RESUMEN
Enterovirus 71 (EV71) and Coxsackie A16 (CVA16) are two major causative agents of hand, foot, and mouth disease (HFMD) in young children. However, the mechanisms regulating the replication and pathogenesis of EV71/CVA16 remain incompletely understood. We performed a genome-wide CRISPR-Cas9 knockout screen and identified Ragulator as a mediator of EV71-induced apoptosis and pyroptosis. The Ragulator-Rag complex is required for EV71 and CVA16 replication. Upon infection, the Ragulator-Rag complex recruits viral 3D protein to the lysosomal surface through the interaction between 3D and RagB. Disruption of the lysosome-tethered Ragulator-Rag-3D complex significantly impairs the replication of EV71/CVA16. We discovered a novel EV71 inhibitor, ZHSI-1, which interacts with 3D and significantly reduces the lysosomal tethering of 3D. ZHSI-1 treatment significantly represses replication of EV71/CVA16 as well as virus-induced pyroptosis associated with viral pathogenesis. Importantly, ZHSI-1 treatment effectively protects against EV71 infection in neonatal and young mice. Thus, our study indicates that targeting lysosome-tethered Ragulator-Rag-3D may be an effective therapeutic strategy for HFMD.
Asunto(s)
Enterovirus Humano A , Enfermedad de Boca, Mano y Pie , Proteínas no Estructurales Virales , Animales , Ratones , Apoptosis , Sistemas CRISPR-Cas , Enterovirus Humano A/genética , Lisosomas , Piroptosis , Proteínas no Estructurales Virales/genética , Replicación Viral , Enfermedad de Boca, Mano y Pie/virología , Modelos Animales de EnfermedadRESUMEN
Interleukin-1 receptor-associated kinase 4 (IRAK4) is a key regulator to control downstream NF-κB and MAPK signals in the innate immune response and has been proposed as a therapeutic target for the treatment of inflammatory and autoimmune diseases. Herein, a series of IRAK4 inhibitors based on a dihydrofuro[2,3-b]pyridine scaffold was developed. Structural modifications of the screening hit 16 (IC50 = 243 nM) led to IRAK4 inhibitors with improved potency but high clearance (Cl) and poor oral bioavailability, as exemplified by compound 21 (IC50 = 6.2 nM, Cl = 43 ml/min/kg, F = 1.6%, LLE = 5.4). Structure modification aimed at improving LLE and reducing clearance identified compound 38. Compound 38 showed significantly improved clearance while maintained excellent biochemical potency against IRAK4 (IC50 = 7.3 nM, Cl = 12 ml/min/kg, F = 21%, LLE = 6.0). Importantly, compound 38 had favorable in vitro safety and ADME profiles. Furthermore, compound 38 reduced the in vitro production of pro-inflammatory cytokines in both mouse iBMDMs and human PBMCs and was orally efficacious in the inhibition of serum TNF-α secretion in LPS-induced mouse model. These findings suggested that compound 38 has development potential as an IRAK4 inhibitor for the treatment of inflammatory and autoimmune disorders.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1 , Transducción de Señal , Humanos , Animales , Ratones , FN-kappa B/metabolismo , Citocinas , Piridinas/farmacologíaRESUMEN
Nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD 1/2) are important cytosolic pattern recognition receptors that initiate host immune response. The dysregulation of NOD signaling is highly associated with inflammatory bowel disease (IBD) that needs novel treatment options. Receptor-interacting protein kinase 2 (RIPK2) is a critical mediator of NOD signaling and considered a promising therapeutic target for IBD treatment. However, there are currently no RIPK2 inhibitors available for clinical use. Here, we report the discovery and characterization of Zharp2-1 as a novel and potent RIPK2 inhibitor that effectively blocks RIPK2 kinase function and NOD-mediated NF-κB/MAPK activation in both human and mouse cell lines. Zharp2-1 exhibits significantly superior solubility compared to the non-prodrug form of the advanced RIPK2 inhibitor prodrug GSK2983559. The improved solubility combined with favorable in vitro metabolic stability translated to excellent in vivo pharmacokinetic profiles for Zharp2-1. In addition, Zharp2-1 demonstrates better effects than GSK2983559 in inhibiting the muramyl dipeptide (MDP)-induced production of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) and MDP-induced peritonitis in mice. Furthermore, Zharp2-1 markedly reduces Listeria monocytogenes infection-induced cytokines release in both human and mouse cells. Importantly, Zharp2-1 significantly ameliorates DNBS-induced colitis in rats and suppressed pro-inflammatory cytokine release in intestinal specimens from IBD patients. Collectively, our findings indicate that Zharp2-1 is a promising RIPK2 inhibitor with the potential to be further developed for IBD therapy.
