New targets for resolution of airway remodeling in obstructive lung diseases

(Other) G protein–coupled receptor ligands

G protein–coupled receptors (GPCRs) play a substantial role in numerous normal physiological functions. Unsurprisingly, they can contribute towards the pathophysiology of various diseases. As noted earlier, GPCR agonists (of the β2-adrenergic receptor) and antagonists (of the mAChRs and CysLTR) are principal drugs in the management of asthma and COPD. In this section, we provide a brief overview of novel targets, the drugs that modulate them, and the potential of such drugs to address AR pathology.

E-prostanoid receptor agonists. The role of prostaglandin E₂ (PGE₂) and E-prostanoid (EP) receptor subtypes in mitigating AR has been a subject of recent research. Early studies demonstrated that autocrine PGE₂, generated as a consequence of cytokine-induced cyclooxygenase-2 induction, significantly suppresses mitogen-induced ASM proliferation in vitro⁷². Furthermore, studies of cultured human ASM in our laboratory have demonstrated that exogenous PGE₂ shows relatively superior anti-mitogenic activity in comparison to multiple β-agonists with anti-mitogenic effects corresponding to drug efficacy in activating the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) axis^73,74. Application of PGE₂ in humans has been hindered by the ability of PGE₂ to signal through multiple receptor subtypes (EP1–4)^75–79, which contributes to undesirable side effects. It is now known that the cough response is mediated by PGE₂ activation of the EP3 receptor on vagal sensory nerves⁸⁰. Additionally, studies in our lab have shown that EP3 receptor signaling has a pro-mitogenic role in ASM⁷⁴. To overcome the heterogeneity of PGE₂ signaling, the development of EP receptor subtype–specific modulators that specifically promote Gs-cAMP-PKA axis activity (via EP2 and EP4 subtypes) has been purposed^84,112–114. Currently, our lab is evaluating various strategies of targeting specific EP receptor subtypes in pre-clinical models of allergen-induced asthma⁸⁴.

Bitter taste receptors. Recently, our laboratory showed that bitter taste receptor (TAS2R) agonists can limit proliferation of ASM cells in vitro^81,82. Mechanistically, TAS2R agonists restrict ASM proliferation by inhibiting (1) the growth factor–activated protein kinase B (Akt) phosphorylation; (2) transcription factors AP-1, STAT3, E2 factor, and NFAT; and (3) genes associated with cell cycle progression.

The anti-mitogenic effects of TAS2R agonists further translate to pre-clinical asthma models as well. In a chronic allergen (ovalbumin or house dust mite) challenge model, treatment with bitter taste compounds (chloroquine and quinine) significantly reversed remodeling features⁸³. Specifically, treatment with bitter compounds inhibited the expression of calponin, smooth muscle alpha-actin, and smooth muscle myosin heavy chain in lungs. Furthermore, levels of matrix metallopeptidase-8 (MMP-8) (neutrophil collagenase), pro-MMP-9 (gelatinase), and MMP-12 (macrophage metalloelastase) were significantly reduced in lungs following treatment with TAS2R agonists. Finally, allergen-induced expression of pro-fibrotic cytokine transforming growth factor-beta (TGF-β) as well as phospho-mothers against decapentaplegic homolog 2 (pSmad2) and fibronectin in the lung tissue was also curtailed by TAS2R agonists. Collectively, these studies indicate that TAS2R agonists, unlike current GPCR ligands used to treat asthma, address multiple features of asthma pathology, including AR. Advancements in the development of selective ligands for TAS2R subtypes will allow for a refined therapeutic approach in the near future.

Bitter tastants have also been shown to modulate function of ciliated epithelial cells¹¹⁵. Specifically, the motile cilia on human airway epithelia express TAS2Rs (T2R4, T2R43, T2R38, and T2R46). The organization of TAS2Rs on cilia with the distribution of the signaling machinery along the ciliary shaft and within the attached epithelial cell presents an interesting mechanical apparatus for signal transduction. Stimulation of TAS2Rs with bitter tastants induces transient Ca²⁺ flux within the epithelial cells and increases ciliary beat frequency. Functionally, promoting increased ciliary movement could be beneficial in the removal of excess mucus from the airways.

