Kinases as Novel Therapeutic Targets in Asthma...

1521-0081/68/3/788–815$25.00 http://dx.doi.org/10.1124/pr.116.012518PHARMACOLOGICAL REVIEWS Pharmacol Rev 68:788–815, July 2016Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: CLIVE PAGE

Kinases as Novel Therapeutic Targets in Asthma andChronic Obstructive Pulmonary Disease

Peter J. Barnes

National Heart and Lung Institute, Imperial College, London, United Kingdom

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789II. Role of Kinases in Asthma and COPD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790III. Kinases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790

A. Kinase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791IV. Nuclear Factor-kB inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791

A. Role in Asthma and Chronic Obstructive Pulmonary Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . 791B. Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793

V. Mitogen-Activated Protein Kinase Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793A. p38 Mitogen-Activated Protein Kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794

1. Role in Asthma and Chronic Obstructive Pulmonary Disease. . . . . . . . . . . . . . . . . . . . . . . . 794B. p38 Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795C. Jun NH2-terminal Kinase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796D. Extracellular Regulating Kinase/MAP Kinase Kinase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . 796E. Apoptosis Signal-Regulating Kinase-1 Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797F. TGFb-Activated Kinase-1 Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797G. MAPKAPK2 Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797

VI. Phosphoinositide-3-kinase Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797A. Role in Asthma and Chronic Obstructive Pulmonary Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . 798B. Inhibiting Phosphoinositide-3-kinase-Akt-Mammalian Target of Rapamycin Signaling . . . 799

1. Phosphoinositide-3-kinase Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7992. Akt-Mammalian Target of Rapamycin Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8003. Activating Endogenous Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800

VII. Janus-Activated Kinase Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801A. Role in Asthma and Chronic Obstructive Pulmonary Disease. . . . . . . . . . . . . . . . . . . . . . . . 801B. Jakinibs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 802

VIII. Spleen Tyrosine Kinase Inhibition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803A. Role in Asthma and Chronic Obstructive Pulmonary Disease. . . . . . . . . . . . . . . . . . . . . . . . 803B. Spleen Tyrosine Kinase Inhibitors.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 804

IX. Receptor Tyrosine Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 804A. Role in Asthma and Chronic Obstructive Pulmonary Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . 804B. Receptor Tyrosine Kinase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805C. c-Kit Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805

X. Transforming Growth Factor-b Receptor Kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805A. Role in Asthma and Chronic Obstructive Pulmonary Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . 805B. T-bRI Kinase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806

XI. Protein Kinase C Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806A. Role in Asthma and Chronic Obstructive Pulmonary Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . 806B. Protein Kinase C Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806

XII. Other Kinase Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807

Address correspondence to: Prof. P. J. Barnes, National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse St,London SW3 6LY, UK. E-mail: [email protected]

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A. Rho-Associated Protein Kinase Inhibition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807B. Interleukin-1 Receptor-Associated Kinase Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807C. Src Family Kinase Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807D. Hematopoietic Cell Kinase Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807E. Bruton’s Tyrosine Kinase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 808

XIII. Future Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 808A. Improving Selectivity and Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 808B. Targeting Several Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809C. Overcoming Kinase Inhibitor Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809D. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809

Abstract——Multiple kinases play a critical role inorchestrating the chronic inflammation and structuralchanges in the respiratory tract of patients with asthmaand chronic obstructive pulmonary disease (COPD). Ki-nases activate signaling pathways that lead to contractionof airway smooth muscle and release of inflammatorymediators (suchas cytokines, chemokines, growth factors)as well as cell migration, activation, and proliferation. Forthis reason there has been great interest in the develop-ment of kinase inhibitors as anti-inflammatory therapies,particular where corticosteroids are less effective, as insevere asthma andCOPD.However, it has proven difficultto develop selective kinase inhibitors that are botheffective and safe after oral administration and this hasled to a search for inhaled kinase inhibitors, which would

reduce systemic exposure. Although many kinases havebeen implicated in inflammation and remodeling ofairway disease, very few classes of drug have reachedthe stage of clinical studies in these diseases. The mostpromisingdrugsarep38MAPkinases, isoenzyme-selectivePI3-kinases, Janus-activated kinases, and Syk-kinases,and inhaled formulations of these drugs are now indevelopment. There has also been interest in developinginhibitors that block more than one kinase, because thesedrugs may be more effective and with less risk of losingefficacy with time. No kinase inhibitors are yet on themarket for the treatment of airway diseases, but as kinaseinhibitors are improved from other therapeutic areasthere is hope that these drugs may eventually proveuseful in treating refractory asthma and COPD.

I. Introduction

Asthma and chronic obstructive pulmonary disease(COPD) both cause obstruction of the airways and involvechronic inflammation of the respiratory tract. Both are veryprevalent diseases andare increasing throughout theworld.Asthma is now the most common chronic disease in

developed countries, affecting up to 20% of the popula-tion, and is increasing, particularly in developingcountries (Beasley et al., 2000). It still causes deathand leads to hospital admissions because of acute

exacerbations. Although asthma is usually well con-trolled with inhaled corticosteroids, many patients withasthma are poorly controlled in the real world asadherence to this treatment is poor and inhaler useoften inadequate (Partridge et al., 2006; Braido et al.,2013). Approximately 20% of asthma patients areparticularly difficult to manage, and 3–5% of patientshave severe disease that is not controlled even bymaximal inhaled treatment with an inhaled corticoste-roid and a long-acting b2-agonist (Chung and Wenzel,

ABBREVIATIONS: AHR, airway hyperresponsiveness; AMPK, adenosine monophosphate kinase; AP-1, activator protein-1; ASK, apoptosissignal-regulatory kinase; ASM, airway smooth muscle; ATP, adenosine triphosphate; AZD2014, 3-(2,4-bis((S)-3-methylmorpholino)pyrido[2,3-d]pyrimidin-7-yl)-N-methylbenzamide; Bay 65-1942, (7E)-7-[2-(cyclopropylmethoxy)-6-oxocyclohexa-2,4-dien-1-ylidene]-5-[(3S)-piperidin-3-yl]-4,8-dihydro-1H-pyrido[2,3-d][1,3]oxazin-2-one; BTK, Bruton’s tyrosine kinase; COPD, chronic obstructive pulmonary disease; CT99021,6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; CXCL8, interleukin-8;EGFR, epidermal growth factor receptor; ERK, extracellular regulating kinase; FEV1, forced expiratory volume in 1 second; FGF(R), fibroblastgrowth factor (receptor); GM-CSF, granulocyte-macrophage colony stimulating factor; GR, glucocorticoid receptor; GSK2269557, 2-(6-(1H-indol-4-yl)-1H-indazol-4-yl)-5-((4-isopropylpiperazin-1-yl)methyl)oxazole hydrochloride; GSK3b, glycogen synthase-3b; Hck, hematopoietic cell kinase; HDAC2,histone deacetylase-2; hsp, heat shock protein; IFN, interferon; IgE, immunoglobulin E; IKK, inhibitor of kB kinase; IL, interleukin; IRAK,interleukin-1 receptor-associated kinase; ITAM, immunoreceptor-tyrosine-based activation motif; JAK, Janus-activated kinase; JNK, Jun NH2-terminal kinase; LAS 189386, 1-{2-[(1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl]pyridin-4-yl}-N-pyrazin-2-yl-1H-indazol-3-amine; LPS, lipopolysaccharide;LY294002, 2-Morpholin-4-yl-8-phenylchromen-4-one; MAPK, mitogen-activated protein kinase; MAPKAPK or MK2, MAP-activated protein kinase;MKP, MAP kinase phosphatase; mTOR, mammalian target of rapamycin; NF-kB, nuclear factor-kappa B; NIK, NF-kB-inducing kinase; PBMC,peripheral blood mononuclear cells; PD-98059, 29-Amino-39-methoxyflavone; PDGF(R), platelet-derived growth factor (receptor); PF-03715455, 1-[5-tertbutyl-2-(3-chloro-4-hydroxyphenyl)pyrazol-3-yl]-3-[[2-[[3-[2-(2-hydroxyethylsulfanyl)phenyl]-[1,2,4]triazolo[4,3-a]pyridin-6-yl]sulfanyl]phenyl]methyl]urea;PH-797804, (aS)-3-{3-bromo-4-[(2,4-difluorobenzyl)oxy]-6-methyl-2-oxopyridin-1(2H)-yl}-N,4-dimethylbenzamde; PI3K, phosphoinositide-3-kinase; PKC, protein kinase C; PTEN, phosphatase and tensin homolog deleted from chromosome 10; ROCK, Rho-associated protein kinase;RTK, receptor tyrosine kinase; S6K, p70 ribosomal S6-kinase; SB-505154, 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride; SD-282, indole-5-carboxamide; SHIP, Src homology 2 domain-containing inositol 59-phosphatase; STAT, signaltransducer of activators of transcription; Syk, spleen tyrosine kinase; TAK, transforming growth factor-b-activated kinase; TG100-115, 3-[2,4-diamino-6-(3-hydroxyphenyl)pteridin-7-yl]phenol (6,7-Bis(3-hydroxyphenyl)pteridine-2,4-diamine); TGF, transforming growth factor; TLR, Toll-likereceptors; TNF, tumor necrosis factor; VEGF(R), vascular-endothelial growth factor (receptor).

