RESUMEN
Radiotherapy is a commonly used conditioning regimen for bone marrow transplantation (BMT). Cytotoxicity limits the use of this life-saving therapy, but the underlying mechanisms remain poorly defined. Here, we use the syngeneic mouse BMT model to test the hypothesis that lethal radiation damages tissues, thereby unleashing signals that indiscriminately activate the inflammasome pathways in host and transplanted cells. We find that a clinically relevant high dose of radiation causes severe damage to bones and the spleen through mechanisms involving the NLRP3 and AIM2 inflammasomes but not the NLRC4 inflammasome. Downstream, we demonstrate that gasdermin D (GSDMD), the common effector of the inflammasomes, is also activated by radiation. Remarkably, protection against the injury induced by deadly ionizing radiation occurs only when NLRP3, AIM2, or GSDMD is lost simultaneously in both the donor and host cell compartments. Thus, this study reveals a continuum of the actions of lethal radiation relayed by the inflammasome-GSDMD axis, initially affecting recipient cells and ultimately harming transplanted cells as they grow in the severely injured and toxic environment. This study also suggests that therapeutic targeting of inflammasome-GSDMD signaling has the potential to prevent the collateral effects of intense radiation regimens.
Asunto(s)
Células de la Médula Ósea/efectos de la radiación , Trasplante de Médula Ósea , Proteínas de Unión al ADN/genética , Inflamasomas/efectos de la radiación , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteínas de Unión a Fosfato/genética , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proteínas de Unión al ADN/deficiencia , Femenino , Fémur/citología , Fémur/metabolismo , Regulación de la Expresión Génica , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/deficiencia , Proteínas de Unión a Fosfato/deficiencia , Piroptosis/genética , Piroptosis/efectos de la radiación , Transducción de Señal , Bazo/metabolismo , Bazo/patología , Bazo/efectos de la radiación , Trasplante Isogénico , Irradiación Corporal Total , Rayos XRESUMEN
OBJECTIVE: The study objective was to evaluate the safety, tolerability, pharmacodynamics, and preliminary efficacy of ATI-450 with methotrexate in patients with rheumatoid arthritis (RA). METHODS: A parallel-assignment, placebo-controlled, investigator-blinded/patient-blinded multicenter study evaluated patients with moderate-to-severe RA aged 18 to 70 years. Eligible patients were randomized (1:1) to ATI-450 50-mg oral tablets twice daily or placebo with a stable weekly dose of methotrexate for 12 weeks. The primary objective was to assess ATI-450 safety and tolerability. The secondary objectives were to assess the median percentage change from baseline high-sensitivity C-reactive protein (hs-CRP) levels, the mean change from baseline in Disease Activity Score in 28 joints based on CRP level (DAS28-CRP) and Rheumatoid Arthritis Magnetic Resonance Imaging Score hand-wrist assessments of synovitis or bone erosion at week 12, and the proportion of patients with American College of Rheumatology 20/50/70 (ACR 20/50/70) and with DAS28-CRP scores of less than 2.6. The exploratory outcomes were change from baseline in endogenous and ex vivo-stimulated cytokine levels. RESULTS: ATI-450 was well tolerated with no severe adverse events reported. ATI-450 reduced median hs-CRP levels by 42% or more at all posttreatment timepoints. In the ATI-450 group, a mean (median) decrease in DAS28-CRP score of 2.0 (2.1) was observed at week 12; proportions of patients with an ACR 20/50/70 response in the per-protocol population were 60%, 33%, and 20%, respectively, at week 12. Endogenous plasma levels of key inflammatory cytokines (tumor necrosis factor α, macrophage inflammatory protein 1ß, interleukin 6, interleukin 8) were reduced across the 12 treatment weeks. CONCLUSION: This is the first clinical study demonstrating that selective mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) pathway blockade leads to a sustained antiinflammatory effect. This suggests that targeting the MK2 pathway mitigates the tachyphylaxis observed with p38 MAPK inhibitors in RA and supports further exploration.