In recent years, the role of mitochondrial dysfunction in disease states, including obstructive lung diseases, has become increasingly clear^116,117. Specifically, a role for autophagy/mitophagy in regulating mitochondrial function in pathophysiology of obstructive lung diseases is emerging. As noted earlier, our studies show that activation of TAS2Rs can promote anti-mitogenic activities. Further explorations into the mechanisms that underlie the anti-mitogenic effects of TAS2R agonists have uncovered an interesting role for bitter tastants in altering mitochondrial function and inducing autophagy⁸². TAS2R agonists can induce changes in mitochondrial membrane potential, increase reactive oxygen species generation, and promote mitochondrial fragmentation. These observations provide insight into the broader therapeutic potential of targeting mitochondrial function and promoting autophagy to restrict cellular proliferation.

Biologics

Asthma pathology is orchestrated by multiple immunologic mediators (cellular and secreted)¹¹⁸. Consequently, in recent years, therapies that target specific cytokines or immune cells to disrupt immune networks responsible for asthma pathology have gained significant interest. Targeting key cytokines with specific antibodies—biologics, including antibodies targeting interleukin-4 (IL-4), IL-5, and IL-13—can significantly limit recruitment of inflammatory cells to the lungs or blunt their pleiotropic effects. For instance, anti–IL-5 treatment can significantly reduce the number of circulating eosinophils in asthmatics and improve lung function^119–121. Anti–IL-5 treatment has been shown to prevent the development of subepithelial fibrosis in a murine model of asthma and reduce incorporation of proteoglycans in the human airway wall^85,86. However, antibodies targeting other cytokines or their receptors have not been studied in the context of AR¹²². Collectively, the data on the effects of biologics on AR are lacking and this is possibly due to the relatively recent development of these drugs. Future longitudinal studies that evaluate biologics in the context of remediation of AR features are needed to address the utility of these drugs as anti-AR agents.

Allergen-specific immunoglobulin E (IgE) isotype antibodies can cross-link on mast cells and basophils, causing degranulation and release of histamine, cytokines, and growth factors¹²³. Biologics that block the interactions of IgE antibodies to the high-affinity FcεRI receptors on mast cells and antigen presenting cells can curtail the sensitization profile in asthmatics and reduce exacerbations. In severe asthmatics evaluated for 36 months, blocking IgE activity was sufficient to reduce the thickening of the reticular lamina, thereby having an impact on AR⁸⁷.

Although biologics have become an increasingly important tool in the management of severe asthma, their application in the clinic has some drawbacks^124–126. Application of cytokine-specific antibody therapy is limited by the heterogeneity of asthma phenotypes¹²⁷. Non-atopic asthmatics are also not suitable for certain therapy (for example, anti-IgE). Biologics can also cause side effects such as hypersensitivity reactions, although the underlying mechanisms are unclear. Finally, there is also significant cost associated with the use of biologics.

Kinase inhibitors

Kinase enzymes modulate multiple cellular functions by regulating various signaling networks, including those regulating cellular proliferation and growth. Consequently, inhibitors that target various kinases have received increasing attention. Modulators of kinase functions account for one third of drugs in the development pipeline, and the majority of these represent cancer therapeutics¹²⁸. In this section, we provide a brief overview of drugs that target distinct kinases. For a more comprehensive discussion of targeting kinases in the context of obstructive lung diseases, the reader is referred to¹²⁹.

Mitogen-activated protein kinase inhibitors. Mitogen-activated protein kinases (MAPKs) have been studied extensively for their contribution to inflammatory gene expression and activation of multiple networks that contribute to the pathophysiology of obstructive lung diseases¹³⁰. Extracellular signal-regulated kinases (ERK1/2) are particularly interesting given that they are activated in multiple cell types that contribute to asthma and COPD pathology^88,131,132. Inhibition of ERK kinase (MAPK1, or MEK1) which is upstream of ERK1/2 can significantly reduce mucin 5AC, oligomeric mucus/gel-forming (MUC5AC) expression in cultured human bronchial epithelial cells subjected to chronic mechanical stress at the air-liquid interface^88,89. Other MAPKs, such as p38, c-Jun N-terminal kinase (JNK), and transforming growth factor beta-activated kinase 1 (TAK1), are activated in asthma and COPD¹²⁹, and inhibitors of these targets can mitigate various features of AR in both cell and animal asthma models^90–95. However, to date, no studies in humans have evaluated these inhibitors. Another limitation of kinase inhibitors is that these compounds are predominantly inhibitors of ATP-competitive and catalytic sites and block all enzymatic activity, including MAPK functions important for normal physiological activity in cells. Given the ubiquitous functions of the MAPK signaling pathway, development of substrate-selective MAPK inhibitors with the goal of targeting specific kinase functions associated with disease, while preserving kinase functions in normal cells, appears necessary to overcome limitations of off-target effects.