Asunto(s)
Colitis , Enfermedades Inflamatorias del Intestino , Humanos , Ratones , Ratas , Animales , Leucocitos Mononucleares/metabolismo , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Transducción de Señal , Citocinas/metabolismoRESUMEN
Interleukin-1 receptor associated kinase-4 (IRAK4) has emerged as a therapeutic target for inflammatory and autoimmune diseases. Through reversing the amide of CA-4948 and computer aided structure-activity relationship (SAR) studies, a series of IRAK4 inhibitors with oxazolo[4,5-b]pyridine scaffold were identified. Compound 32 showed improved potency (IC50 = 43 nM) compared to CA-4948 (IC50 = 115 nM), but suffered from hERG inhibition (IC50 = 5.7 µM). Further optimization led to compound 42 with reduced inhibition of hERG (IC50 > 30 µM) and 13-fold higher activity (IC50 = 8.9 nM) than CA-4948. Importantly, compound 42 had favorable in vitro ADME and in vivo pharmacokinetic properties. Furthermore, compound 42 significantly reduced LPS-induced production of serum TNF-α and IL-6 cytokines in the mouse model. The overall profiles of compound 42 support it as a lead for the development of IRAK4 inhibitors for the treatment of inflammatory and autoimmune disorders.
Asunto(s)
Citocinas , Quinasas Asociadas a Receptores de Interleucina-1 , Animales , Ratones , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Lipopolisacáridos/farmacología , Síndrome de Respuesta Inflamatoria Sistémica , Relación Estructura-ActividadRESUMEN
Intestinal epithelial cells (IECs) are implicated in the propagation of T-cell-mediated inflammatory diseases, including graft-versus-host disease (GVHD), but the underlying mechanism remains poorly defined. Here, we report that IECs require receptor-interacting protein kinase-3 (RIPK3) to drive both gastrointestinal (GI) tract and systemic GVHD after allogeneic hematopoietic stem cell transplantation. Selectively inhibiting RIPK3 in IECs markedly reduces GVHD in murine intestine and liver. IEC RIPK3 cooperates with RIPK1 to trigger mixed lineage kinase domain-like protein-independent production of T-cell-recruiting chemokines and major histocompatibility complex (MHC) class II molecules, which amplify and sustain alloreactive T-cell responses. Alloreactive T-cell-produced interferon gamma enhances this RIPK1/RIPK3 action in IECs through a JAK/STAT1-dependent mechanism, creating a feed-forward inflammatory cascade. RIPK1/RIPK3 forms a complex with JAK1 to promote STAT1 activation in IECs. The RIPK1/RIPK3-mediated inflammatory cascade of alloreactive T-cell responses results in intestinal tissue damage, converting the local inflammation into a systemic syndrome. Human patients with severe GVHD showed highly activated RIPK1 in the colon epithelium. Finally, we discover a selective and potent RIPK1 inhibitor (Zharp1-211) that significantly reduces JAK/STAT1-mediated expression of chemokines and MHC class II molecules in IECs, restores intestinal homeostasis, and arrests GVHD without compromising the graft-versus-leukemia (GVL) effect. Thus, targeting RIPK1/RIPK3 in IECs represents an effective nonimmunosuppressive strategy for GVHD treatment and potentially for other diseases involving GI tract inflammation.
