ABSTRACT
All living organisms are charged with repair after injury particularly at epithelial barrier sites, but in some cases this response leads instead to structural remodeling and long-term disease. Identifying the molecular and cellular control of this divergence is key to disease modification. In that regard, stress kinase control of epithelial stem cells is a rational entry point for study. Here we examine the potential for mitogen-activated protein kinase 13 (MAPK13) regulation of epithelial stem cells using models of respiratory viral injury and post-viral lung disease. We show that Mapk13 gene-knockout mice handle acute infectious illness as expected but are protected against structural remodeling manifest as basal-epithelial stem cell (basal-ESC) hyperplasia-metaplasia, immune activation, and mucinous differentiation. In corresponding cell models, Mapk13-deficiency directly attenuates basal-ESC growth and organoid formation. Extension to human studies shows marked induction/activation of basal-cell MAPK13 in clinical samples of comparable remodeling found in asthma and COPD. Here again, MAPK13 gene-knockdown inhibits human basal-ESC growth in culture. Together, the data identify MAPK13 as a control for structural remodeling and disease after epithelial injury and as a suitable target for down-regulation as a disease-modifying strategy.
ABSTRACT
Respiratory viral infections are a major public health problem, with much of their morbidity and mortality due to post-viral lung diseases that progress and persist after the active infection is cleared. This paradigm is implicated in the most common forms of chronic lung disease, such as asthma and COPD, as well as other virus-linked diseases including progressive and long-term coronavirus disease 2019. Despite the impact of these diseases, there is a lack of small-molecule drugs available that can precisely modify this type of disease process. Here we will review current progress in understanding the pathogenesis of post-viral and related lung disease with characteristic remodelling phenotypes. We will also develop how this data leads to mitogen-activated protein kinase (MAPK) in general and MAPK13 in particular as key druggable targets in this pathway. We will also explore recent advances and predict the future breakthroughs in structure-based drug design that will provide new MAPK inhibitors as drug candidates for clinical applications. Each of these developments point to a more effective approach to treating the distinct epithelial and immune cell based mechanisms, which better account for the morbidity and mortality of post-viral and related types of lung disease. This progress is vital given the growing prevalence of respiratory viruses and other inhaled agents that trigger stereotyped progression to acute illness and chronic disease.
Subject(s)
Asthma , Lung Diseases , Virus Diseases , Viruses , Humans , Mitogen-Activated Protein Kinases/pharmacology , Lung Diseases/drug therapy , Lung , Virus Diseases/drug therapy , Drug DiscoveryABSTRACT
Common respiratory diseases continue to represent a major public health problem, and much of the morbidity and mortality is due to airway inflammation and mucus production. Previous studies indicated a role for mitogen-activated protein kinase 14 (MAPK14) in this type of disease, but clinical trials are unsuccessful to date. Our previous work identified a related but distinct kinase known as MAPK13 that is activated in respiratory airway diseases and is required for mucus production in human cell-culture models. Support for MAPK13 function in these models came from effectiveness of MAPK13 versus MAPK14 gene-knockdown and from first-generation MAPK13-14 inhibitors. However, these first-generation inhibitors were incompletely optimized for blocking activity and were untested in vivo. Here we report the next generation and selection of a potent MAPK13-14 inhibitor (designated NuP-3) that more effectively downregulates type-2 cytokine-stimulated mucus production in air-liquid interface and organoid cultures of human airway epithelial cells. We also show that NuP-3 treatment prevents respiratory airway inflammation and mucus production in new minipig models of airway disease triggered by type-2 cytokine challenge or respiratory viral infection. The results thereby provide the next advance in developing a small-molecule kinase inhibitor to address key features of respiratory disease.NEW & NOTEWORTHY This study describes the discovery of a potent mitogen-activated protein kinase 13-14 (MAPK13-14) inhibitor and its effectiveness in models of respiratory airway disease. The findings thereby provide a scheme for pathogenesis and therapy of lung diseases [e.g., asthma, chronic obstructive pulmonary disease (COPD), Covid-19, postviral, and allergic respiratory disease] and related conditions that implicate MAPK13-14 function. The findings also refine a hypothesis for epithelial and immune cell functions in respiratory disease that features MAPK13 as a possible component of this disease process.
