ABSTRACT
Introduction: Atopic dermatitis, a chronic, pruritic skin disease, affects 10-30% of children and up to 14% of adults in developed countries. ATI-1777, a potent and selective Jak1/3 inhibitor, was designed with multiple sites of metabolism to deliver local efficacy in the skin and limit systemic exposure. In preclinical studies, ATI-1777 selectively inhibited Jak1/3 with limited systemic exposure and without any adverse effects. Primary objective: The primary goal of this study was to assess the preliminary clinical efficacy of ATI-1777 topical solution in adults with moderate or severe atopic dermatitis. Design: ATI-1777-AD-201, a phase 2a, first-in-human, randomized, double-blind, vehicle-controlled, parallel-group study, evaluated the efficacy, safety, tolerability, and pharmacokinetics of ATI-1777 topical solution in 48 participants with atopic dermatitis over 4 weeks. Primary endpoint: The primary endpoint was a reduction of a modified Eczema Area and Severity Index score from baseline. Results: Reduction was significantly greater in the ATI-1777-treated group on day 28 than in vehicle-treated group (percentage reduction from baseline = 74.45% [standard error = 6.455] and 41.43% [standard error = 6.189], respectively [P < .001]). Average plasma concentrations of ATI-1777 were <5% of the half-maximal inhibitory concentration of ATI-1777 for inhibiting Jak1/3. No deaths or serious adverse events were reported. Conclusion: Topical ATI-1777 does not lead to pharmacologically relevant systemic drug exposure and may reduce clinical signs of atopic dermatitis. Trial Registration: The study was registered at ClinicalTrials.gov with the number NCT04598269.
ABSTRACT
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.
Subject(s)
Neoplasms , Mice , Animals , T-Lymphocytes, Cytotoxic , CD4-Positive T-Lymphocytes , Immunotherapy , Macrophages , Tumor Microenvironment , Cell Line, TumorABSTRACT
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.
ABSTRACT
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.
Subject(s)
Adenocarcinoma , Pancreatic Neoplasms , Animals , Cell Line, Tumor , DNA Damage , HSP27 Heat-Shock Proteins/genetics , HSP27 Heat-Shock Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Mice , Pancreatic Neoplasms/drug therapy , Protein Serine-Threonine KinasesABSTRACT
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.
Subject(s)
Inflammasomes/immunology , Inflammation/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Phosphate-Binding Proteins/immunology , Pore Forming Cytotoxic Proteins/immunology , Animals , Cells, Cultured , Inflammation/pathology , Mice , Mice, Congenic , Mice, Knockout , Mice, Transgenic , Phosphate-Binding Proteins/deficiency , Pore Forming Cytotoxic Proteins/deficiencyABSTRACT
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.
ABSTRACT
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.
Subject(s)
Bone Marrow Cells/radiation effects , Bone Marrow Transplantation , DNA-Binding Proteins/genetics , Inflammasomes/radiation effects , Intracellular Signaling Peptides and Proteins/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Phosphate-Binding Proteins/genetics , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , DNA-Binding Proteins/deficiency , Female , Femur/cytology , Femur/metabolism , Gene Expression Regulation , Inflammasomes/metabolism , Intracellular Signaling Peptides and Proteins/deficiency , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/deficiency , Phosphate-Binding Proteins/deficiency , Pyroptosis/genetics , Pyroptosis/radiation effects , Signal Transduction , Spleen/metabolism , Spleen/pathology , Spleen/radiation effects , Transplantation, Isogeneic , Whole-Body Irradiation , X-RaysABSTRACT
Chemotherapy is important for cancer treatment, however, toxicities limit its use. While great strides have been made to ameliorate the acute toxicities induced by chemotherapy, long-term comorbidities including bone loss remain a significant problem. Chemotherapy-driven estrogen loss is postulated to drive bone loss, but significant data suggests the existence of an estrogen-independent mechanism of bone loss. Using clinically relevant mouse models, we showed that senescence and its senescence-associated secretory phenotype (SASP) contribute to chemotherapy-induced bone loss that can be rescued by depleting senescent cells. Chemotherapy-induced SASP could be limited by targeting the p38MAPK-MK2 pathway, which resulted in preservation of bone integrity in chemotherapy-treated mice. These results transform our understanding of chemotherapy-induced bone loss by identifying senescent cells as major drivers of bone loss and the p38MAPK-MK2 axis as a putative therapeutic target that can preserve bone and improve a cancer survivor's quality of life. SIGNIFICANCE: Senescence drives chemotherapy-induced bone loss that is rescued by p38MAPK or MK2 inhibitors. These findings may lead to treatments for therapy-induced bone loss, significantly increasing quality of life for cancer survivors.