Asunto(s)
Enfermedad Injerto contra Huésped , Intestinos , Ratones , Humanos , Animales , Mucosa Intestinal/metabolismo , Inflamación/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Enfermedad Injerto contra Huésped/prevención & control , Enfermedad Injerto contra Huésped/metabolismo , Homeostasis , Proteína Serina-Treonina Quinasas de Interacción con ReceptoresRESUMEN
The NOD1/2 (nucleotide-binding oligomerization domain-containing protein 1/2) signaling pathways are involved in innate immune control and host defense. NOD dysfunction can result in a variety of autoimmune disorders. NOD-induced generation of inflammatory cytokines is mediated by receptor-interacting protein kinase 2 (RIPK2), which has been considered as a promising therapeutic target. Herein, we disclose the design, synthesis, and SAR study of a series of RIPK2 inhibitors. The lead compound 17 displayed a high affinity for RIPK2 (Kd = 5.9 nM) and was capable of inhibiting RIPK2 kinase function in an ADP-Glo assay. In vitro DMPK studies showed that compound 17 had good metabolic stability and no CYP inhibition. Compound 17 effectively suppressed inflammatory cytokine production in both cells and animal model.
Asunto(s)
Citocinas , Yohexol , Adenosina Difosfato , Animales , Citocinas/metabolismo , Yohexol/análogos & derivados , Relación Estructura-ActividadRESUMEN
Human enterovirus A71 (EV-A71), a member of the Picornaviridae family, is one of the main etiological viruses that lead to hand, foot, and mouth disease (HFMD). We utilized a multiplex tandem mass tag-based quantitative proteomic technique to monitor the alternation of the whole cell proteome and phosphoproteome of human rhabdomyosarcoma cells over the course of EV-A71 infection. We successfully quantified more than 7000 host proteins and 17,000 phosphosites, of which 80 proteins and nearly 1700 phosphosites were significantly regulated upon viral infection. We found that Myc proto-oncogene protein level decreased significantly, benefiting EV-A71 replication. Multiple signaling pathways were regulated in phosphorylation events that converge for protein translation, cell cycle control, and cell survival. Numerous host factors targeted by virus proteins are phosphoproteins. These factors are involved in host translational initiation, unfolded protein response, endoplasmic reticulum stress, and stress granule formation, and their phosphorylation may play key roles in the virus life cycle. Notably, we identified three conserved phosphorylation sites on viral polyproteins that have not been previously reported. Our study provides valuable resources for a systematic understanding of the interaction between the host cells and the EV-A71 at the protein and the post-translational level.
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Enterovirus Humano A , Infecciones por Enterovirus , Enterovirus , Antígenos Virales/metabolismo , Enterovirus Humano A/fisiología , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Poliproteínas , Proteoma/genética , Proteoma/metabolismo , Proteómica , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Receptor-interacting protein kinase-1 (RIPK1) is involved in the necroptosis pathway, which regulates inflammatory signaling and cell death in a variety of diseases, including inflammatory and neurodegenerative disorders. We identified a novel hit compound 36 by a cell-based screening assay (anti-necroptosis EC50 = 58 nM). Starting from compound 36, we designed a series of scaffolds to improve anti-necroptosis activity, physicochemical properties and metabolic stability. The isothiazolo[5,4-b]pyridine backbone proved to be a promising scaffold which provided a number of potent necroptosis inhibitors. Compound 56, for example, effectively blocked necroptosis in both human and mouse cells (EC50 = 1-5 nM). A binding assay showed that compound 56 potently binds to RIPK1 (Kd = 13 nM), but not RIPK3 (Kd > 10,000 nM). Kinase functional assay (ADP-Glo) confirmed that compound 56 inhibits RIPK1 phosphorylation with an IC50 at 5.8 nM. Importantly, compound 56 displayed excellent cross-species liver microsomal metabolic stability (t1/2 > 90 min). Furthermore, compound 56 exhibited favorable in vitro safety profiles in hERG and CYP assays. Finally, pre-treatment with 56 significantly reduced hypothermia and lethal shock in the systemic inflammatory response syndrome mice model. Taken together, compound 56 represented a promising prototype for the development of therapeutic agent to treat inflammation-related diseases.