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Metastatic breast cancer is an intractable disease that responds poorly to immunotherapy. We show that p38MAPKα inhibition (p38i) limits tumor growth by reprogramming the metastatic tumor microenvironment in a CD4+ T cell-, IFNγ-, and macrophage-dependent manner. To identify targets that further increased p38i efficacy, we utilized a stromal labeling approach and single-cell RNA sequencing. Thus, we combined p38i and an OX40 agonist that synergistically reduced metastatic growth and increased overall survival. Intriguingly, patients with a p38i metastatic stromal signature had better overall survival that was further improved by the presence of an increased mutational load, leading us to ask if our approach would be effective in antigenic breast cancer. The combination of p38i, anti-OX40, and cytotoxic T-cell engagement cured mice of metastatic disease and produced long-term immunologic memory. Our findings demonstrate that a detailed understanding of the stromal compartment can be used to design effective antimetastatic therapies. SIGNIFICANCE: Immunotherapy is rarely effective in breast cancer. We dissected the metastatic tumor stroma, which revealed a novel therapeutic approach that targets the stromal p38MAPK pathway and creates an opportunity to unleash an immunologic response. Our work underscores the importance of understanding the tumor stromal compartment in therapeutic design. This article is highlighted in the In This Issue feature, p. 1275.
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Neoplasias , Ratones , Animales , Linfocitos T Citotóxicos , Linfocitos T CD4-Positivos , Inmunoterapia , Macrófagos , Microambiente Tumoral , Línea Celular TumoralRESUMEN
The aim of this study is to investigate whether PI3K (phosphatidylinositol-3-kinase) is involved in IL-1ß (interleukin-1ß)-induced IL-6 production in A549 (human lung adenocarcinoma epithelial cell) and human RASF (rheumatoid arthritis synovial fibroblast). PI3K inhibitor, LY294002 significantly reduced IL-1ß-induced IL-6 production in A549 cells but not in RASF, indicating that IL-1ß-induced IL-6 production was partially mediated by PI3Kin A549 cells but not in RASF. siRNA (small interfering RNA) of IRAK4 (IL-1 receptor-associated kinase 4) treatment decreased IRAK4 mRNA level by up to 90% in A549 cells. In this condition, IL-1ß-induced increase of IL-6 mRNA and protein level was decreased by up to 93% and 70%, respectively. Furthermore, the combination of IRAK4 siRNA and LY294002 treatment decreased protein induction level of IL-6 in A549 cells compared with that of IRAK4 siRNA or LY294002 alone. These results indicate that IL-1ß-induced IL-6 production in A549 cells is mediated by both PI3K and IRAK4 and suggest that involvement of PI3K in the IL-1-induced IL-6 production is cell type specific.
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Células Epiteliales/inmunología , Quinasas Asociadas a Receptores de Interleucina-1/inmunología , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Pulmón/citología , Fosfatidilinositol 3-Quinasa/inmunología , Artritis Reumatoide/inmunología , Línea Celular , Línea Celular Tumoral , Fibroblastos/inmunología , HumanosRESUMEN
A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-α in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.
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Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinonas/uso terapéutico , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Administración Oral , Animales , Artritis/tratamiento farmacológico , Artritis Experimental , Células CACO-2 , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Masculino , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Pirimidinonas/química , Ratas , Ratas Endogámicas Lew , Relación Estructura-ActividadRESUMEN
A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.
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Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Piridonas/síntesis química , Piridonas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Masculino , Microsomas Hepáticos/enzimología , Estructura Molecular , Piridazinas/química , Piridazinas/farmacología , Piridonas/química , Pirimidinas/química , Pirimidinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The synthesis and SAR studies of a novel N-aryl pyridinone class of p38 kinase inhibitors are described. Systematic structural modifications to the HTS lead, 5, led to the identification of (-)-4a as a clinical candidate for the treatment of inflammatory diseases. Additionally, the chiral synthesis and properties of (-)-4a are described.