Kinase Inhibitors in Airway Disease 789

2014; Hekking et al., 2015). There is a need to developmore effective therapies for these patients who consumea disproportionate amount of medical resources.COPD is also very prevalent, affecting about 10% of

people over 45 years in developed countries and in-creasing especially in developing countries (Barneset al., 2015). The increasing world-wide prevalence ofCOPD reflects aging of the population because it is adisease of elderly people. COPD has a high morbidityand is one of the leading causes of hospitalization andloss of time from work. It has a rising mortality and isnow the third most common cause of death in developedcountries (Lozano et al., 2012). No current treatments,including corticosteroids, reduce disease progression ormortality and have relatively little effect (;20% re-duction) in preventing exacerbations, reflecting theirlack of anti-inflammatory effects in this disease. Thereis therefore an enormous unmet need for the develop-ment of new treatments for COPD that target theunderlying inflammatory process and aberrant repairmechanisms (Barnes, 2013b).Although asthmaandCOPDare distinct inflammatory

diseases, some patients with COPDmay have features ofasthma such as eosinophilic inflammation and greaterairway reversibility, whereas some patients with asthmamay have features of COPD, such as neutrophilic in-flammation, reduced reversibility, and reduced respon-siveness to corticosteroids. These patients have beendescribed as having asthma-COPD overlap syndrome,although several phenotypes are included in this label(Bateman et al., 2015; Postma and Rabe, 2015; Barnes,2016). Improved treatment of patients with asthma-COPD overlap is needed (Barnes, 2015b).Thus, asthma and COPD are very common chronic

inflammatory diseases of the respiratory tract withseveral unmet needs for more effective medication.Because chronic inflammation involves the activationof multiple kinase signaling pathways (Cohen, 2002),this has provided a rationale for the development ofselective kinase inhibitors for the treatment of asthmaand COPD, particularly in patients with severe diseasethat cannot be controlled by existing therapies. In viewof the role of protein kinases in chronic inflammationand tissue remodeling, the use of kinase inhibitors is alogical approach. Furthermore, there is increasingevidence that kinases are also involved in corticosteroidresistance, which is a feature of severe asthma andCOPD (Barnes and Adcock, 2009). Development ofkinase inhibitors for inflammatory diseases has provento be problematic because of the adverse effects whenthese drugs are given systemically. Theoretically, thiscould be overcome by the development of inhaledinhibitors, but it has proven difficult to develop kinaseinhibitors that have local activity in the airways. Thisreview summarizes the current evidence for the effectsof protein kinase inhibitors in inflammatory airwaydiseases.

II. Role of Kinases in Asthma and COPD

Both asthma and COPD are characterized by achronic inflammatory response in the respiratory tractthat involves the recruitment and activation of severalinflammatory and immune cells and the secretion ofmultiple inflammatory mediators, including lipid me-diators, cytokines, chemokines, and growth factors, andwell as enzymes that generate inflammatory productsor are involved in tissue remodeling (Barnes, 2008a,b).Many of these inflammatory proteins are regulated byproinflammatory transcription factors, such as nuclearfactor-kB (NF-kB) and activator protein-1 (AP-1) thatare regulated by kinase signaling pathways. Further-more, thesemediators act on surface receptors on targetcells in the airways that also activate kinase pathways.In the case of asthma, allergen may be an importantdriver of the allergic inflammatory response and thisinvolves the crosslinking of allergens with immunoglob-ulin E (IgE) bound to mast cells, with the activation ofkinase s that result in release of preformed mediators,such as histamine and the synthesis and release of lipidmediators, such as cysteinyl-leukotrienes and prosta-glandins (Barnes, 2011; Galli and Tsai, 2012). Allergenis also taken up by dendritic cells and this activateskinase pathways, leading to the recruitment of T-helper2 (Th2) lymphocytes to the airways that drive eosino-philic inflammation and airway hyperresponsiveness,which are the defining features of asthma (Barnes,2011). Innate immunity also plays a key role in asthma,particularly severe and nonallergic asthma that is drivenin innate lymphoid-2 cells (Scanlon and McKenzie,2012). Innate immune mechanisms and the activa-tion of pattern recognition receptors, such as Toll-likereceptors (TLR) that are activated by cigarette smokeand wood smoke (which are key risk factors for thedevelopment of COPD), and by pathogens, such ascolonizing bacteria. Mitogen-activated protein kinases(MAPK) play a key role in the activation of both innateand adaptive immune cells, resulting in the secretionof multiple cytokines, chemokines, and growth factorsthat then activate further waves of inflammation(Arthur and Ley, 2013). COPD represents an acceler-ation of the normal aging process in lungs and thesepathways of cellular senescence are also regulated bymultiple interacting kinase pathways (Mercado et al.,2015).

III. Kinases

Kinases are enzymes that transfer the g-phosphate ofadenosine-triphosphate (ATP) to hydroxyl-bearing sub-strates and thus phosphorylate proteins, lipids, andsugars, which results in activation of enzymes, cellulartranslocation, or interaction with other proteins andplay a critical role in signal transduction. Over 500kinase genes have now been recognized in humans,

790 Barnes

making up ;2% of the genome (the “kinome”), and over30% of proteins are regulated by phosphorylation(Manning et al., 2002). These are organized into distinctfamilies, such as mitogen-activated protein kinases(MAPK). The majority of kinases phosphorylate serine(Ser) or threonine (Thr) residues (serine/threoninekinases); some act on tyrosine (Tyr) residues (tyrosinekinases), whereas other act on both types (dual-specificity kinases). Tyrosine-kinases may act as recep-tors for ligands such as growth factors, such as epidermalgrowth factor receptor (EGFR). Because kinases playsuch a critical role in signaling, they are carefullyregulated by phosphatases, which remove phosphategroups and thus reverse the effects of a specific kinase.Kinases are comprised of several domains, including awell conserved catalytic domain that binds ATP. Al-though the majority of kinases target proteins, sometarget lipids (such as phosphatidylinositol and sphin-gosine), carbohydrates (such as hexokinase), or nucleo-tides, such as thymidine kinase.Protein kinases are selective for Ser/Thr or Tyr res-

idues in their target proteins and recognize a con-sensus sequence around these residues rather than allof the residues on a particular target protein (Fabbroet al., 2015). However, a particular kinase is usually notspecific for a particular protein but may act on severalproteins sharing this consensus sequence. Pseudosub-strates bind to the kinase but lack the amino acid thatis phosphorylated, so that they effectively act as aninhibitor of the kinase. Kinases are organized intosignaling pathways, whereby one kinase phosphory-lates a downstream kinase, which then phosphorylatesthe next downstream kinase in the sequence. Eachkinase may phosphorylate different kinases so thatseveral signaling cascades may be activated and theremay be crosstalk between different kinase signalingpathways, resulting in amplification or inhibition ofsignaling. This makes signaling pathways highly com-plex, and it is often difficult to predict the effects ofinhibiting a single kinase. Furthermore, there arecomplex feedback inhibitory loops that may result inunexpected effects of interrupting kinase pathways.