Subject(s)
Antineoplastic Agents/adverse effects , Cellular Senescence/drug effects , MAP Kinase Signaling System/drug effects , Neoplasms/drug therapy , Osteoporosis/chemically induced , Animals , Disease Models, Animal , Doxorubicin/adverse effects , Femur/cytology , Femur/diagnostic imaging , Femur/pathology , Humans , Injections, Intraperitoneal , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Transgenic , Osteoporosis/diagnosis , Osteoporosis/pathology , Paclitaxel/adverse effects , Protein Serine-Threonine Kinases/metabolism , X-Ray Microtomography , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
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.
Subject(s)
Colitis/enzymology , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Administration, Oral , Animals , Cluster Analysis , Disease Models, Animal , Female , Gene Expression Profiling , Inflammation , Mass Spectrometry , Mice , Mice, Inbred C57BL , Phosphorylation , Principal Component Analysis , Proteomics , Rats , Signal Transduction , Terminology as Topic , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
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.
Subject(s)
Antineoplastic Agents/adverse effects , Bone and Bones/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Administration, Oral , Animals , Bone Neoplasms/secondary , Bone and Bones/pathology , Breast Neoplasms/pathology , Cell Line, Tumor , Disease Progression , Drug Therapy , Female , HEK293 Cells , Humans , Induction Chemotherapy , MAP Kinase Signaling System , Macrophages/metabolism , Mice , Neoplasm Metastasis , Osteoclasts/metabolism , Paclitaxel/pharmacology , Prognosis , Quality of Life , Stromal Cells/metabolism , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Inflammasomes/metabolism , Inflammation/pathology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Signal Transduction , Animals , Arthritis/pathology , Bone and Bones/pathology , Cytokines/biosynthesis , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Joints/pathology , Male , Mice , Mitogen-Activated Protein Kinase 14/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/metabolism , RNA Stability , Rats, Inbred LewABSTRACT
UNLABELLED: Neoplastic cells rely on the tumor microenvironment (TME) for survival and progression factors. Indeed, senescent and cancer-associated fibroblasts (CAF) express factors that promote tumorigenesis that are collectively referred to as the senescence-associated secretory phenotype (SASP). Despite their importance in tumorigenesis, the mechanisms that control TME-derived factor expression remain poorly understood. Here, we address a key unanswered question: how the SASP is sustained in senescent fibroblasts and CAFs. We find that the mitogen-activated protein kinase p38 (p38MAPK) controls AUF1 occupancy on SASP mRNAs and thus controls their stability. The importance of this regulatory mechanism is underscored by our findings that stromal-specific p38MAPK inhibition abrogates the tumor-promoting activities of CAFs and senescent fibroblasts. Our data suggest that targeting SASP mRNA stability through inhibition of p38MAPK will significantly aid the development of clinical strategies to target the TME. SIGNIFICANCE: The TME plays a key role in tumorigenesis. We demonstrate that p38MAPK governs a posttranscriptional mechanism that sustains the protumorigenic SASP. Inhibition of p38MAPK abrogates the tumor-promoting activities of CAFs and senescent fibroblasts. Thus, p38MAPK is a TME-specific Achilles' heel that may be exploited as a new therapeutic target.
