representing a transitional state between injured epithelial cells and newly regenerated alveoli. DATPs express profibrotic genes, suggesting that they might promote idiopathic pulmonary fibrosis (IPF). However, the molecular pathways that induce and/or maintain DATPs are incompletely understood. Here we show that the bifunctional kinase/RNase-IRE1α-a central mediator of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress is a critical promoter of DATP abundance and function. Administration of a nanomolar-potent, monoselective kinase inhibitor of IRE1α (KIRA8)-or conditional epithelial IRE1α gene knockout-both reduce DATP cell number and fibrosis in the bleomycin model, indicating that IRE1α cell-autonomously promotes transition into the DATP state. IRE1α enhances the profibrotic phenotype of DATPs since KIRA8 decreases expression of integrin αvß6, a key activator of transforming growth factor ß (TGF-ß) in pulmonary fibrosis, corresponding to decreased TGF-ß-induced gene expression in the epithelium and decreased collagen accumulation around DATPs. Furthermore, IRE1α regulates DNA damage response (DDR) signaling, previously shown to promote the DATP phenotype, as IRE1α loss-of-function decreases H2AX phosphorylation, Cdkn1a (p21) expression, and DDR-associated secretory gene expression. Finally, KIRA8 treatment increases the differentiation of Krt19CreERT2-lineage-traced DATPs into type 1 alveolar epithelial cells after bleomycin injury, indicating that relief from IRE1α signaling enables DATPs to exit the transitional state. Thus, IRE1α coordinates a network of stress pathways that conspire to entrap injured cells in the DATP state. Pharmacological blockade of IRE1α signaling helps resolve the DATP state, thereby ameliorating fibrosis and promoting salutary lung regeneration.
Asunto(s)
Endorribonucleasas , Fibrosis Pulmonar Idiopática , Apoptosis/fisiología , Estrés del Retículo Endoplásmico/fisiología , Endorribonucleasas/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
BACKGROUND: Inositol-requiring enzyme 1α (IRE1α), along with protein kinase R-like endoplasmic reticulum kinase (PERK), is a principal regulator of the unfolded protein response (UPR). Recently, the 'mono'-specific IRE1α inhibitor, kinase-inhibiting RNase attenuator 6 (KIRA6), demonstrated a promising effect against multiple myeloma (MM). Side-stepping the clinical translation, a detailed UPR phenotype in patients with MM and the mechanisms of how KIRA8 works in MM remains unclear. METHODS: We characterized UPR phenotypes in the bone marrow of patients with newly diagnosed MM. Then, in human MM cells we analyzed the possible anti-tumor mechanisms of KIRA8 and a Food and Drug Administration (FDA)-approved drug, nilotinib, which we recently identified as having a strong inhibitory effect against IRE1α activity. Finally, we performed an RNA-sequence analysis to detect key IRE1α-related molecules against MM. RESULTS: We illustrated the dominant induction of adaptive UPR markers under IRE1α over the PERK pathway in patients with MM. In human MM cells, KIRA8 decreased cell viability and induced apoptosis, along with the induction of C/EBP homologous protein (CHOP); its combination with bortezomib exhibited more anti-myeloma effects than KIRA8 alone. Nilotinib exerted a similar effect compared with KIRA8. RNA-sequencing identified Polo-like kinase 2 (PLK2) as a KIRA8-suppressed gene. Specifically, the IRE1α overexpression induced PLK2 expression, which was decreased by KIRA8. KIRA8 and PLK2 inhibition exerted anti-myeloma effects with apoptosis induction and the regulation of cell proliferation. Finally, PLK2 was pathologically confirmed to be highly expressed in patients with MM. CONCLUSION: Dominant activation of adaptive IRE1α was established in patients with MM. Both KIRA8 and nilotinib exhibited anti-myeloma effects, which were enhanced by bortezomib. Adaptive IRE1α signaling and PLK2 could be potential therapeutic targets and biomarkers in MM.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Endorribonucleasas/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Terapia Molecular Dirigida , Mieloma Múltiple/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Adulto , Anciano , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Movimiento Celular , Proliferación Celular , Estudios Transversales , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/patología , Pronóstico , Pirazinas/administración & dosificación , Pirimidinas/administración & dosificación , Estudios Retrospectivos , Células Tumorales CultivadasRESUMEN
Under endoplasmic reticulum stress, unfolded protein accumulation leads to activation of the endoplasmic reticulum transmembrane kinase/endoRNase (RNase) IRE1α. IRE1α oligomerizes, autophosphorylates and initiates splicing of XBP1 mRNA, thus triggering the unfolded protein response (UPR). Here we show that IRE1α's kinase-controlled RNase can be regulated in two distinct modes with kinase inhibitors: one class of ligands occupies IRE1α's kinase ATP-binding site to activate RNase-mediated XBP1 mRNA splicing even without upstream endoplasmic reticulum stress, whereas a second class can inhibit the RNase through the same ATP-binding site, even under endoplasmic reticulum stress. Thus, alternative kinase conformations stabilized by distinct classes of ATP-competitive inhibitors can cause allosteric switching of IRE1α's RNase--either on or off. As dysregulation of the UPR has been implicated in a variety of cell degenerative and neoplastic disorders, small-molecule control over IRE1α should advance efforts to understand the UPR's role in pathophysiology and to develop drugs for endoplasmic reticulum stress-related diseases.