A. Kinase Inhibitors

The recognition that kinases are key regulators of cellproliferation, apoptosis, and inflammation and thatmutation of kinase genes is an important mechanismin many immune and inflammatory diseases hasprompted a search for selective small molecule inhibi-tors in many human diseases (Cohen, 2002). Manysmall molecule kinase inhibitors have now been ap-proved for use in cancer (Wu et al., 2015c), and althoughmost of the drug discovery efforts have focused onmalignancy, kinase inhibitors are now seen as impor-tant potential therapies for many chronic inflammatoryand immune disorders (Cohen, 2014). Indeed, kinaseinhibitors are now the second largest group of drugs in

development after G-protein-coupled receptor ligandsand account for ;30% of drug discovery projects inmajor pharmaceutical companies (Wu et al., 2015c).Because both asthma and COPD involve inflammatoryand immune mechanisms, there have been great effortsto develop kinase inhibitors as new therapies, particu-larly targeting areas of unmet need (Adcock et al., 2006).Development of specific kinase inhibitors has proven tobe very difficult because the catalytic site is wellconserved between different kinases and most inhibi-tors target the ATP-binding site. This means that manykinase inhibitors are poorly selective and often haveseveral off-target effects on structurally related kinases,which may increase the risks of adverse effects. Afurther problem with kinase inhibitors in cancer ther-apy is the development of resistance due to sitemutations at the ATP-binding pocket. With chronictreatment of inflammatory diseases there may also bea gradual wearing off of efficacy of kinase inhibitors dueto the upregulation of compensatory pathways. Forexample, p38MAPK inhibitors for rheumatoid arthritismay lose efficacy because of upregulation of otherproinflammatory kinase pathways (Firestein et al.,2008). This might be less of a problem if more upstreamkinases are targeted, although the risks of side effectsmay be even greater.

Most kinase inhibitors so far have been given orally,so that systemic side effects are common and often dose-limiting. As a means of reducing adverse effects, therehas been great interest in the development of allostericinhibitors that target less conserved sites in the kinasedomain or remote sites (Wu et al., 2015b). These drugsmay not need to be of such high potency that is needed tocompete with the high concentrations of ATP at thecatalytic site and may also be more resistant tomutations. Allosteric kinase inhibitors, such as trame-tinib, are now in clinical development, at least for thetreatment of cancer. For example, allosteric inhibitorsthat bind to a distal site have now been discovered forp38 MAPK (Comess et al., 2011). Inhaled delivery mayalso reduce the risk of adverse effects by targeting thedrug to the airways. So far it has proven difficult todevelop inhaled kinase inhibitors that are effectivebecause they do not have topical activity. This is relatedto the fact that kinases are intracellular targets, so thedrug properties that target cell penetration also in-crease oral bioavailability and rapid transport acrossthe lung into the systemic circulation.

IV. Nuclear Factor-kB inhibition

A. Role in Asthma and Chronic ObstructivePulmonary Disease.

The proinflammatory transcription factor NF-kB isactivated in asthma and COPD and switches on multi-ple inflammatory genes, including cytokines, chemo-kines, proteases, and inhibitors of apoptosis, resulting


in amplification of the inflammatory response (Barnesand Karin, 1997; Hart et al., 1998; Di Stefano et al.,2002; Caramori et al., 2003). NF-kB activation, mea-sured by nuclear localization of p65 (RelA), is seenpredominantly in airway epithelial cells and macro-phages andmay be activated by inflammatory cytokines(such as TNF-a and IL-1b), oxidative stress, allergens,and bacteria. NF-kB may play an important amplifyingrole in acute exacerbations of asthma and COPD, whichare commonly triggered by upper respiratory tractviruses, such as rhinovirus (Caramori et al., 2003).NF-kB is activated by rhinovirus in airway epithelialcells (Footitt et al., 2016; Papi et al., 2013) and there ismarked activation of NF-kB in the lungs of patients whodied from asthma (Caramori et al., 2009).The molecular pathways involved in NF-kB activa-

tion include several kinases, which are targets forinhibition. The classic (canonical) pathway for inflam-matory stimuli and infections to activate NF-kB signal-ing involve the IKK (inhibitor of kB kinase) complex,which is composed of two catalytic subunits, IKK-a andIKK-b, and a regulatory subunit, IKK-g (or NF-kBessential modulator) (Hayden and Ghosh, 2012). TheIKK complex phosphorylates NF-kB-bound IkBs, tar-geting them for degradation by the proteasome andthereby releasing NF-kB dimers that are composed ofp65 and p50 subunits, which translocate to the nucleus

where they bind to kB recognition sites in the promoterregions of inflammatory and immune genes, resultingin their transcriptional activation (Fig. 1). This re-sponse depends mainly on the catalytic subunit IKK-b(also known as IKK2), which carries out IkB phosphor-ylation. Although the canonical pathway has beenstudied most extensively in asthma and COPD, thereis a noncanonical (alternative) pathway, which involvesthe upstream kinase NF-kB-inducing kinase (NIK) thatphosphorylates IKK-a homodimers and releases RelBand processes p100 to p52 in response to certainmembers of the TNF family, such as lymphotoxin-b(Sun, 2012). This pathway switches on different genesets and may mediate different immune functionsfrom the canonical pathway. Dominant-negative IKK-binhibitsmost of the proinflammatory functions ofNF-kB,whereas inhibiting IKK-a has a role only in re-sponse to limited stimuli and in certain cells suchas B-lymphocytes. The noncanonical pathway is in-volved in development of the immune system and inadaptive immune responses, but its role in asthma andCOPD has not been explored in detail. The coactivatormolecule CD40 is expressed on antigen-presenting cells,such as dendritic cells and macrophages, activates thenoncanonical pathway when it interacts with CD40Lexpressed on lymphocytes, and may amplify allergicresponses in asthma (Lombardi et al., 2010). CD40

Fig. 1. Nuclear factor-kB signaling pathway. The canonical (classic) pathway is activated by inflammatory cytokines, such as tumor necrosis factor(TNF)-a, interleukin(IL)-1b, and lipopolysaccharide (LPS), leading to phosphorylation of inhibitor of kB kinases [IKKa, IKKb, which are in a complexwith NF-kB essential modulator (NEMO)], resulting in phosphorylation of inhibitor of kB (IkB-b), which is ubiquitinated and degraded by theproteasome releasing p65 and p50 to translocate to the nucleus, where they bind to a kB DNA recognition sequence, leading to the activation ofcytokines, chemokine, and protease genes. The noncanonical pathway is activated by CD40 and lymphotoxin(LT)-b, which activate NF-kB-inducingkinase (NIK), resulting in activation IKKa homodimers, which phosphorylate RelB/p100, generating RelB/p50 complexes that translocate to thenucleus and switches on immune genes. IKK-b may be inhibited by several compounds (shown in the green box), but no inhibitors of the non-canonicalpathway have been identified.

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stimulation induces emphysema in mice, and solubleCD40L is increased in plasma of COPD patients andcorrelates with the degree of emphysema, suggesting apossible pathogenic role (Shigeta et al., 2012). Indirectevidence for the involvement of the noncanonical path-way is the activation of NIK and phosphorylation ofIKK-a in airway epithelial cells after exposure tocigarette smoke and oxidative stress (Chung et al.,2011). On the other hand, RelB overexpression in miceinhibits cigarette smoke neutrophilic inflammationthrough stimulation of cyclooxygenase-2 (McMillanet al., 2011).