Asunto(s)
Necroptosis , Piridinas , Humanos , Ratones , Animales , Fosforilación , Muerte Celular , Piridinas/farmacología , Síndrome de Respuesta Inflamatoria Sistémica , Apoptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/farmacologíaRESUMEN
XIAP-associated factor 1 (XAF1) is an interferon (IFN)-stimulated gene (ISG) that enhances IFN-induced apoptosis. However, it is unexplored whether XAF1 is essential for the host fighting against invaded viruses. Here, we find that XAF1 is significantly upregulated in the host cells infected with emerging RNA viruses, including influenza, Zika virus (ZIKV), and SARS-CoV-2. IFN regulatory factor 1 (IRF1), a key transcription factor in immune cells, determines the induction of XAF1 during antiviral immunity. Ectopic expression of XAF1 protects host cells against various RNA viruses independent of apoptosis. Knockout of XAF1 attenuates host antiviral innate immunity in vitro and in vivo, which leads to more severe lung injuries and higher mortality in the influenza infection mouse model. XAF1 stabilizes IRF1 protein by antagonizing the CHIP-mediated degradation of IRF1, thus inducing more antiviral IRF1 target genes, including DDX58, DDX60, MX1, and OAS2. Our study has described a protective role of XAF1 in the host antiviral innate immunity against RNA viruses. We have also elucidated the molecular mechanism that IRF1 and XAF1 form a positive feedback loop to induce rapid and robust antiviral immunity. IMPORTANCE Rapid and robust induction of antiviral genes is essential for the host to clear the invaded viruses. In addition to the IRF3/7-IFN-I-STAT1 signaling axis, the XAF1-IRF1 positive feedback loop synergistically or independently drives the transcription of antiviral genes. Moreover, XAF1 is a sensitive and reliable gene that positively correlates with the viral infection, suggesting that XAF1 is a potential diagnostic marker for viral infectious diseases. In addition to the antitumor role, our study has shown that XAF1 is essential for antiviral immunity. XAF1 is not only a proapoptotic ISG, but it also stabilizes the master transcription factor IRF1 to induce antiviral genes. IRF1 directly binds to the IRF-Es of its target gene promoters and drives their transcriptions, which suggests a unique role of the XAF1-IRF1 loop in antiviral innate immunity, particularly in the host defect of IFN-I signaling such as invertebrates.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Factor 1 Regulador del Interferón , Infecciones por Virus ARN , Virus ARN , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/inmunología , Humanos , Inmunidad Innata , Factor 1 Regulador del Interferón/inmunología , Ratones , Ratones Noqueados , Infecciones por Virus ARN/inmunología , Replicación ViralRESUMEN
Mixed lineage kinase domain-like protein (MLKL) is the terminal effector of necroptosis, a form of regulated necrosis. Optimal activation of necroptosis, which eliminates infected cells, is critical for antiviral host defense. MicroRNAs (miRNAs) regulate the expression of genes involved in various biological and pathological processes. However, the roles of miRNAs in necroptosis-associated host defense remain largely unknown. We screened a library of miRNAs and identified miR-324-5p as the most effective suppressor of necroptosis. MiR-324-5p downregulates human MLKL expression by specifically targeting the 3'UTR in a seed region-independent manner. In response to interferons (IFNs), miR-324-5p is downregulated via the JAK/STAT signaling pathway, which removes the posttranscriptional suppression of MLKL mRNA and facilitates the activation of necroptosis. In influenza A virus (IAV)-infected human primary macrophages, IFNs are induced, leading to the downregulation of miR-324-5p. MiR-324-5p overexpression attenuates IAV-associated necroptosis and enhances viral replication, whereas deletion of miR-324-5p potentiates necroptosis and suppresses viral replication. Hence, miR-324-5p negatively regulates necroptosis by manipulating MLKL expression, and its downregulation by IFNs orchestrates optimal activation of necroptosis in host antiviral defense.
Asunto(s)
Virus de la Influenza A , MicroARNs , Antivirales , Humanos , Interferones , MicroARNs/genética , MicroARNs/metabolismo , Necroptosis , Replicación Viral/fisiologíaRESUMEN
Background: Necroptosis is an important form of regulated cell death involved in inflammatory diseases, degenerative diseases and cancer. RIPK3 is an interesting target for intervention of necroptosis-associated diseases. Methodology: Herein the authors report the synthesis of a series RIPK3 inhibitors under the guidance of structure-based drug design which leads to the identification of compound 37. Results: Compound 37 potently rescued human and mouse cells from necroptotic stimuli TNF-α, Smac mimetic, z-VAD and LPS + z-VAD, displayed high affinity to RIPK3 (Kd = 14 nM) but no observable affinity to RIPK1 and inhibited RIPK3 kinase function. Importantly, compound 37 significantly alleviated TNF-induced systemic inflammatory response syndrome in the mouse model. Conclusion: These results support compound 37 as a prototype RIPK3 inhibitor for lead optimization.