Asunto(s)
Endorribonucleasas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Catálisis , Células Cultivadas , Reactivos de Enlaces Cruzados , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Estrés del Retículo Endoplásmico/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Conformación Molecular , Mutación/genética , Mutación/fisiología , Fosforilación , Empalme del ARN/efectos de los fármacos , Factores de Transcripción del Factor Regulador X , Ribonucleasas/metabolismo , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Proteína 1 de Unión a la X-BoxRESUMEN
Pathogenic COPA variants cause a Mendelian syndrome of immune dysregulation with elevated type I interferon signaling. COPA is a subunit of coat protein complex I (COPI) that mediates Golgi to ER transport. Missense mutations of the COPA WD40 domain impair binding and sorting of proteins targeted for ER retrieval, but how this causes disease remains unknown. Given the importance of COPA in Golgi-ER transport, we speculated that type I interferon signaling in COPA syndrome involves missorting of STING. We show that a defect in COPI transport causes ligand-independent activation of STING. Furthermore, SURF4 is an adapter molecule that facilitates COPA-mediated retrieval of STING at the Golgi. Activated STING stimulates type I interferon-driven inflammation in CopaE241K/+ mice that is rescued in STING-deficient animals. Our results demonstrate that COPA maintains immune homeostasis by regulating STING transport at the Golgi. In addition, activated STING contributes to immune dysregulation in COPA syndrome and may be a new molecular target in treating the disease.
Asunto(s)
Proteína Coatómero/genética , Proteína Coatómero/metabolismo , Enfermedades del Sistema Inmune/genética , Proteínas de la Membrana/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Técnicas de Sustitución del Gen , Aparato de Golgi/metabolismo , Células HEK293 , Homeostasis/inmunología , Humanos , Interferón Tipo I/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Transporte de Proteínas/genética , Transducción de Señal/genética , Síndrome , TransfecciónRESUMEN
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
Endoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1α is a UPR sensor/effector that promotes apoptosis if ER stress remains high and irremediable (i.e., a "terminal" UPR). Using multiple small molecule inhibitors against IRE1α, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1α and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1α kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the contribution of IRE1α activation to bleomycin-induced pulmonary fibrosis. One such KIRA-KIRA7-provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1α may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1α may eventually be developed into efficacious anti-fibrotic drugs.
Asunto(s)
Células Epiteliales Alveolares/efectos de los fármacos , Endorribonucleasas/antagonistas & inhibidores , Fibrosis/tratamiento farmacológico , Pulmón/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fibrosis/metabolismo , Fibrosis/patología , Pulmón/metabolismo , Pulmón/patología , Ratones , Inhibidores de Proteínas Quinasas/uso terapéutico , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Master regulators of the unfolded protein response (UPR), IRE1α and PERK, promote adaptation or apoptosis depending on the level of endoplasmic reticulum (ER) stress. Although the UPR is activated in many cancers, its effects on tumor growth remain unclear. Derived from endocrine cells, pancreatic neuroendocrine tumors (PanNET) universally hypersecrete one or more peptide hormones, likely sensitizing these cells to high ER protein-folding stress. To assess whether targeting the UPR is a viable therapeutic strategy, we analyzed human PanNET samples and found evidence of elevated ER stress and UPR activation. Genetic and pharmacologic modulation of IRE1α and PERK in cultured cells, xenograft, and spontaneous genetic (RIP-Tag2) mouse models of PanNETs revealed that UPR signaling was optimized for adaptation and that inhibiting either IRE1α or PERK led to hyperactivation and apoptotic signaling through the reciprocal arm, thereby halting tumor growth and survival. These results provide a strong rationale for therapeutically targeting the UPR in PanNETs and other cancers with elevated ER stress. SIGNIFICANCE: The UPR is upregulated in pancreatic neuroendocrine tumors and its inhibition significantly reduces tumor growth in preclinical models, providing strong rationale for targeting the UPR in these cancers.