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Benzamidas/síntesis química , Benzamidas/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Pironas/síntesis química , Pironas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Benzamidas/química , Modelos Animales de Enfermedad , Perros , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Macaca fascicularis , Masculino , Estructura Molecular , Piridonas , Pironas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/farmacologíaRESUMEN
The aim of this study is to investigate the induction of interleukin-34 (IL-34) and macrophage colony-stimulating factor (M-CSF) mRNA by inflammatory cytokines and the involvement of mitogen-activated protein kinases (MAPKs) in this signaling pathway in human osteoblasts as both IL-34 and M-CSF bind to the same receptor c-FMS. Among four inflammatory cytokines [(IL-1ß, IL-6, IL-17, and tumor necrosis factor-α (TNF-α)], IL-34 mRNA expression level was dramatically induced by IL-1ß (17-fold) and TNF-α (74-fold). IL-1ß and TNF-α activated the intracellular mitogen-activated protein kinases (MAPKs): p44/42 MAPK, p38, and c-Jun N-terminal kinase (JNK) as well as nuclear factor-κB (NF-κB) in osteoblasts. IL-1ß- and TNF-α-mediated induction of IL-34 mRNA expression was decreased by JNK inhibitor. Interestingly, although treatment of MEK-1/2 inhibitor showed no reduction in the increase of IL-34 mRNA expression by cytokines, combination of MEK-1/2 inhibitor and JNK inhibitor significantly inhibited IL-1ß- and TNF-α-mediated IL-34 mRNA expression level compared to those by each inhibitor alone. On the other hand, M-CSF mRNA expression level was significantly induced by both IL-1ß and TNF-α by up to 7- and 11-fold, respectively. IL-1ß- and TNF-α-mediated induction of M-CSF mRNA was not affected by p38, JNK, and MEK-1/2 inhibitors. However, NF-κB inhibitor completely inhibited the elevation of M-CSF mRNA expression by these cytokines. These results showed that proinflammatory cytokines, IL-1ß and TNF-α, induced the expression of IL-34 mRNA via JNK and p44/42 MAPK but not p38 in human osteoblasts while p38, JNK, and p44/42 MAPK were not involved in the induction of M-CSF mRNA expression by these cytokines.
Asunto(s)
Citocinas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Osteoblastos/metabolismo , Transducción de Señal/fisiología , Células Cultivadas , Citocinas/genética , Citocinas/farmacología , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1beta/farmacología , Interleucina-1beta/fisiología , Interleucinas/genética , Interleucinas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Osteoblastos/efectos de los fármacos , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
PURPOSE: ATI-450 is an oral, small-molecule inhibitor of the p38α mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2 (MK2) inflammatory signaling pathway. This phase 1, single and multiple ascending dose (SAD, MAD) study evaluated ATI-450 safety, tolerability, pharmacokinetics, and pharmacodynamics. PATIENTS AND METHODS: Healthy adults were randomly assigned to SAD (10, 30, 50, 100 mg; n=24) and MAD (10, 30, 50 mg twice daily [BID] for 7 days; n=24) cohorts of ATI-450 or placebo (n=14). Safety and tolerability were evaluated through clinical and laboratory assessments. Pharmacokinetic parameters were evaluated in plasma samples; pharmacodynamic assessments included quantification of cytokine levels (tumor necrosis factor α [TNF-α], interleukin [IL]-1ß, IL-6, IL-8) and phosphorylation of the MK2 downstream substrate, heat shock protein 27 (p-HSP27). RESULTS: The most common adverse events were headache (10/48, 20.8%), dizziness (6/48, 12.5%), upper respiratory tract infection (3/48, 6.3%), and constipation (3/48, 6.3%). Pharmacokinetics were dose-proportional, with a terminal half-life of 9â12 hours in the MAD cohorts on day 7. Dose- and concentration-dependent inhibition of ex vivo stimulated cytokines and target biomarker was observed. On day 7, patients in the 50 mg BID dose cohort recorded mean trough drug levels that were 1.4, 2.2, 2.3, and 2.4 times greater than the IC80 for TNF-α, IL-1ß, IL-8, and p-HSP27, respectively. Mean Cmax was 3.5, 5.4, 5.6, and 6.0 times greater than the IC80 for TNF-α, IL-1ß, IL-8, and p-HSP27, respectively. IL-6 inhibition >50% was noted for part of the dosing interval. CONCLUSION: ATI-450 was well tolerated at the doses investigated, exhibited dose- and time-independent (ie, linear) pharmacokinetics, and dose-related pharmacodynamic effects. These results support further study of ATI-450 in immunoinflammatory diseases in phase 2 trials.