Asunto(s)
Diseño de Fármacos , Necroptosis , Animales , Modelos Animales de Enfermedad , Ratones , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Factor de Necrosis Tumoral alfaRESUMEN
RIPK1 plays a key role in the necroptosis pathway that regulates inflammatory signaling and cell death in various diseases, including inflammatory and neurodegenerative diseases. Herein, we report a series of potent RIPK1 inhibitors, represented by compound 70. Compound 70 efficiently blocks necroptosis induced by TNFα in both human and mouse cells (EC50 = 17-30 nM). Biophysical assay demonstrates that compound 70 potently binds to RIPK1 (Kd = 9.2 nM), but not RIPK3 (Kd > 10,000 nM). Importantly, compound 70 exhibits greatly improved metabolic stability in human and rat liver microsomes compared to compound 6 (PK68), a RIPK1 inhibitor reported in our previous work. In addition, compound 70 displays high permeability in Caco-2 cells and excellent in vitro safety profiles in hERG and CYP assays. Moreover, pre-treatment of 70 significantly ameliorates hypothermia and lethal shock in SIRS mice model. Lastly, compound 70 possesses favorable pharmacokinetic parameters with moderate clearance and good oral bioavailability in SD rat. Taken together, our work supports 70 as a potent RIPK1 inhibitor and highlights its potential as a prototypical lead for further development in necroptosis-associated inflammatory disorders.
Asunto(s)
Acetamidas/farmacología , Antiinflamatorios no Esteroideos/farmacología , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Tiazoles/farmacología , Acetamidas/síntesis química , Acetamidas/química , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Ratas , Ratas Sprague-Dawley , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/químicaRESUMEN
Interferon-γ-inducible factor 16 (IFI16) triggers stimulator of interferon (IFN) genes (STING)-dependent type I IFN production during host antiviral immunity and facilitates p53-dependent apoptosis during suppressing tumorigenesis. We have previously reported that STING-mediated IFI16 degradation negatively regulates type I IFN production. However, it is unknown whether STING also suppresses IFI16/p53-dependent apoptosis via degradation of IFI16. Here, our results from flow cytometry apoptosis detection and immunoblot assays show that IFI16 and nutlin-3, a p53 pathway activator, synergistically induce apoptosis in U2OS and A549 cells. Protein kinase R-triggered phosphorylation of p53 at serine 392 is critical for the IFI16-p53-dependent apoptosis. However, overexpression of STING suppresses p53 serine 392 phosphorylation, p53 transcriptional activity, expression of p53 target genes, and p53-dependent mitochondrial depolarization and apoptosis. In summary, our current study demonstrates that STING-mediated IFI16 degradation negatively regulates IFI16-mediated p53-dependent apoptosis in osteosarcoma and non-small cell lung cancer cells, which suggests a protumorigenic role for STING in certain cancer types because of its potent ability to degrade upstream IFI16.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteína p53 Supresora de Tumor , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas , Línea Celular Tumoral , Proteínas de Drosophila , Humanos , Inmunidad Innata , Neoplasias Pulmonares , Fosforilación , Transducción de SeñalRESUMEN
Necroptosis is a form of regulated necrotic cell death that is independent of caspases. Receptor-interacting protein kinase 3 (RIPK3) has been identified as a key regulator for necroptosis, and has been proposed as a potential therapeutic target for the treatment of diseases associated with necroptosis. In this report, we describe the design, synthesis, and evaluation of a series of novel RIPK3 inhibitors. The lead compound 38 exhibited potent activity (EC50 = 0.42 µM) in blocking TNFα, Smac mimetic and z-VAD (TSZ) induced cell death in HT-29 cells. Mechanistic studies showed that compound 38 bound to RIPK3 with high affinity (Kd = 7.1 nM), and inhibited RIPK3 kinase activity in a ADP-Glo functional assay. In addition, compound 38 displayed good selectivity over another necroptosis regulator RIPK1 (Kd = 6000 nM). Furthermore, compound 38 demonstrated excellent in vitro safety profiles with minimal inhibition of CYP isozymes and hERG potassium channel. Lastly, compound 38 efficiently blocked hypothermia and death in mice in the TNFα-induced systemic inflammatory response syndrome model.