Asunto(s)
Endorribonucleasas/antagonistas & inhibidores , Tumores Neuroendocrinos/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , eIF-2 Quinasa/antagonistas & inhibidores , Adenina/análogos & derivados , Adenina/farmacología , Adenina/uso terapéutico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Endorribonucleasas/metabolismo , Femenino , Humanos , Indoles/farmacología , Indoles/uso terapéutico , Ratones , Ratones Transgénicos , Tumores Neuroendocrinos/genética , Tumores Neuroendocrinos/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , eIF-2 Quinasa/metabolismoRESUMEN
Dipeptidyl peptidase IV (DPP4) inhibitors are emerging as a new class of therapeutic agents for the treatment of type 2 diabetes. They exert their beneficial effects by increasing the levels of active glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are two important incretins for glucose homeostasis. Starting from a high-throughput screening hit, we were able to identify a series of piperidinone- and piperidine-constrained phenethylamines as novel DPP4 inhibitors. Optimized compounds are potent, selective, and have good pharmacokinetic profiles.
Asunto(s)
Inhibidores de la Adenosina Desaminasa , Inhibidores de la Dipeptidil-Peptidasa IV , Glicoproteínas/antagonistas & inhibidores , Fenetilaminas/síntesis química , Piperidinas/síntesis química , Animales , Disponibilidad Biológica , Cristalografía por Rayos X , Dipeptidil Peptidasa 4 , Humanos , Conformación Molecular , Fenetilaminas/farmacocinética , Fenetilaminas/farmacología , Piperidinas/farmacocinética , Piperidinas/farmacología , Piperidonas/síntesis química , Piperidonas/farmacocinética , Piperidonas/farmacología , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
TGF-ß1 signaling is a critical driver of collagen accumulation and fibrotic disease but also a vital suppressor of inflammation and epithelial cell proliferation. The nature of this multifunctional cytokine has limited the development of global TGF-ß1 signaling inhibitors as therapeutic agents. We conducted phenotypic screens for small molecules that inhibit TGF-ß1-induced epithelial-mesenchymal transition without immediate TGF-ß1 receptor (TßR) kinase inhibition. We identified trihydroxyphenolic compounds as potent blockers of TGF-ß1 responses (IC50 ~50 nM), Snail1 expression, and collagen deposition in vivo in models of pulmonary fibrosis and collagen-dependent lung cancer metastasis. Remarkably, the functional effects of trihydroxyphenolics required the presence of active lysyl oxidase-like 2 (LOXL2), thereby limiting effects to fibroblasts or cancer cells, the major LOXL2 producers. Mechanistic studies revealed that trihydroxyphenolics induce auto-oxidation of a LOXL2/3-specific lysine (K731) in a time-dependent reaction that irreversibly inhibits LOXL2 and converts the trihydrophenolic to a previously undescribed metabolite that directly inhibits TßRI kinase. Combined inhibition of LOXL2 and TßRI activities by trihydrophenolics resulted in potent blockade of pathological collagen accumulation in vivo without the toxicities associated with global inhibitors. These findings elucidate a therapeutic approach to attenuate fibrosis and the disease-promoting effects of tissue stiffness by specifically targeting TßRI kinase in LOXL2-expressing cells.