B. Inhibitors.

Inhibition of NF-kB is a logical approach to treatinginflammation in asthma and COPD because there isevidence for its activation and increased activation ofmultiple proinflammatory genes. However, deletion ofNF-kB related genes inmice is lethal or animals developsevere septicemia, indicating that there are risks ofNF-kB inhibition (Hayden and Ghosh, 2012). Cortico-steroids inhibit NF-kB activation in patients withasthma (Hart et al., 2000), but there is resistance tothis effect in patients with severe asthma and COPD(Barnes and Adcock, 2009). Theophylline is also report-ed to inhibit NF-kB in human airway epithelial cellsthrough IKK inhibition, although high concentrationsare needed for this effect (Ichiyama et al., 2001). Severalnaturally occurring compounds, such as plant polyphe-nols, have also been reported to have inhibitory effectson NF-kB. For example, resveratrol inhibits NF-kB andinflammatory cytokine release from COPD epithelialcells in vitro (Donnelly et al., 2004). The green teaextract epigallocatechin-3-gallate inhibits IKK activity(IKK-a as well as IKK-b) in epithelial cells in vitro(Yang et al., 2001).Several IKK-b inhibitors of differing chemical classes

have been developed and in vitro have demonstratedinhibition of p65 nuclear translocation and the activa-tion of inflammatory genes after various stimuli as wellas anti-inflammatory effects in in vivo animal models(Suzuki et al., 2011). Both direct and allosteric inhibitorshave been developed. A decoy oligodeoxyribonucleotideagainst NF-kB markedly inhibits allergic lung inflam-mation in an ovalbumin-sensitized mice with markedinhibition of Th2 cytokine release (Desmet et al., 2004).A selective IKK-b inhibitor IMD-0354 [N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide]also reduced allergic inflammation and airway hyper-responsiveness in this murine model, predominantlythrough inhibition of T cell trafficking (Sugita et al.,2009; Maslanka et al., 2016). Another IKK-b inhibitor[Bay 65-1942 ((7E)-7-[2-(cyclopropylmethoxy)-6-oxocy-clohexa-2,4-dien-1-ylidene]-5-[(3S)-piperidin-3-yl]-4,8-dihydro-1H-pyrido[2,3-d][1,3]oxazin-2-one)] is also ef-fective at inhibiting pulmonary allergic inflammationin a rat model (Ziegelbauer et al., 2005). Yet another

IKK-b inhibitor TPCA-1 is effective in a mouse modelof neutrophilic inflammation induced by inhaled endo-toxin, although it was not effective in a neutrophilelastase-induced inflammation model (Birrell et al.,2006). IKK-b inhibitors have also been ineffective incigarette smoke-induced lung inflammation in mice(Rastrick et al., 2013). Several IKK-b inhibitors sup-pressed the release of cytokines and chemokines fromhuman airway smooth muscle and epithelial cells andwere mimicked by adenovirus mediated delivery of adominant-negative IKK-b inhibitor (Catley et al.,2006; Newton et al., 2007). To avoid potential systemicexposure, an inhaled IKK-b inhibitor (PF-104) wasdeveloped to inhibit neutrophilic lung inflammationinduced by inhaled endotoxin in mice (Sommers et al.,2009). Ainsliadimer-A is found in a Chinese herbalproduct that has been described as a potent andselective inhibitor of IKKa/b, thus inhibiting theclassic and alternative pathways of NF-kB (Donget al., 2015). It inhibits the release of inflammatorycytokines after LPS injection in mice, so has potentialas an anti-inflammatory drug.

Because NF-kB plays a critical role in host defenseagainst pathogens, there are concerns that inhibitorsmay increase the risk of infection. There is also anuncertain relationship to cancer, because NF-kB maydrive tumorigenisis, but its inhibitionmay also increasecell proliferation. Limited numbers of clinical trialshave been conducted. IMD-1041, a prodrug of theIKK-b inhibitor IMD-0354, was tested in patients withCOPD for anti-inflammatory effects, but the trial wasterminated because of side effects of the medication(ClinicalTrials.gov: NCT00883584). Other IKK-b inhib-itors are in clinical trials for other inflammatorydiseases, but no results have been published and noproducts have been approved. It is likely that an inhaledformulation will be necessary to reduce adverse effects.

V. Mitogen-Activated Protein Kinase Inhibition

MAPK are a large family of highly conserved serine/threonine protein kinases that are involved in signaltransduction (Johnson and Lapadat, 2002). Althoughoriginally shown to be activated by mitogens, they areactivated by many stimuli, including cytokines, growthfactors, and various stresses (osmotic, oxidative, heatshock) and result in diverse cellular responses, such asproliferation, differentiation, mitosis, cell survival, ap-optosis, and inflammatory gene expression. MAPK arenormally inactive in the resting cell and become acti-vated by phosphorylation, usually of a threonine and atyrosine residue. Several scaffold proteins are associ-ated with MAPK pathways and appear to direct signal-ing within the cell by directing proteins to particularcellular compartments and may also modulate signal-ing. Typically MAPKs operate in a three-tiered cascaderegulated by successive phosphorylation starting with


activation of MAP kinase kinase kinases (MAP3K), thenMAP kinase kinases (MAP2K), and finally MAPK (Fig.2). ThemajorMAPK pathways involved in inflammatorydiseases are ERK (extracellular regulating kinase), p38MAPK, and JNK (c-Jun NH2-terminal kinase), but mostattention has focused on inhibition of the p38 MAPKpathway, because several small molecule inhibitors werediscovered and there has been particular interest in p38inhibitors for the treatment of asthma and COPD.

A. p38 Mitogen-Activated Protein Kinase

The p38 MAPK family comprises a-, b, g-, and d-isoforms, which are highly conserved proline-directedprotein kinases that are activated by several extracel-lular stresses, including oxidative and osmotic stressand inflammatory cytokines. P38 MAPK is activated byphosphorylation of Thr180 and Tyr182 in the Thr-Gly-Tyrmotif of the activation loop, which allows p38 tobind ATP at its catalytic binding site to transfer theg-phosphate group from ATP to the protein targeted forphosphorylation. P38 MAPK is activated by upstreamkinases MKK3, MKK4, and MKK6, which are them-selves activated by MAP3Ks, such as ASK1(apoptosissignal-regulatory kinase 1), TAK1 (transforminggrowth factor-b-activated kinase-1), MLK3 (mixed-lineage kinase 3), and MAP kinase kinase kinase(MEKK3), which are activated by many stimuli,

including inflammatory cytokines, growth factors, andoxidative stress. P38 MAPK target many proteins,including other kinases, transcription factors, andcytoskeletal proteins, resulting in multiple inflamma-tory effects. P38 MAPK activates over 60 downstreamtargets, so may be a key point for inhibition (Trempolecet al., 2013). P38a and p38b isoforms are widelydistributed and target a downstream target MAP-activated protein kinase 2 (MAPKAPK2 orMK2), whichmediates many of these effects. P38a, via activation ofMK2, increases inflammation by stabilizing mRNAtranscripts of some cytokines, such as TNFa, by phos-phorylating adenosine-uracil–binding proteins, such astristeraprolin, that bind to the adenosine-uracil–rich 39-untranslated regions of mRNAs to regulate their sta-bility (Dean et al., 2004; Brook et al., 2006). MK2 alsophosphorylates heat shock protein(hsp)-27, which hasbeen used as a biomarker of p38a activation. P38apotentiates the proinflammatory effects of NF-kB byphosphorylation of histone H3 at the promoter sites onproinflammatory cytokine and chemokine genes to in-crease binding of NF-kB binding to their promoterregions (Saccani et al., 2002). P38g and p38d isoformshave a more restricted distribution, particularly mac-rophages, and target mainly transcription factors.

1. Role in Asthma and Chronic Obstructive Pulmo-nary Disease. There is considerable evidence that p38

Fig. 2. Mitogen-activated protein kinase signaling pathways. MAPK signaling is activated by external stimuli, such as growth factors and cellularstress, and activates a three-tiered cascade with MAPK kinase kinases (MAP3K), activating MAPK kinases (MAP2K) and finally MAPK. The majorMAPK pathways involved in inflammatory diseases are ERK (extracellular regulating kinase), p38 MAPK, and JNK (c-Jun NH2-terminal kinase).Upstream kinases include TGFb-activated kinase-1 (TAK1) and apoptosis signal-regulating kinase-1 (ASK1). Downstream pf p38 MAPK is MAPKactivated protein kinase 2 (MAPKAPK2 or MK2). Several classes of inhibitor are now developed and shown in green boxes.