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Hipotermia/tratamiento farmacológico , Hipotermia/metabolismo , Inyecciones Intravenosas , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Relación Estructura-Actividad , Síndrome de Respuesta Inflamatoria Sistémica/tratamiento farmacológico , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Factor de Necrosis Tumoral alfa/administración & dosificación , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Necroptosis is a form of regulated necrosis that requires the activation of receptor-interacting kinase 3 (RIPK3 or RIP3) and its phosphorylation of the substrate MLKL (mixed lineage kinase domain-like protein). Necroptosis has emerged as important cell death involved in the pathogenesis of various diseases including inflammatory diseases, degenerative diseases, and cancer. Here, we discovered a small molecule Zharp-99 as a potent inhibitor of necroptosis through blocking the kinase activity of RIPK3. Zharp-99 efficiently blocks necroptosis induced by ligands of the death receptor and Toll-like receptor as well as viral infection in human, rat and mouse cells. Zharp-99 strongly inhibits cellular activation of RIPK3, and MLKL upon necroptosis stimuli. Zharp-99 directly blocks the kinase activity of RIPK3 without affecting RIPK1 kinase activity at the tested concentration. Importantly, Zharp-99 exerts effective protection against TNF-α induced systemic inflammatory response syndrome in the mouse model. Zharp-99 displays favorable in vitro safety profiles and in vivo pharmacokinetic parameters. Thus, our study demonstrates Zharp-99 as a potent inhibitor of RIPK3 kinase and also highlights its potential for further development of new approaches for treating necroptosis-associated inflammatory disorders.
RESUMEN
The chemokine receptor CXCR4 has been proposed as a drug target based on its important functions in HIV infection, inflammation/autoimmune diseases and cancer metastasis. Herein we report the design, synthesis and evaluation of novel CXCR4 antagonists based on a pyrrolidine scaffold. The structural exploration/optimization identified numerous potent CXCR4 antagonists, represented by compound 46, which displayed potent binding affinity to CXCR4 receptor (IC50 = 79 nM competitively displacing fluorescent 12G5 antibody) and inhibited CXCL12 induced cytosolic calcium flux (IC50 = 0.25 nM). Moreover, in a transwell invasion assay, compound 46 significantly mitigated CXCL12/CXCR4 mediated cell migration. Compound 46 exhibited good physicochemical properties (MW 367, logD7.4 1.12, pKa 8.2) and excellent in vitro safety profiles (e.g., hERG patch clamp IC50 > 30 µM and minimal CYP isozyme inhibition). Importantly, 46 displayed much improved metabolic stability in human and rat liver microsomes. Lastly, 46 demonstrated marked efficacy in a cancer metastasis model in mice. These results strongly support 46 as a prototypical lead for the development of promising CXCR4 antagonists as clinical candidates.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Diseño de Fármacos , Pirrolidinas/síntesis química , Pirrolidinas/farmacología , Receptores CXCR4/antagonistas & inhibidores , Animales , Antineoplásicos/química , Calcio/metabolismo , Línea Celular Tumoral , Técnicas de Química Sintética , Citosol/efectos de los fármacos , Citosol/metabolismo , Humanos , Ratones , Metástasis de la Neoplasia , Pirrolidinas/química , RatasRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0069911.].