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Combination chemotherapies remain the cornerstone treatment for pancreatic ductal adenocarcinoma (PDAC), but de novo and acquired resistance is common. In this study, we aimed to identify and characterize resistance mechanisms to a FIRINOX chemotherapy regimen (a combination of 5-fluorouracil, irinotecan, and oxaliplatin) because it is the most aggressive regimen currently used clinically for patients with PDAC. Using an unbiased reverse-phase protein array, we detected phospho-activation of heat shock protein 27 (Hsp27) as the most up-regulated event after FIRINOX treatment in PDAC cells. Silencing HSP27 by RNA interference or by a small-molecule inhibitor enhanced apoptosis caused by FIRINOX in vitro. Mechanistically, FIRINOX up-regulated tumor necrosis factorα (TNFα), causing autocrine phosphorylation and activation of transforming growth factorßactivated kinase 1 (TAK1), MAPK activated protein kinase 2 (MAPKAPK2 or MK2), and, ultimately, Hsp27. Targeting MK2, the kinase that directly phosphorylates Hsp27, abrogated Hsp27 activation, sensitized PDAC cells to apoptosis, and suppressed SN-38induced protective autophagy in vitro, in part by blocking phospho-activation of Beclin1. In an autochthonous PDAC mouse model, the MK2 inhibitor ATI-450 decreased PDAC development and progression. When combined with FIRINOX, ATI-450 eliminated most PDAC foci and marked prolonged mouse survival without causing additional toxicity. Last, we found that high phospho-MK2 expression in tumors was associated with poorer survival of patients with PDAC. Our study identified MK2 as a mediator of genotoxic stressinduced activation of prosurvival pathways and provides preclinical support for combining an MK2 inhibitor with FIRINOX-based chemotherapies to treat PDAC.
Asunto(s)
Adenocarcinoma , Neoplasias Pancreáticas , Animales , Línea Celular Tumoral , Daño del ADN , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas Serina-Treonina QuinasasRESUMEN
NOD-like receptor (NLR), family pyrin domain containing 3 (NLRP3) assembles a protein complex known as the NLRP3 inflammasome upon sensing certain pathogen products or sterile danger signals. Gain-of-function mutations such as the D301N substitution in NLRP3, which cause its constitutive activation (NLRP3CA) also results in inflammasome assembly. This inflammasome processes prointerleukin-1 ß (proIL-1ß) and proIL-18 into bioactive IL-1ß and IL-18, respectively, and cleaves gasdermin D (GSDMD). GSDMD amino-terminal fragments form plasma membrane pores that facilitate the secretion of IL-1ß and IL-18 and lead to the inflammatory cell death pyroptosis. Accordingly, GSDMD inactivation results in negligible spontaneous inflammation in various experimental models such as in Nlrp3CA/+ mice lacking GSDMD (Nlrp3CA/+;Gsdmd−/− mice). Here, we found that Nlrp3CA/+;Gsdmd−/− mice, when challenged with LPS or TNF-α, still secreted IL-1ß and IL-18, indicating inflammasome activation independent of GSDMD. Accordingly, Gsdmd−/− macrophages failed to secrete IL-1ß and undergo pyroptosis when briefly exposed to NLRP3 inflammasome activators but released these cytokines when persistently activated. Sustained NLRP3 inflammasome induced caspase-8/-3 and GSDME cleavage and IL-1ß maturation in vitro in Gsdmd−/− macrophages. Thus, a salvage inflammatory pathway involving caspase-8/-3GSDME was activated after NLRP3 activation when the canonical NLRP3-GSDMD signaling was blocked. Consistent with genetic data, the active metabolite of FDA-approved disulfiram CuET, which inhibited GSDMD and GSDME cleavage in macrophages, reduced the severe inflammation and tissue damage that occurred in the Nlrp3CA/+ mice. Thus, NLRP3 inflammasome activation overwhelms the protection afforded by GSDMD deficiency, rewiring signaling cascades through mechanisms that include GSDME to propagate inflammation.