Subject(s)
Mitogen-Activated Protein Kinase 14 , Pulmonary Disease, Chronic Obstructive , Animals , Humans , Swine , Mitogen-Activated Protein Kinase 14/metabolism , Swine, Miniature/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Inflammation/drug therapy , Inflammation/metabolism , Mucus/metabolism , Cytokines/metabolism , Mitogen-Activated Protein Kinase 13/metabolismABSTRACT
Common respiratory diseases continue to represent a major public health problem, and much of the morbidity and mortality is due to airway inflammation and mucus production. Previous studies indicated a role for mitogen-activated protein kinase 14 (MAPK14) in this type of disease, but clinical trials are unsuccessful to date. Our previous work identified a related but distinct kinase known as MAPK13 that is activated in respiratory airway diseases and is required for mucus production in human cell-culture models. Support for MAPK13 function in these models came from effectiveness of MAPK13 versus MAPK14 gene-knockdown and from first-generation MAPK13-14 inhibitors. However, these first-generation inhibitors were incompletely optimized for blocking activity and were untested in vivo. Here we report the next generation and selection of a potent MAPK13-14 inhibitor (designated NuP-3) that more effectively down-regulates type-2 cytokine-stimulated mucus production in air-liquid interface and organoid cultures of human airway epithelial cells. We also show that NuP-3 treatment prevents respiratory airway inflammation and mucus production in new minipig models of airway disease triggered by type-2 cytokine challenge or respiratory viral infection. The results thereby provide the next advance in developing a small-molecule kinase inhibitor to address key features of respiratory disease.
ABSTRACT
New studies of chronic obstructive pulmonary disease (COPD) are revealing the key role of airway epithelial cells and innate immune cells in the initiation, exacerbation, and progression of airway disease. An emerging scheme focuses on expansion of airway progenitor epithelial cells that feed forward for a type 2 immune response and consequent IL-13-driven mucus production that is linked to the morbidity and mortality of COPD. Analysis of human airway progenitor epithelial cells and airway tissue shows that IL-13 signaling to MUC5AC mucin gene expression relies on specific activation of mitogen-activated protein kinase 13, providing a druggable target for attenuating mucus production in the setting of viral infection and other inhaled stimuli of airway inflammation. Moreover, structure-based drug design is delivering highly potent, selective, and nontoxic small-molecule kinase inhibitors of mitogen-activated protein kinase 13 that offer a therapeutic strategy to downregulate excess mucus production to a physiological level and thereby achieve a precision medicine solution to the major health care problem of COPD and related airway diseases.
Subject(s)
Endothelial Progenitor Cells/cytology , Immunity, Innate , Inflammation/immunology , Mucin 5AC/genetics , Pulmonary Disease, Chronic Obstructive/immunology , Animals , Cell Communication , Drug Discovery , Gene Expression , Humans , Inflammation/complications , Interleukin-13/immunology , Mitogen-Activated Protein Kinase 13/immunology , Mucin 5AC/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , Respiratory Mucosa/immunology , Respiratory Mucosa/pathology , Signal TransductionABSTRACT
BACKGROUND: P38 MAP kinases are centrally involved in mediating extracellular signaling in various diseases. While much attention has previously been focused on the ubiquitously expressed family member MAPK14 (p38α), recent studies indicate that family members such as MAPK13 (p38δ) display a more selective cellular and tissue expression and might therefore represent a specific kinase to target in certain diseases. METHODS: To facilitate the design of potent and specific inhibitors, we present here the structural, biophysical, and functional characterization of two new MAPK13-inhibitor complexes, as well as the first comprehensive structural, biophysical, and functional analysis of MAPK13 complexes with four different inhibitor compounds of greatly varying potency. RESULTS: These inhibitors display IC50 values either in the nanomolar range or micromolar range (>800-fold range). The nanomolar inhibitors exhibit much longer ligand-enzyme complex half-lives compared to the micromolar inhibitors as measured by biolayer interferometry. Crystal structures of the MAPK13 inhibitor complexes reveal that the nanomolar inhibitors engage MAPK13 in the DFG-out binding mode, while the micromolar inhibitors are in the DFG-in mode. Detailed structural and computational docking analyses suggest that this difference in binding mode engagement is driven by conformational restraints imposed by the chemical structure of the inhibitors, and may be fortified by an additional hydrogen bond to MAPK13 in the nanomolar inhibitors. CONCLUSIONS: These studies provide a structural basis for understanding the differences in potency exhibited by these inhibitors. GENERAL SIGNIFICANCE: They also provide the groundwork for future studies to improve specificity, potency, pharmacodynamics, and pharmacokinetic properties.
Subject(s)
Mitogen-Activated Protein Kinase 13/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Binding Sites , Humans , Mitogen-Activated Protein Kinase 13/chemistry , Mitogen-Activated Protein Kinase 13/metabolism , Protein Binding , Protein Kinase Inhibitors/pharmacology , Quantitative Structure-Activity RelationshipABSTRACT
The p38 MAP kinases (p38 MAPKs) represent an important family centrally involved in mediating extracellular signaling. Recent studies indicate that family members such as MAPK13 (p38δ) display a selective cellular and tissue expression and are therefore involved in specific diseases. Detailed structural studies of all p38 MAPK family members are crucial for the design of specific inhibitors. In order to facilitate such ventures, the structure of MAPK13 was determined in both the inactive (unphosphorylated; MAPK13) and active (dual phosphorylated; MAPK13/pTpY) forms. Here, the first preparation, crystallization and structure determination of MAPK13/pTpY are presented and the structure is compared with the previously reported structure of MAPK13 in order to facilitate studies for structure-based drug design. A comprehensive analysis of inactive versus active structures for the p38 MAPK family is also presented. It is found that MAPK13 undergoes a larger interlobe configurational rearrangement upon activation compared with MAPK14. Surprisingly, the analysis of activated p38 MAPK structures (MAP12/pTpY, MAPK13/pTpY and MAPK14/pTpY) reveals that, despite a high degree of sequence similarity, different side chains are used to coordinate the phosphorylated residues. There are also differences in the rearrangement of the hinge region that occur in MAPK14 compared with MAPK13 which would affect inhibitor binding. A thorough examination of all of the active (phosphorylated) and inactive (unphosphorylated) p38 MAPK family member structures was performed to reveal a common structural basis of activation for the p38 MAP kinase family and to identify structural differences that may be exploited for developing family member-specific inhibitors.