RESUMEN
Renal cell carcinoma (RCC) is the most common type of kidney cancer. It has a poor prognosis, with approximately 20-30% of patients developing recurrent and/or metastatic diseases that is relatively high resistant to conventional therapy. Resisting cell death is a hallmark of cancer cells. Apoptosis is a form of programmed cell death mediated by the activation of caspases. Necroptosis is a form of regulated necrosis that relies on the activation of receptor-interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like protein (MLKL), the substrate of RIPK3. Cancer cells often display apoptosis resistance via upregulation of anti-apoptotic genes and defective necroptosis due to the epigenetic silence of Ripk3. MicroRNAs (miRNAs) are non-coding small RNAs that are involved in numerous biological processes including cell proliferation, differentiation and death. In this study, we screened a set of â¼120 miRNAs for apoptosis-regulating miRNAs and identified miR-381-3p as a suppressor of TNF-induced apoptosis in various cancer cells. Ectopic expression of miR-381-3p inhibits the activation of caspase-8 and caspase-3. The expression level of miR-381-3p inversely correlates with the sensitivity of cancer cells to TNF-induced apoptosis. Moreover, we found that overexpression of miR-381-3p blocks TNF-induced necroptosis by inhibiting the activation of RIPK3 and MLKL. Of note, Kaplan-Meier Plotter analysis demonstrates that papillary RCC patients with high miR-381-3p expression have a lower overall survival than those with low expression level of miR-381-3p. Importantly, miR-381-3p overexpression promotes colony formation in human renal cancer cells. Thus, miR-381-3p acts as an oncogenic miRNA that counteracts both apoptotic and necroptotic signaling pathways. Our findings highlight miR-381-3p as a biomarker for predicting sensitivity to apoptosis and necroptosis, and as a possible therapeutic target for RCC.
RESUMEN
Smac/Diablo is a pro-apoptotic protein via interaction with inhibitors of apoptosis proteins (IAPs) to relieve their inhibition of caspases. Smac mimetic compounds (also known as antagonists of IAPs) mimic the function of Smac/Diablo and sensitize cancer cells to TNF-induced apoptosis. However, the majority of cancer cells are resistant to Smac mimetic alone. Doxorubicin is a widely used chemotherapeutic drug and causes adverse effect of cardiotoxicity in many patients. Therefore, it is important to find strategies of combined chemotherapy to increase chemosensitivity and reduce the adverse effects. Here, we report that doxorubicin synergizes with Smac mimetic to trigger TNF-mediated apoptosis, which is mechanistically distinct from doxorubicin-induced cell death. Doxorubicin sensitizes cancer cells including human pancreatic and colorectal cancer cells to Smac mimetic treatment. The combined treatment leads to synergistic induction of TNFα to initiate apoptosis through activating NF-κB and c-Jun signaling pathways. Knockdown of caspase-8 or knockout of FADD significantly blocked apoptosis synergistically induced by Smac mimetic and doxorubicin, but had no effect on cell death caused by doxorubicin alone. Moreover, Smac mimetic and doxorubicin-induced apoptosis requires receptor-interacting protein kinase 1 (RIPK1) and its deubiquitinating enzyme cylindromatosis (CYLD), not A20. These in vitro findings demonstrate that combination of Smac mimetic and doxorubicin synergistically triggers apoptosis through the TNF/CYLD/RIPK1/FADD/caspase-8 signaling pathway. Importantly, the combined treatment induced in vivo synergistic anti-tumor effects in the xenograft tumor model. Thus, the combined therapy using Smac mimetic and doxorubicin presents a promising apoptosis-inducing strategy with great potential for the development of anti-cancer therapy.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Materiales Biomiméticos/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Enzima Desubiquitinante CYLD/genética , Doxorrubicina/farmacología , Proteínas Mitocondriales/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Animales , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Caspasa 8/genética , Caspasa 8/metabolismo , Línea Celular Tumoral , Enzima Desubiquitinante CYLD/metabolismo , Sinergismo Farmacológico , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Masculino , Ratones , Ratones Desnudos , Proteínas Mitocondriales/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Análisis de Supervivencia , Factor de Necrosis Tumoral alfa/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chemokine receptor CXCR4 and its natural ligand CXCL12 (also known as stromal cell-derived factor-1, or SDF-1) regulate a broad range of physiological functions. Dysregulation of the CXCL12/CXCR4 axis is involved in numerous pathological conditions such as HIV infection, inflammation and cancer. Herein, we report the design, synthesis, and characterization of novel CXCR4 antagonists based on cyclic amine scaffolds. Compound 24 was identified as a potent CXCR4 receptor antagonist (competitive inhibition of 12G5 binding, IC50 =24â nM; functional inhibition of CXCL12-induced cytosolic calcium increase, IC50 =0.1â nM). In addition, compound 24 potently inhibited cell migration in CXCR4/CXCL12-mediated chemotaxis in a matrigel invasion assay. The absolute configuration of compound 24 was elucidated by X-ray crystallography.