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MAPK is activated in asthma and COPD and playsan important role in driving chronic inflammation aswell as corticosteroid insensitivity (Chung, 2011). P38ais activated in airway epithelial cells and macrophagesby lipopolysaccharide (LPS) and cigarette smoke andmediates the release of inflammatory proteins, suchas CXCL8 (IL-8), IL-6, TNF-a, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Se-lective inhibitors of p38a inhibit the release of thesecytokines from human macrophages activated by ciga-rette smoke and LPS (Koch et al., 2004; Smith et al.,2006). However, p38a inhibitors did not cause maximalinhibition in macrophages and it is possible that otherisoforms, such as p38d are important in macrophages(Smith et al., 2006). Alveolar macrophages from pa-tients with severe asthma show increased activation ofp38, which may reflect a reduction in the endogenousinhibitor of p38, MAPK phosphatase-1 (MKP1, alsoknown as dual specificity phosphatase-1) (Bhavsaret al., 2008). In mice exposed to cigarette smoke and toozone, a p38a inhibitor SD-282 significantly suppressedthe neutrophilic inflammatory response, whereas corti-costeroids were ineffective (Medicherla et al., 2007;Williams et al., 2008). This suggests that p38a mayplay an important role in pulmonary neutrophilic in-flammation in COPD. Indeed, phosphorylated p38ashows increased expression in alveolar macrophages,epithelial cells, and CD4+ and CD8+ lymphocytes inlungs from COPD patients (Renda et al., 2008; Gaffeyet al., 2013).P38a is also an important mediator of inflammation

in response to upper respiratory tract virus infectionsand so may play a role in acute exacerbations of asthmaand COPD. Rhinovirus-induced release of chemokinesfrom alveolar macrophages is associated with activa-tion of p38a (Hall et al., 2005) and a p38a inhibitorsuppresses release of inflammatory cytokines fromhuman airway epithelial cells infected with rhinovirus(Griego et al., 2000). Haemophilus influenzae, bacteriacommonly colonizing the lower airways and causingacute exacerbations of COPD patients, also activatesp38 in airway epithelia cells andmediates the release ofCXCL8 (Wang et al., 2003). H. influenzae also stimu-lates the secretion of the mucin MUC5AC from epithe-lial cells via p38a activation, suggesting that inhibitionof this pathway may reduce mucus hypersecretion inCOPD patients (Shen et al., 2008).P38 activation is also important in mediating allergic

inflammation. An inhaled p38a antisense oligonucleo-tide was effective in inhibiting eosinophilic inflamma-tion and airway hyperresponsiveness after allergenchallenge in an ovalbumin-sensitized mouse model(Duan et al., 2005b). A selective p38a inhibitor, indole-5-carboxamide (SD-282), similarly reduced allergic in-flammation, airway hyperresponsiveness and airwayremodeling in a chronic allergen exposure mouse model(Nath et al., 2006). P38a may play an important role in

allergic inflammation through the activation of thetranscription factor GATA3, which regulates the Th2cytokines IL-4, IL-5, and IL-13, because p38 phosphory-latesGATA3 in the cytoplasmof Th2 cells so that it is ableto translocate to the nucleus to activate Th2 cytokinegenes (Maneechotesuwan et al., 2007). A p38 inhibitor, bypreventing GATA3 phosphorylation, inhibits the expres-sion of Th2 cytokines (Maneechotesuwan et al., 2009).P38 also prolongs eosinophil survival by enhancing theeffects of IL-5 and a p38 inhibitor promotes apoptosis(Kankaanranta et al., 1999).

P38a appears to play an important role in the corti-costeroid insensitivity that occurs in severe asthmaand COPD (Barnes, 2013a). The corticosteroid insensi-tivity induced by IL-2 and IL-4 or by IL-13 in peripheralblood mononuclear cells (PBMC) is reversed by a p38ainhibitor (Matthews et al., 1999; Irusen et al., 2002).Corticosteroids increase the expression of MKP-1 andthus inhibit p38 activation. In mice with deletion ofMKP-1 corticosteroid lose their anti-inflammatory ef-fects (Abraham et al., 2006). In addition, TNF-a andIL-1b result in phosphorylation of the glucocorticoidreceptor (GRa) at Ser211 via p38a activation, preventingtranslocation to the nucleus (Szatmary et al., 2004).P38a is activated in PBMC from patients with severeand steroid-resistant asthma and is correlated withGRa phosphorylation and reduced responsiveness tocorticosteroids (Mercado et al., 2012; Li et al., 2015).Furthermore, a p38a inhibitor is able to restore cortico-steroid responsiveness in these cells. Similarly, in PBMCfrom COPD patients the reduced responsiveness tocorticosteroids was reversed by treatment with a p38ainhibitor (Khorasani et al., 2015). P38g also phosphory-lates GRa and reduces nuclear translocation, resulting incorticosteroid insensitivity (Mercado et al., 2011b). P38gis increased in PBMC of patients with severe asthma andmay be reduced by treatment with the long-acting b2-agonist formoterol, which thereby reduces corticosteroidresistance in these cells (Mercado et al., 2011b).

B. p38 Inhibitors

There is a strong rationale for the development of p38inhibitors to reduce chronic inflammation (both neutro-philic and eosinophilic), to reduce corticosteroid resis-tance, and to reduce structural changes, particularly inpatients with severe asthma and COPD. Selective p38ainhibitors have been rather effective in asthmatic andCOPD cells in vitro and in in vivo animal models of thesediseases. Several p38 inhibitors have been developed forclinical use, and these drugs aremainly selective for p38aand p38b isoforms (Norman, 2015). These drugs havebeen studied largely in patients with rheumatoid arthri-tis, but have been disappointing because they have beendose-limited by side effects and have lost effusivenessafter repeated administration due to the development ofcompensatory signaling pathways. Most of the clinicalstudies have been inCOPD,where there is a great need to


develop effective anti-inflammatory treatments becausecorticosteroids are largely ineffective, and eight p38ainhibitors are known to have entered clinical trials inCOPD (Norman, 2015). An oral p38a inhibitor dilmapi-mod (SB-681323) significantly reduced phosphorylatedhsp-27 (demonstrating target engagement) and TNF-arelease from circulating leukocytes of COPD patients,demonstrating proof of mechanism (Singh et al., 2010)Losmapimod is an oral dual p38a/b inhibitor that had noeffect on sputum neutrophils in a phase II study of about300 COPD patients over 12 weeks but reduced phosphor-ylation of hsp-27 and reduced plasma fibrinogen byapproximately 11% (Lomas et al., 2012). In the largestclinical trial of about 600 COPD patients studied over24 weeks, three doses of losmapimod were ineffective onexercise tolerance (6 minute walking distance), althoughthere was a trend for reduction in exacerbations;however, there were side effects at the highest dose,particularly dermatological effects (Watz et al., 2014).In a post hoc analysis in patients with low bloodeosinophils (,2%), there was a dose-related reductionin exacerbations with a 55% reduction at the highestdose and a small improvement in FEV1 (15mg)(Marks-Konczalik et al., 2015). PH-797804 [(aS)-3-{3-bromo-4-[(2,4-difluorobenzyl)oxy]-6-methyl-2-oxopyridin-1(2H)-yl}-N,4-dimethylbenzamdePF-03715455=1-[5-tertbutyl-2-(3-chloro-4-hydroxyphenyl)pyrazol-3-yl]-3-[[2-[[3-[2-(2-hydroxyethylsulfanyl)phenyl]-[1,2,4]triazolo[4,3-a]pyridin-6-yl]sulfanyl]phenyl]methyl]urea] is an oralp38a-selective inhibitor, which increased trough FEV1

values and dyspnea at some doses in a 6-week study of230 patients with COPD (MacNee et al., 2013). Because ofthe problems of side effects at high oral doses of p38inhibitors, several inhaled drugs have been developed, butno results in airway disease have been reported (Millanet al., 2011). An inhaled p38 inhibitor, PF-03715455[1-[5-tertbutyl-2-(3-chloro-4-hydroxyphenyl)pyrazol-3-yl]-3-[[2-[[3-[2-(2-hydroxyethylsulfanyl)phenyl]-[1,2,4]tri-azolo[4,3-a]pyridin-6-yl]sulfanyl]phenyl]methyl]urea],had no significant effect on sputum inflammatory bio-markers although an oral p38 inhibitor had some sup-pressive effect in the same population (Singh et al., 2015).RV-568 is a potent inhaled p38 inhibitor that inhibits allfour isoforms of p38 and also the Src family kinase Hckand is referred to as a narrow spectrum kinase inhibitor,which significantly increased FEV1 and reduced sputummalondealdehyde after 2weeks of administration (Russellet al., 2013).No clinical studies of p38 inhibitors in asthmahave been reported, andmost of the drugs in developmentfor COPD have now been discontinued.