Asunto(s)
Inhibidores Enzimáticos , Transición Epitelial-Mesenquimal , Fibroblastos/metabolismo , Neoplasias Pulmonares , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Fibrosis Pulmonar , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Células A549 , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Animales , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fibroblastos/patología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Fenoles/química , Fenoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
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
Dipeptidyl peptidase IV (DPP4) deactivates glucose-regulating hormones such as GLP-1 and GIP, thus, DPP4 inhibition has become a useful therapy for type 2 diabetes. Optimization of the high-throughput screening lead 6 led to the discovery of 25 (ABT-341), a highly potent, selective, and orally bioavailable DPP4 inhibitor. When dosed orally, 25 dose-dependently reduced glucose excursion in ZDF rats. Amide 25 is safe in a battery of in vitro and in vivo tests and may represent a new therapeutic agent for the treatment of type 2 diabetes.
Asunto(s)
Compuestos de Bifenilo/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Dipeptidil Peptidasa 4/metabolismo , Hipoglucemiantes/farmacología , Inhibidores de Serina Proteinasa/farmacología , Triazoles/farmacología , Animales , Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/farmacocinética , Ciclohexenos/química , Diabetes Mellitus Tipo 2/genética , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Femenino , Hipoglucemiantes/síntesis química , Hipoglucemiantes/farmacocinética , Modelos Moleculares , Ratas , Ratas Zucker , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacocinética , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/farmacocinética , Difracción de Rayos XRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Endorribonucleasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Regulación Alostérica , Unión Competitiva , Estrés del Retículo Endoplásmico , Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/química , Humanos , Ligandos , Estructura Molecular , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Ribonucleasas/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
Enzymatic activity in the fecal droppings from the house dust mite has been postulated to contribute to the elicited allergic response. Screening dust mite extracts through 137,180 tetrapeptide fluorogenic substrates allowed for the characterization of proteolytic substrate specificity from the potential cysteine and serine proteases in the extract. The extract was further screened against a 4000 member peptide nucleic acid (PNA) encoded inhibitor library designed to target cysteine proteases using microarray detection. Affinity chromatography coupled with mass spectrometry identified Der p 1 as one of the proteases targeted by the PNA inhibitors in the dust mite lysate. A phenotypic readout of Der p 1 function in allergy progression was demonstrated by the inhibition of CD25 cleavage from T cells by dust mite extract that had been treated with the Der p 1 inhibitor identified from the PNA-encoded inhibitor library.
Asunto(s)
Antígenos Dermatofagoides/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Biblioteca de Péptidos , Secuencia de Aminoácidos , Antígenos Dermatofagoides/aislamiento & purificación , Antígenos Dermatofagoides/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Mapeo de Interacción de Proteínas/métodos , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato/genéticaRESUMEN
Diastereoselective enolate alkylation reactions of N-acylsulfinamides and conversion of the alkylation products to a variety of enantiopure products are reported. Several sulfinamides were prepared in solution followed by acylation to provide N-acylsulfinamides. The N-acylsulfinamides were then evaluated in diastereoselective enolate alkylation reactions. Of the sulfinamides evaluated, tert-butanesulfinamide provided the highest diastereoselectivity. To establish the potential utility of sulfinamides as versatile auxiliaries, methods were developed for (1) the racemization-free acylation of tert-butanesulfinamide to prepare optically pure N-acyl-tert-butanesulfinamides, (2) the diastereoselective C-alkylation of N-acyl-tert-butanesulfinamides, (3) the conversion of the N-acyl-tert-butanesulfinamides to the active ester equivalent by N-alkylation and S-oxidation, and (4) the cleavage of the N-alkyl-N-acyl-tert-butanesulfonamides to give chiral alcohol, ester, amide, and carboxylic acid target compounds. These studies provide the groundwork for the development of sulfinamides as dual chiral auxiliaries and linkers for the multistep solid-phase synthesis of enantioenriched compounds.
RESUMEN
Small molecules encoded with peptidonucleic acid (PNA) were used to probe protein function in a microarray format. The PNA tag served to encode the synthetic history of the small molecule and to positionally encode the identity of the small molecule by its location upon hybridization to an oligonucleotide microarray.
RESUMEN
When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to cause programmed cell death. We discovered that thioredoxin-interacting protein (TXNIP) is a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1ß (IL-1ß) secretion. Txnip gene deletion reduces pancreatic ß cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule IRE1α RNase inhibitors suppress TXNIP production to block IL-1ß secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.