Receptor tyrosine kinase inhibitors. Receptor tyrosine kinases (RTKs) occupy a central role in critical signaling networks that promote asthma pathology, including remodeling¹³³. With inflammation, distinct RTKs and their ligands (for example, epidermal growth factor) are upregulated in human asthmatic airways and show a strong correlation with disease severity^134–143. RTKs can stimulate pathophysiological functions in ASM and epithelial cells. Thus, significant interest in advancing tyrosine kinase inhibitors for targeting RTKs has developed.

Activation of epidermal growth factor receptor (EGFR) is essential for mucus secretion and goblet cell metaplasia¹⁴⁴. It is also responsible for sustaining oxidative damage in the epithelial compartment through recruitment of neutrophils in a TGF-β–dependent manner^145–148. EGFR inhibitors tyrphostin AG1478 and BIBX1522 have been evaluated in vitro and in animal models of lung inflammation^99–101. Collectively, these studies report significant reductions in expression of mucus-associated MUC5AC gene and mucin secretion. More importantly, there is a concomitant reduction in collagen deposition and ASM proliferation^96–98. Although these observations are encouraging, some inhibitors of EGFR have failed to produce similar outcomes in clinical studies¹⁴⁹. Activation of platelet-derived growth factor receptor (PDGFR) has been shown to stimulate ASM proliferation in vitro and in vivo^138,150,151. Multiple drugs targeting PDGFR can mitigate ASM proliferation in vitro, although animal and human studies that address AR are lacking¹⁰⁴. In severe corticosteroid-dependent asthmatics, treatment with the tyrosine kinase inhibitor mastinib has shown improved outcomes; however, some adverse effects, including skin rash and edema, have also been reported¹⁵². During airway inflammation, multiple cell types (immune and resident) can be stimulated to secrete angiogenic factors, including vascular endothelial growth factor (VEGF)^153–159. These angiogenic factors can further stimulate increase in formation of new blood vessels from endothelial cells in the subepithelial mucosa^160–162. Although the contribution of neovascularization in AR is unclear, it has been suggested that newly formed vasculature is permeable, thus contributing to edema and limiting airflow^{71,161,163,164}. An antagonist of VEGFR-1 and VEGFR-2 (SU5416) has been shown to limit inflammatory responses in animals¹⁶⁵, although its impact on AR is unknown and studies in humans are lacking.

Because multiple RTKs contribute to pathology of AR, a novel strategy that targets multiple RTKs has gained momentum. In a pre-clinical murine model of ovalbumin-induced asthma, treatment with nintedanib—a small-molecule inhibitor that targets multiple RTKs (VEGFR, fibroblast growth factor receptor, and PDGFR)—significantly improved indices of remodeling and airway inflammation¹⁶⁶. This approach could be useful in targeting multiple redundant RTK networks that contribute to AR. Finally, inhibitors of non-RTKs, such as stem cell growth factor receptor (c-kit), spleen tyrosine kinase (SYK), the proto-oncogene tyrosine-protein kinase Src, and Janus kinase (JAK), have also been investigated in rodent models of asthma but have not yet progressed to studies in humans^{105,106,167–171}.