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Inflamasomas/inmunología , Inflamación/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Proteínas de Unión a Fosfato/inmunología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Animales , Células Cultivadas , Inflamación/patología , Ratones , Ratones Congénicos , Ratones Noqueados , Ratones Transgénicos , Proteínas de Unión a Fosfato/deficiencia , Proteínas Citotóxicas Formadoras de Poros/deficienciaRESUMEN
The aim of this study is to investigate if macrophage-colony stimulating factor (M-CSF) or interleukin-34 (IL-34) induces cytokines or chemokines using human whole blood (HWB) and if an M-CSF- or IL-34-induced cytokine or chemokine production from HWB is inhibited by soluble M-CSF receptor or c-FMS kinase inhibitors. Among eight cytokines or growth factors tested, only IL-6 level was increased by up to 6-fold by M-CSF or IL-34 in HWB. In contrast, chemokine levels (IP-10/CXCL10, IL-8/CXCL8, and MCP-1/CCL2) were dramatically increased by M-CSF or IL-34 in HWB while exhibiting a large variation among donors. Variability of the MCP-1 signal induced by M-CSF or IL-34 was relatively less among donors compared to the IP-10 and IL-8 signals. The elevation of these chemokine levels was significantly decreased by soluble M-CSF receptor, indicating the elevation of these chemokines was mediated by M-CSF or IL-34. Furthermore, GW2580, a c-FMS kinase inhibitor, inhibited the induction of MCP-1 by M-CSF or IL-34 in a concentration dependent manner. These indicate MCP-1 is the most appropriate chemokine target for a chemokine release assay to evaluate the potency of c-FMS kinase inhibitors and MCP-1 release assay using HWB would be useful, relevant tool for translational pharmacology of c-FMS kinase inhibitors.
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Quimiocinas/biosíntesis , Interleucinas/fisiología , Factor Estimulante de Colonias de Macrófagos/fisiología , Anisoles/farmacología , Quimiocinas/sangre , Humanos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Pirimidinas/farmacologíaRESUMEN
Starting from an initial HTS screening lead, a novel series of C(5)-substituted diaryl pyrazoles were developed that showed potent inhibition of p38alpha kinase. Key to this outcome was the switch from a pyridyl to pyrimidine at the C(4)-position leading to analogs that were potent in human whole blood based cell assay as well as in a number of animal efficacy models for rheumatoid arthritis. Ultimately, we identified a clinical candidate from this substrate; SD-0006.
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Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Dominio Catalítico , Humanos , Modelos Moleculares , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.
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Enfermedades Gastrointestinales/inducido químicamente , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Enfermedades Linfáticas/inducido químicamente , Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Linfocitos B/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colon/efectos de los fármacos , Colon/patología , Perros , Femenino , Enfermedades Gastrointestinales/patología , Hemorragia Gastrointestinal/inducido químicamente , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Lineales , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Enfermedades Linfáticas/patología , Macaca fascicularis , Masculino , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Bazo/citología , Bazo/metabolismo , Linfocitos T/metabolismo , Pruebas de Toxicidad Aguda , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
PH-797804 is a diarylpyridinone inhibitor of p38alpha mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38alpha inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38alpha kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180 degrees rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38alpha kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38alpha kinase inhibitors.