Subject(s)
Mitogen-Activated Protein Kinase 13/chemistry , p38 Mitogen-Activated Protein Kinases/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Enzyme Activation , Humans , Mitogen-Activated Protein Kinase 13/metabolism , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Conformation , Sequence Alignment , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The process of conducting cell-based phenotypic screens can result in data sets from small libraries or portions of large libraries, making accurate hit picking from multiple data sets important for efficient drug discovery. Here, we describe a screen design and data analysis approach that allow for normalization not only between quadrants and plates but also between screens or batches in a robust, quantitative fashion, enabling hit selection from multiple data sets. We independently screened the MicroSource Spectrum and NCI Diversity Set II libraries using a cell-based phenotypic high-throughput screening (HTS) assay that uses an interferon-stimulated response element (ISRE)-driven luciferase-reporter assay to identify interferon (IFN) signal enhancers. Inclusion of a per-plate, per-quadrant IFN dose-response standard curve enabled conversion of ISRE activity to effective IFN concentrations. We identified 45 hits based on a combined z score ≥2.5 from the two libraries, and 25 of 35 available hits were validated in a compound concentration-response assay when tested using fresh compound. The results provide a basis for further analysis of chemical structure in relation to biological function. Together, the results establish an HTS method that can be extended to screening for any class of compounds that influence a quantifiable biological response for which a standard is available.
Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays , Drug Discovery/methods , Gene Expression , Gene Expression Regulation/drug effects , Genes, Reporter , Humans , Interferon Regulatory Factors/metabolism , Reproducibility of Results , Response Elements , Small Molecule LibrariesABSTRACT
The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.
Subject(s)
Chloride Channels/metabolism , Ion Channel Gating/physiology , Metalloproteases/metabolism , Proteolysis , Cell Line , Chloride Channels/genetics , Humans , Ion Transport/physiology , Metalloproteases/genetics , Protein Structure, TertiaryABSTRACT
Increased mucus production is a common cause of morbidity and mortality in inflammatory airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. However, the precise molecular mechanisms for pathogenic mucus production are largely undetermined. Accordingly, there are no specific and effective anti-mucus therapeutics. Here, we define a signaling pathway from chloride channel calcium-activated 1 (CLCA1) to MAPK13 that is responsible for IL-13-driven mucus production in human airway epithelial cells. The same pathway was also highly activated in the lungs of humans with excess mucus production due to COPD. We further validated the pathway by using structure-based drug design to develop a series of novel MAPK13 inhibitors with nanomolar potency that effectively reduced mucus production in human airway epithelial cells. These results uncover and validate a new pathway for regulating mucus production as well as a corresponding therapeutic approach to mucus overproduction in inflammatory airway diseases.
Subject(s)
Epithelial Cells/metabolism , Interleukin-13/physiology , Mitogen-Activated Protein Kinase 13/antagonists & inhibitors , Mucus/metabolism , Respiratory System/metabolism , Binding Sites , Cells, Cultured , Chloride Channels/genetics , Chloride Channels/metabolism , Chloride Channels/physiology , Crystallography, X-Ray , Drug Design , Epithelial Cells/drug effects , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Hydrogen Bonding , Kinetics , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 13/chemistry , Mitogen-Activated Protein Kinase 13/genetics , Mitogen-Activated Protein Kinase 13/metabolism , Models, Molecular , Mucins/genetics , Mucins/metabolism , Naphthalenes/chemistry , Naphthalenes/pharmacology , Protein Binding , Pulmonary Disease, Chronic Obstructive/metabolism , Pyrazoles/chemistry , Pyrazoles/pharmacology , RNA Interference , Respiratory System/pathology , Secretory Pathway/drug effectsABSTRACT
An acyl iminium ion-initiated tandem cyclization gave an unexpected dienone product, a seco-azasteroid (2). The factors governing the formation of 2 were investigated in an attempt to optimize its formation. The reaction was applied to a more elaborate system, resulting in the synthesis of the full steroid skeleton of 13-azaandrosta-1,4-diene-3,17-dione (3), which contains the unusual substitution of a chlorine atom for the axial 19-methyl.