C. Jun NH2-terminal Kinase Inhibitors

JNK is an important activator of the proinflammatorytranscription factor activator protein-1 (AP-1), whichregulates many inflammatory genes in asthma andCOPD. Sensitized mice exposed to inhaled allergenshow increased activation of JNK in airway epithelial

cells and lung. Furthermore, a JNK inhibitor SP600125(1,9-pyrazoloanthrone) suppressed the inflammatoryresponse to allergen and reduced mucus hypersecretionand airway hyperresponsiveness, suggesting a role forJNK in allergic inflammation (Nath et al., 2005; Wuet al., 2015a). TNF-a prolongs the survival of humaneosinophils via activation of JNK, which thereby sus-tains eosinophilic inflammation (Kankaanranta et al.,2014). JNK may also play a role in airway remodeling,because SP600125 also reduces airway smooth muscleproliferation after chronic inhaled allergen exposure inmice (Eynott et al., 2003). In JNK-deficient mice, thefibrosis after chronic exposure to allergen (house dustmite) was significantly reduced (van der Velden et al.,2014). JNK regulates airway smooth muscle prolifera-tion and increased collagen synthesis through theactivation of transforming growth factor (TGF)-b (Xieet al., 2007) and this is mediated through the activationof AP-1 (Lee et al., 2006b). JNK also plays a role inCD40-mediated class switching in B cells, andSP600125 inhibits the formation of IgE transcripts inB cells (Jabara and Geha, 2005). JNK activation is alsoinvolved in corticosteroid resistance in severe asthmapatient, because AP-1 competes with GRa and there isevidence for JNK activation in PBMC of patients withsteroid-resistant asthma (Adcock et al., 1995; Lane et al.,1998). The corticosteroid resistance induced by IL-2 andIL-4 in PBMC ismediated by activation of JNK, resultingin phosphorylation of Ser226 in GR-a to prevent itsnuclear localization (Kobayashi et al., 2012). JNK activa-tion is also involved in the corticosteroid resistanceinduced by rhinovirus infect ion of airway epithelial cells,which is reduced by a JNK inhibitor and completelyreversed by a combination of JNK and IKK-b inhibitors(Papi et al., 2013). JNK activation may also play a role inCOPD. Cigarette smoke cytotoxicity in airway epithelialcells is mediated via activation of JNK (Lee et al., 2008).

The activation of JNK and AP-1 in models of asthmaandCOPDand its role in stimulating cytokine release andcorticosteroid resistance suggests that JNK inhibitorsmay have therapeutic value in the treatment of asthmaandCOPD,which is supported by thebeneficial effects of aselective JNK inhibitor SP600125 in these models.SP600125 is a competitive inhibitor of ATP for bothJNK1 and JNK2. No clinical trials of JNK inhibitors havebeen undertaken, presumably because of toxicity issues.

Curcumin is a plant polyphenol found in turmeric,which inhibits JNK, and is currently in clinical trials forchronic inflammatory diseases, such as psoriasis; how-ever, it is nonselective and targets several other signal-ing pathways, such as NF-kappaB and nuclear factor(erythroid-derived 2)-like 2 (Hatcher et al., 2008).

D. Extracellular Regulating Kinase/MAP KinaseKinase Inhibitors

Several studies demonstrated activation of ERK1 andERK2 in asthma and COPD models. Activation of mast

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cells may release IL-1 and IL-6 through activation ofERK1/2, which is inhibited by genistein, a flavonoid thathas additional pharmacological effects (Kim et al., 2014).IL-13 causes mucus hypersecretion as a result of in-creasedMUC5AC via ERK activation (Kono et al., 2010).Epithelial growth factor receptor (EGFR)-inducedmucussecretion is alsomediated via ERK and inhibited by theERK kinase (MEK1) inhibitor PD-98059 [29-Amino-39-methoxyflavone] (Li et al., 2012). Although p38 MAPKappears to be the dominant regulator of cytokine re-lease from monocytes, in lung and alveolar macro-phage ERK appears to be predominant and ERKinhibitors are more effective than p38 inhibitors insuppressing cytokine secretion (Koch et al., 2004;Tudhope et al., 2008). The ERK pathway is activated bygrowth factors and may be involved in proliferation ofairway smooth muscle cells and other structural changesin the airways.The proinflammatory effect of ERK suggest that an

ERK inhibitor may be of potential benefit, particularlyin reducing mucus secretion. The MEK1/ERK inhibitorPD-98059 is effective as an inhibitor of this pathwayand acts as a noncompetitive allosteric inhibitor with ahigh degree of specificity. However, this compound isnot suitable for in vivo or clinical studies because of itsvery low solubility. Several other MEK-1 inhibitors,such as trametinib and solumetinib, have been tested inclinical trials in cancer and have shown some efficacy insolid tumors and are relatively well-tolerated, but so farno studies in COPD or asthma have been reported(Wang et al., 2007; Fremin and Meloche, 2010). In-terestingly, macrolide antibiotics inhibit cigarettesmoke-induced CXCL8 secretion by epithelial cells,which is mediated via ERK phosphorylation, althoughmacrolides have other effects on signaling such asinhibition of NF-kB (Shinkai et al., 2007).

E. Apoptosis Signal-Regulating Kinase-1 Inhibition

Apoptosis signal-regulating kinase-1 (ASK1) is anupstream kinase (MAP3K5) that activates both p38MAPK and JNK and is activated by oxidative andendoplasmic reticulum stress (Hattori et al., 2009).Thioredoxin is normally bound to ASK1 to suppressits activity, and ASK becomes activated when thiore-doxin is reduced by oxidative stress. ASK1mediates thecytotoxicity of cigarette smoke in airway epithelial cells,which is inhibited by the closely associated antioxidantthioredoxin (Lee et al., 2008). ASK1 is a good potentialtarget for COPD, and several small molecule inhibitorshave been identified (Starosyla et al., 2015). Interest-ingly, ASK1 knockout mice are viable, whereas p38aand JNK knockout mice are embryonically lethal.Clinical trials are underway with ASK1 inhibitors, suchas GS-4997, in diabetic renal disease (Lin et al., 2015),but so far these drugs have not been explored in COPDor asthma. Thioredoxin is the endogenous inhibitor ofASK1 but is difficult to deliver intracellularly.