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Benzamidas/farmacología , Biología Computacional , Inhibidores de Proteínas Quinasas/farmacología , Pironas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Benzamidas/química , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Estructura Molecular , Fosforilación , Inhibidores de Proteínas Quinasas/química , Piridonas , Pironas/química , Especificidad por Sustrato , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Signal transduction through the p38 mitogen-activated protein (MAP) kinase pathway is central to the transcriptional and translational control of cytokine and inflammatory mediator production. p38 MAP kinase inhibition hence constitutes a promising therapeutic strategy for treatment of chronic inflammatory diseases, based upon its potential to inhibit key pathways driving the inflammatory and destructive processes in these debilitating diseases. The present study describes the pharmacological properties of the N-phenyl pyridinone p38 MAP kinase inhibitor benzamide [3- [3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2- oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (-)-(9CI); PH-797804]. PH-797804 is an ATP-competitive, readily reversible inhibitor of the alpha isoform of human p38 MAP kinase, exhibiting a K(i) = 5.8 nM. In human monocyte and synovial fibroblast cell systems, PH-797804 blocks inflammation-induced production of cytokines and proinflammatory mediators, such as prostaglandin E(2), at concentrations that parallel inhibition of cell-associated p38 MAP kinase. After oral dosing, PH-797804 effectively inhibits acute inflammatory responses induced by systemically administered endotoxin in both rat and cynomolgus monkeys. Furthermore, PH-797804 demonstrates robust anti-inflammatory activity in chronic disease models, significantly reducing both joint inflammation and associated bone loss in streptococcal cell wall-induced arthritis in rats and mouse collagen-induced arthritis. Finally, PH-797804 reduced tumor necrosis factor-alpha and interleukin-6 production in clinical studies after endotoxin administration in a dose-dependent manner, paralleling inhibition of the target enzyme. Low-nanomolar biochemical enzyme inhibition potency correlated with p38 MAP kinase inhibition in human cells and in vivo studies. In addition, a direct correspondence between p38 MAP kinase inhibition and anti-inflammatory activity was observed with PH-797804, thus providing confidence in dose projections for further human studies in chronic inflammatory disease.
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Antiinflamatorios no Esteroideos/uso terapéutico , Benzamidas/uso terapéutico , Pironas/uso terapéutico , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Adolescente , Adulto , Animales , Antiinflamatorios no Esteroideos/sangre , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/enzimología , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/enzimología , Artritis Reumatoide/inmunología , Benzamidas/sangre , Benzamidas/química , Benzamidas/farmacología , Densidad Ósea/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Línea Celular , Citocinas/biosíntesis , Citocinas/sangre , Dinoprostona/biosíntesis , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Lipopolisacáridos/farmacología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos DBA , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Monocitos/enzimología , Monocitos/inmunología , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Osteoclastos/inmunología , Piridonas , Pironas/sangre , Pironas/química , Pironas/farmacología , Ratas , Ratas Endogámicas Lew , Síndrome de Respuesta Inflamatoria Sistémica/tratamiento farmacológico , Síndrome de Respuesta Inflamatoria Sistémica/enzimología , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Adulto JovenRESUMEN
The identification and evolution of a series of potent and selective p38 inhibitors is described. p38 inhibitors based on a N-benzyl pyridinone high-throughput screening hit were prepared and their SAR explored. Their design was guided by ligand bound co-crystals of p38alpha. These efforts resulted in the identification of 12r and 19 as orally active inhibitors of p38 with significant efficacy in both acute and chronic models of inflammation.