Other kinase inhibitors. Diverse stimuli (cytokines, viruses, growth factors, free radicals, and so on) can activate the transcription factor nuclear factor-kappa B (NF-κB) in multiple airway cell types. This transcription factor plays a key role in orchestrating immune responses and thus multiple intra- and inter-cell inflammatory signals¹²⁹. Although inhibitors that target activation of NF-κB have been shown to suppress certain synthetic functions of ASM¹⁷² and modulate pro-inflammatory outcomes in epithelial cells¹⁷³, specific NF-κB inhibitors have not translated into clinical trials for asthma and this is due to their multiple side effects¹²⁹. Inhibitors of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI-3K) that regulate cellular lipids and coordinate inflammatory pathways have undergone extensive investigation in asthma and COPD^174,175. However, data assessing AR indices are lacking. TGF-β plays an important role in cellular proliferation and differentiation and its expression increases in asthmatic airways, especially in the submucosal compartment^176–178. TGF-β has also been implicated in AR and can promote proliferation in ASM^179,180. TGF-β activates TGF-β receptor type I (T-βRI) kinase, which in turn activates Smad-dependent signaling that regulates expression of various genes. Small-molecule inhibitors of (T-βRI) kinase have yielded mixed results in studies assessing their effects on mechanisms mediating AR. T-βRI kinase inhibitors have been shown to diminish collagen deposition in lungs of rats challenged repeatedly with an allergen¹⁰⁸. In vitro, T-βRI inhibitors have demonstrated the ability to limit ASM proliferation, although their use in animal studies failed to inhibit TGF-β–induced increases in ASM mass^108,181. Clinical application of these inhibitors has been limited due to adverse effects, including cardiotoxicity in clinical trials for cancer therapy¹⁸². Protein kinase C (PKC) is another target of interest given its ability to promote contractile signaling in ASM following activation of Gq-coupled GPCRs. PKC is also relevant to AR because of its role in ASM proliferation¹⁸³ and mucus secretion in epithelium¹⁴⁸. Owing to severe toxicity, non-selective inhibitors of PKC have not progressed to clinical trials^129,184. Selective inhibitors of PKC isoforms have been developed for clinical studies for treatment of cancer, metabolic diseases, and psychiatric disorders, although adverse effects have been problematic and no clinical trials have been conducted for treatment of obstructive lung diseases in humans^129,184. Finally, Rho-associated protein kinase (ROCK) inhibitors have been shown to mitigate multiple features of asthma, including AR in guinea pig and murine models^109,185. Although ROCK inhibitors have been approved for certain indications, clinical trials in asthma or COPD are lacking¹²⁹ as issues regarding selectivity and toxicity have limited progression of these inhibitors to clinical application in airway diseases¹²⁹.

In summary, in vitro studies of pharmacological inhibition of multiple kinases that contribute to dysfunction in ASM and epithelium have yielded promising results^181,186. However, certain limitations have stalled progression of many drugs for clinical use. Specificity, efficacy, solubility issues, and poor pharmacokinetic profiles plague drug development¹⁸⁷. With chronic inhibitor treatment, compensatory signaling by other kinases may limit drug efficacy; this appears to be the case with p38 isoform inhibitors^129,188. Inhibition of any widely expressed kinase runs the risk of adverse effects. For example, given that NF-κB is crucial for mounting an immune response to microbial pathogens, blocking its activation could render patients susceptible to life-threatening infections¹²⁹. Current challenges in developing effective and safe kinase inhibitors hinge on improving the poor solubility, selectivity, and targeting of the current versions of these drugs¹²⁹.

Other small-molecule inhibitors

Phosphodiesterase (PDE) inhibitors have a beneficial effect of promoting ASM relaxation by increasing intracellular cAMP resulting in PKA-mediated ASM relaxation. In murine models of asthma, PDE inhibitors have also been shown to curtail inflammation and reduce AR¹¹⁰. Inhibition of PDE3 (but not PDE4) has anti-proliferative action on mitogen-activated human ASM cells in vitro, but as with most potential anti-AR drugs, useful in vivo data are lacking¹¹¹. More recently, an inhibitor of PDE8 (PF-04957325) has been shown to regulate proliferation of ASM cells by enhancing cAMP accumulation generated specifically from the β2-AR/AC6 pathway¹⁸⁹.

The rapamycin derivative SAR-943 has been shown to limit the mitogen-induced proliferation of human ASM cells (but not human epithelial cells) in vitro and mitigate inflammation and AR in vivo in ovalbumin-challenged mice¹⁹⁰, yet no clinical studies for asthma or COPD have been reported.