F. TGFb-Activated Kinase-1 Inhibition

TGFb-activated kinase-1 (TAK1) is another up-streamMAP kinase (MAP3K7) that is tightly regulatedand activates both ERK and p38a MAPK pathways aswell as NF-kB, thus playing a pivotal role in innateimmunity in response to TGFb, TNFa, IL-1b, and TLRvia the adapter protein MyD88 (Sato et al., 2005;Ajibade et al., 2013). Proliferation of human airwaysmooth muscle in response to PDGF is significantlyreduced by the selective TAK1 inhibition, with adominant-negative inhibitor and small molecule inhib-itor, 5Z-7-oxozeaenol (Pera et al., 2011). TAK1 inhibi-tion also inhibits cigarette smoke-induced release ofCXCL8 from airway smooth muscle cells (Pera et al.,2012). H. influenzae activates NF-kB and p38 MAPK inhuman airway epithelial cells via the activation of TAK1and NIK (Shuto et al., 2001). TAK1may also activate theadaptive immune system through the activation ofNF-kB in T-lymphocytes (Sun et al., 2004). TAK1 istherefore an attractive target for inhibition, because bothMAPK and NF-kB pathways would be suppressed.Several small molecule TAK1 inhibitors are in develop-ment (mainly for cancer therapy), but there are limiteddata on kinase selectivity and they have not yet advancedto clinical studies (Kilty and Jones, 2015).

G. MAPKAPK2 Inhibition

MAPKAPK2 (MK2) is activated directly via p38aMAPK, and inhibition of this downstream kinase mightavoid some of the toxicity problems seen with p38ainhibitors (Gaestel, 2013). MK2 appears to play a role inprolonging NF-kB activation in Th2 cells and thereforeamplifies allergic inflammation (Gorska et al., 2007). MK2is also important in mediating the increased release ofCXCL8 in response to cigarette smoke in human airwaysmooth muscle cells (Moretto et al., 2012). However, ATP-competitive MK2 inhibitors have poor selectivity and lowsolubility (Schlapbach and Huppertz, 2009). By contrast,noncompetitive inhibitors lookmore attractive and severalcompounds are in development but have not yet enteredclinical trials (Anderson et al., 2009; Fiore et al., 2016).

VI. Phosphoinositide-3-kinase Inhibition

PI3Ks are a family of lipid kinases that generate lipidsecond messengers that regulate a number of cellularevents, including innate and adaptive immune responses,growth, differentiation, cell motility and survival, andintracellular trafficking (Vanhaesebroeck et al., 2012;Hawkins and Stephens, 2015). PI3Ks catalyze the phos-phorylation of the inositol ring of cell membrane phos-phatidylinositols to generate lipid second messengers.They are classified into three classes based on thephosphorylated lipids generated. Class I PI3Ks catalyzethe production of phosphatidylinositol-(3,4,5)-trisphos-phate (PIP3) from PIP2 and are heterodimers comprising


a p110 catalytic subunit with four isoforms (designatedP110a, b, g, and d) together with an adaptor subunitof either p85 (class IA) that are triggered by recep-tor tyrosine kinases (RTKs) and include PI3Ka, b, andd, or of p110/p84 (class IB) that are mainly triggeredby G-protein-coupled receptors that include PI3Kg(Vanhaesebroeck et al., 2016). PIP3 accumulates in theplasma membrane and recruits pleckstrin homologydomain-containing proteins, including phosphoinositide-dependent kinase-1 (PDK1) and Akt (also called proteinkinase B). PIP3-activated PDK1 then phosphorylates Aktat Thr308 site and, together with Ser473 phosphorylationbymammalian target of rapamycin complex 2 (mTORC2),this ensures full activation of Akt. P-Akt then triggersmultiple downstream signaling pathways (Fig. 3). Down-stream signaling fromp-Akt is complex but amajor targetin mTORC1, which activates cellular senescence and isinvolved in accelerated aging (Johnson et al., 2013).PI3Ka and PI3Kb are expressed in most cell types,whereas PI3Kg and PI3Kd are localized predominantlyin leukocytes. By contrast, there is relatively little in-formation about the function of Class II and III PI3Ks.Class II PI3Ks include PI3K-C2a, PI3K-C2b, and PI3K-C2g. PI3K-C2a and PI3K-C2g are the best characterizedisoforms, and they appear to contribute to vesiculartrafficking by regulating the production of PtdIns(3)P,

but because there are no selective inhibitors they aredifficult to investigate. Class III includes only one mem-ber, Vps34, which is involved in vesicular trafficking, TLRsignaling, and autophagy via PtdIns(3)P generation.

The PI3K pathway is tightly regulated by phos-phatases, particularly PTEN (phosphatase and ten-sin homolog deleted from chromosome 10), the majorendogenous PI3K inhibitor, which is expressed inall cell types and dephosphorylates PIP3 to PIP2,thus turning off PI3K activation (Carracedo andPandolfi, 2008; Worby and Dixon, 2014). PTEN actsas a tumor suppressor and deletions are commonlyfound in cancers such as lung cancer, but it is nowrecognized as an important regulator of inflamma-tion and cell proliferation. The Src homology 2domain-containing inositol 59-phosphatase (SHIP) alsoinhibits PI3K signaling and is found mainly in hema-topoietic cells, including macrophages (Backers et al.,2003).

A. Role in Asthma and Chronic ObstructivePulmonary Disease

There is increasing evidence that PI3K plays a keyrole in asthma and COPD through activation of in-flammation, corticosteroid resistance, and cellularsenescence, resulting in accelerated aging (Fig. 3)

Fig. 3. Phosphoinositide-3-kinase signaling pathways. PI3K is activated by extracellular signals, Including growth factors and chemokines, resultingin activation of Akt (protein kinase B), then tuberous sclerosis complex (TSC)1/2, then mammalian target of rapamycin complex-1 (mTORC1), which inturn activates ribosomal S6-kinase (S6K), which reduces sirtuin-1 (SIRT1) and histone deacetylase(HDAC)2 and impairs autophagy. There is crosstalkto other signaling pathways because Akt1 activates IKKb and mTORC1 activates JNK. Oxidative stress activates PI3K signaling though inhibition ofPTEN (phosphatase and tensin homolog deleted from chromosome 10) and SHIP1 (Src homology 2 domain-containing inositol 59-phosphatase-1). AMPkinase (AMPK) inhibits mTORC1 via an effect on TSC1/2. Several classes of inhibitor (shown in green boxes) may reduce PI3K signaling.

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(Marwick et al., 2010b). These effects are mediated viaClass I PI3Ks, and the potential role of Class II and IIPI3Ks is unknown. A nonselective PI3K inhibitorLY294002 [2-Morpholin-4-yl-8-phenylchromen-4-one]given by inhalation inhibits the allergic inflammatoryresponse to allergen in sensitized mice, with reduction ineosinophils, Th2 cytokines, and airway hyperresponsive-ness (AHR) (Duan et al., 2005a). Similar results wereobtained with a dominant-negative inhibitor of the regu-latory subunit p85, indicating that Class IA PI3Ks areinvolved and may determine the switch from Th1 to Th2cytokines (Myou et al., 2003). More specifically Pi3Kdplays a key role in mast cell degranulation after cross-linking of IgE receptors, an effect blocked by inactivationof PI3Kd andby the selectivePI3Kd inhibitor IC87114 (Aliet al., 2008; Kim et al., 2008). In the murine model ofasthma, intratracheal administration of IC87114 sup-pressed the allergic response, Th2 cytokines, and AHR(Lee et al., 2006a), and these effects are mirrored inPI3Kd-inactivated mice (Nashed et al., 2007). In anAspergillus fumigatus model of allergic inflammation inmice, PI3Kd inhibition suppressed the allergic response,and this effect was mediated through inhibition ofendoplasmic reticulum stress, which is increased inasthma and results in NF-kB activation (Lee et al.,2016). PI3Kmayalso play a role in increasing contractilityof canine airway smoothmuscle (Halayko et al., 2004) andthe increased proliferation of airway smooth muscle inresponse to growth factors such as TGF-b (Goldsmithet al., 2006).PI3K activation is also important in COPD. Total