Asunto(s)
Antiinflamatorios/química , Inhibidores de Proteínas Quinasas/química , Piridonas/química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Administración Oral , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacocinética , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Microsomas Hepáticos/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Piridonas/síntesis química , Piridonas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Inflammatory bowel disease (IBD) is a chronic and debilitating disorder that has few treatment options due to a lack of comprehensive understanding of its molecular pathogenesis. We used multiplexed mass spectrometry to collect high-content information on protein phosphorylation in two different mouse models of IBD. Because the biological function of the vast majority of phosphorylation sites remains unknown, we developed Substrate-based Kinase Activity Inference (SKAI), a methodology to infer kinase activity from phosphoproteomic data. This approach draws upon prior knowledge of kinase-substrate interactions to construct custom lists of kinases and their respective substrate sites, termed kinase-substrate sets that employ prior knowledge across organisms. This expansion as much as triples the amount of prior knowledge available. We then used these sets within the Gene Set Enrichment Analysis framework to infer kinase activity based on increased or decreased phosphorylation of its substrates in a dataset. When applied to the phosphoproteomic datasets from the two mouse models, SKAI predicted largely non-overlapping kinase activation profiles. These results suggest that chronic inflammation may arise through activation of largely divergent signaling networks. However, the one kinase inferred to be activated in both mouse models was mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2 or MK2), a serine/threonine kinase that functions downstream of p38 stress-activated mitogen-activated protein kinase. Treatment of mice with active colitis with ATI450, an orally bioavailable small molecule inhibitor of the MK2 pathway, reduced inflammatory signaling in the colon and alleviated the clinical and histological features of inflammation. These studies establish MK2 as a therapeutic target in IBD and identify ATI450 as a potential therapy for the disease.
Asunto(s)
Colitis/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Administración Oral , Animales , Análisis por Conglomerados , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Inflamación , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Fosforilación , Análisis de Componente Principal , Proteómica , Ratas , Transducción de Señal , Terminología como Asunto , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
p38α activation of multiple effectors may underlie the failure of global p38α inhibitors in clinical trials. A unique inhibitor (CDD-450) was developed that selectively blocked p38α activation of the proinflammatory kinase MK2 while sparing p38α activation of PRAK and ATF2. Next, the hypothesis that the p38α-MK2 complex mediates inflammasome priming cues was tested. CDD-450 had no effect on NLRP3 expression, but it decreased IL-1ß expression by promoting IL-1ß mRNA degradation. Thus, IL-1ß is regulated not only transcriptionally by NF-κB and posttranslationally by the inflammasomes but also posttranscriptionally by p38α-MK2. CDD-450 also accelerated TNF-α and IL-6 mRNA decay, inhibited inflammation in mice with cryopyrinopathy, and was as efficacious as global p38α inhibitors in attenuating arthritis in rats and cytokine expression by cells from patients with cryopyrinopathy and rheumatoid arthritis. These findings have clinical translation implications as CDD-450 offers the potential to avoid tachyphylaxis associated with global p38α inhibitors that may result from their inhibition of non-MK2 substrates involved in antiinflammatory and housekeeping responses.
Asunto(s)
Inflamasomas/metabolismo , Inflamación/patología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transducción de Señal , Animales , Artritis/patología , Huesos/patología , Citocinas/biosíntesis , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Articulaciones/patología , Masculino , Ratones , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad del ARN , Ratas Endogámicas LewRESUMEN
The role of the stromal compartment in tumor progression is best illustrated in breast cancer bone metastases, where the stromal compartment supports tumor growth, albeit through poorly defined mechanisms. p38MAPKα is frequently expressed in tumor cells and surrounding stromal cells, and its expression levels correlate with poor prognosis. This observation led us to investigate whether inhibition of p38MAPKα could reduce breast cancer metastases in a clinically relevant model. Orally administered, small-molecule inhibitors of p38MAPKα or its downstream kinase MK2 each limited outgrowth of metastatic breast cancer cells in the bone and visceral organs. This effect was primarily mediated by inhibition of the p38MAPKα pathway within the stromal compartment. Beyond effectively limiting metastatic tumor growth, these inhibitors reduced tumor-associated and chemotherapy-induced bone loss, which is a devastating comorbidity that drastically affects quality of life for patients with cancer. These data underscore the vital role played by stromal-derived factors in tumor progression and identify the p38MAPK-MK2 pathway as a promising therapeutic target for metastatic disease and prevention of tumor-induced bone loss.Significance: Pharmacologically targeting the stromal p38MAPK-MK2 pathway limits metastatic breast cancer growth, preserves bone quality, and extends survival. Cancer Res; 78(19); 5618-30. ©2018 AACR.