Subject(s)
Fibroblasts/metabolism , Neoplasms/metabolism , Tumor Microenvironment , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Line , Cells, Cultured , Cellular Senescence , Female , Heterogeneous Nuclear Ribonucleoprotein D0 , Heterogeneous-Nuclear Ribonucleoprotein D/metabolism , Humans , Imidazoles/pharmacology , Lipopolysaccharides , Mice, Nude , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Tumor Necrosis Factor-alpha/blood , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
PH-797804 ((aS)-3-{3-bromo-4-[(2,4-difluorobenzyl)oxy]-6-methyl-2-oxopyridin-1(2H)-yl}-N,4-dimethylbenzamde) is a diarylpyridinone inhibitor of p38 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. Due to steric constraints imposed by the pyridinone carbonyl group and the 6- and 6'-methyl substituents of PH-797804, rotation around the connecting bond of the pyridinone and the N-phenyl ring is restricted. Density functional theory predicts a remarkably high rotational energy barrier of >30â kcal mol(-1), corresponding to a half-life of more than one hundred years at room temperature. This gives rise to discrete conformational spaces for the N-phenylpyridinone group, and as a result, two atropic isomers that do not interconvert under ambient conditions. Molecular modeling studies predict that the two isomers should differ in their binding affinity for p38α kinase; whereas the atropic S (aS) isomer binds favorably, the opposite aR isomer incurs significant steric interference with p38α kinase. The two isomers were subsequently identified and separated by chiral chromatography. IC50 values from p38α kinase assays confirm that one atropisomer is >100-fold more potent than the other. It was ultimately confirmed by small-molecule X-ray diffraction that the more potent atropisomer, PH-797804, is the aS isomer of the racemic pair. Extensive pharmacological characterization supports that PH-797804 carries most activity both inâ vitro and inâ vivo, and it has a stability profile compatible with oral formulation and delivery options.
Subject(s)
Benzamides/chemistry , Protein Kinase Inhibitors/chemistry , Pyridones/chemistry , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Arthritis/drug therapy , Benzamides/pharmacology , Benzamides/therapeutic use , Computer Simulation , Crystallography, X-Ray , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Female , Lipopolysaccharides/toxicity , Male , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Structure, Tertiary , Pyridones/pharmacology , Pyridones/therapeutic use , Quantum Theory , Rats , Receptors, Cell Surface , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
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.
Subject(s)
Benzamides/chemical synthesis , Benzamides/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Pyrones/chemical synthesis , Pyrones/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Benzamides/chemistry , Disease Models, Animal , Dogs , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Macaca fascicularis , Male , Molecular Structure , Pyridones , Pyrones/chemistry , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , p38 Mitogen-Activated Protein Kinases/chemistry , p38 Mitogen-Activated Protein Kinases/pharmacologyABSTRACT
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.
Subject(s)
Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Pyridones/chemical synthesis , Pyridones/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Disease Models, Animal , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Male , Microsomes, Liver/enzymology , Molecular Structure , Pyridazines/chemistry , Pyridazines/pharmacology , Pyridones/chemistry , Pyrimidines/chemistry , Pyrimidines/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Protein Kinase Inhibitors/therapeutic use , Pyrimidinones/therapeutic use , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Administration, Oral , Animals , Arthritis/drug therapy , Arthritis, Experimental , Caco-2 Cells , Crystallography, X-Ray , Humans , Inhibitory Concentration 50 , Male , Models, Molecular , Protein Kinase Inhibitors/chemistry , Pyrimidinones/chemistry , Rats , Rats, Inbred Lew , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Epithelial Cells/immunology , Interleukin-1 Receptor-Associated Kinases/immunology , Interleukin-1beta/immunology , Interleukin-6/immunology , Lung/cytology , Phosphatidylinositol 3-Kinase/immunology , Arthritis, Rheumatoid/immunology , Cell Line , Cell Line, Tumor , Fibroblasts/immunology , HumansABSTRACT
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.
Subject(s)
Cytokines/physiology , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Osteoblasts/metabolism , Signal Transduction/physiology , Cells, Cultured , Cytokines/genetics , Cytokines/pharmacology , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/drug effects , Humans , Interleukin-1beta/pharmacology , Interleukin-1beta/physiology , Interleukins/genetics , Interleukins/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System/physiology , Macrophage Colony-Stimulating Factor/genetics , Macrophage Colony-Stimulating Factor/metabolism , Osteoblasts/drug effects , RNA, Messenger/metabolism , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Tumor Necrosis Factor-alpha/physiologyABSTRACT
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.