PI3K activity measured as p-Akt is markedly in-creased in peripheral lungs and macrophages of pa-tients with COPD, and there is increased expression ofthe PI3Kd isoform (Marwick et al., 2009; To et al.,2010). In vitro oxidative stress increases p-Akt inperipheral bloodmonocytes and alveolarmacrophages,which is prevented by a PI3Kd but not PI3Kg inhibitor(Marwick et al., 2010a). PI3Kg knockout mice showreduced neutrophil migration and activation, as wellas impaired T-lymphocyte and macrophage function(Medina-Tato et al., 2007), and PI3Kg is the pivotalsignaling pathway mediating neutrophil recruitment tothe lung after instillation of chemokines (Thomas et al.,2005). PI3Kg plays an important role in chronic in-flammatory and immune diseases (Costa et al., 2011).PI3Kd is also involved in the inflammatory responseand may cooperate with PI3Kg in the activation ofneutrophils, for example (Condliffe et al., 2005). BothPI3Kg and PI3Kd inhibitors improve the accuracy ofmigration in COPD neutrophils (Sapey et al., 2014). Anaerosolized PI3Kg/d inhibitor [TG100-115 (3-[2,4-diamino-6-(3-hydroxyphenyl)pteridin-7-yl]phenol (6,7-Bis(3-hydroxyphenyl)pteridine-2,4-diamine))] waseffective in cigarette smoke-induced pulmonary inflam-mation in mice, which is corticosteroid resistant, andalso in the allergic inflammation model (Doukas et al.,

2009). PI3Kd is also involved in corticosteroid resis-tance after oxidative stress, which is mediated byreduced histone deacetylase-2 (HDAC2), and PI3Kdinhibitors may reverse corticosteroid resistance inpatients with COPD by increasing HDAC2 expres-sion. Inhibition of PI3Kd with IC87114 reversescorticosteroid-resistance to cigarette smoke exposurein mice (To et al., 2010), and PI3Kd-inactivated mice donot develop corticosteroid-resistance after cigarettesmoke exposure, whereas PI3Kg-inactivated animalsdevelop corticosteroid-resistant inflammation as nor-mal (Marwick et al., 2009). PI3Ka is involved in cellproliferation and survival and is activated in early lungcancer cells and so may contribute to the increased riskof developing lung cancer in patients with COPD. PI3Kactivation in COPD also activates mTOR, measured byphosphorylation of p70 ribosomal S6-kinase (S6K),which is increased in COPD lungs and PBMC (Mitaniet al., 2015). S6Knot only reducesHDAC2 but also leadsto activation of JNK, increased c-Jun, and activation ofAP-1, which also contributes to corticosteroid resis-tance. PI3K activation also drives cellular senescenceand accelerated aging in COPD lungs through theactivation of mTOR, which plays a pivotal role inaccelerated aging (Johnson et al., 2013; Mercadoet al., 2015). This leads to a reduction in the keyantiaging molecule sirtuin-1, which is reduced in thelungs of patients with COPD (Rajendrasozhan et al.,2008; Nakamaru et al., 2009). The same PI3K-mTORpathway may also lead to the comorbidities associatedwith COPD, such as ischemic heart disease, type2 diabetes, osteoporosis, chronic renal disease, anddementia (Barnes, 2015a). For example, endothelialprogenitor cells, which are important for repairingdamage to endothelial cells to maintain normal vascu-lar function, show accelerated aging and decreasedsirtuin-1 due to activation of PI3K (Paschalaki et al.,2013).

B. Inhibiting Phosphoinositide-3-kinase-Akt-Mammalian Target of Rapamycin Signaling

In view of the critical role of PI3K-Akt-mTORsignaling it is not surprising that there has been anintense effort to discover drugs that inhibit thispathway. This has been mainly in the field of cancerin view of the key role of PI3K in cell proliferation, andthe PI3K-Akt-mTOR pathways are the most fre-quently dysregulated in human solid tumors. Thepathways are also relevant to chronic degenerativediseases that involve accelerated aging (Johnson et al.,2013) and in the treatment of inflammatory andimmune diseases (Hawkins and Stephens, 2015).There has been great interest in the potential forinhibitors of the PI3K pathways in the treatment ofsevere asthma and COPD (Ito et al., 2007).

1. Phosphoinositide-3-kinase Inhibitors. Nonselectiveinhibitors of Class I PI3K include wortmannin and


LY294002, which are useful in in vitro and in vivoanimal studies but are not suitable for clinical usebecause of their poor pharmacokinetics and high tox-icity. Several pan-class I PI3K inhibitors have beendeveloped for use in malignancy, including hematologicmalignancies and solid tumors, but so far none havebeen approved for clinical use, mainly because oftoxicity issues (Dienstmann et al., 2014). This has ledto a search for isoform-selective inhibitors. However,PI3K isoforms may compensate for each other, at leastin cancer, through feedback mechanisms, where block-ing one isoform leads to a compensatory increased in thenoninhibited isoforms. For example a PI3Kb-selectiveinhibitor leads to an increase in PI3Ka (Schwartz et al.,2015). PI3Ka inhibitors have been developed for treat-ing solid tumors but may result in hyperglycemia,because they interfere with insulin signaling. PI3Kginhibitors have been considered, but so far no drugshave been developed for clinical use (Ruckle et al., 2006)as potential anti-inflammatory treatments (Ameriksand Venable, 2009). PI3Kd inhibitors have been de-veloped for the treatment of B cell malignancies and thePI3Kd-selective inhibitor idelalisib (CAL-101) has beenapproved for the treatment of some B cell malignancies(Vanhaesebroeck and Khwaja, 2014). However, in thetreatment of chronic lymphocytic leukemia, side effectssuch as leukopenia, colitis, and skin rashes may developwith idelalisib (Brown, 2015; Louie et al., 2015). There isa good rationale for combining inhibition of PI3Kg andd, and several combined PI3Kg/d inhibitors are indevelopment, such as IPI-145 (duvelisib), which iseffective in several inflammation and autoimmuneanimalmodels and is now in early clinical development(Winkler et al., 2013). To overcome dose-limiting sideeffects for the treatment of asthma and COPD, theseinhibitors have been developed for inhaled delivery. Acombined PI3Kg/d inhibitor TG100-115 is effective inmodels of allergic asthma and cigarette smoke-inducedinflammation in mice and is not in clinical development(Doukas et al., 2009). There is a good rationale for thiscombination because PI3Kg inhibition should be anti-inflammatory and inhibit cell trafficking into the lungs,whereas PI3Kd inhibition should be effective in reversingcorticosteroids resistance in COPD and severe asthma.InhaledPI3Kd inhibitors, suchasGSK2269557 [2-(6-(1H-indol-4-yl)-1H-indazol-4-yl)-5-((4-isopropylpiperazin-1-yl)methyl)oxazole hydrochloride], are also in developmentfor the treatment of COPD and severe asthma (Wilsonet al., 2013). Another approach to inhibition of PI3Kd isthe use of existing therapies. Theophylline was shown toincreaseHDAC2 and reverse corticosteroids resistance inmacrophages form COPD patients (Ito et al., 2002; Cosioet al., 2004) and is effective through the selective in-hibition of oxidant-activated PI3Kd in relatively lowconcentrations that are lower than those used forits bronchodilator effect (To et al., 2010). A clinicaltrial of low-dose theophylline combined with inhaled

corticosteroids is now underway in COPD patients(Barnes, 2013c; Devereux et al., 2015). Nortriptyline, atricyclic antidepressant, and solithromycin, a novelmacrolide antibiotic, are also effective in reversingcorticosteroid-resistance in COPD cells through inhibit-ing PI3Kd (Mercado et al., 2011a; Kobayashi et al., 2013).

2. Akt-Mammalian Target of Rapamycin Inhibitors.Akt is a downstream target of PI3K activation, andseveral oral inhibitors have been developed for use incancer therapy. Perfosine blocks p-Akt and is in severalclinical trials for malignancy, but this drug appears toalso target several other pathways, leading to thedevelopment of more selective inhibitors (Alexander,2011). Rapamycin (sirolimus) is a macrolide that bindsto FK506-binding protein-12, which functions as anallosteric inhibitor of mTORC1. It is already in clinicaluse as an immunosuppressant in transplantation andautoimmunity, although it has frequent and severeadverse effects that limit its chronic use. There isparticular interest in finding less toxic analogs ofrapamycin with improved pharmacokinetics (rapalogs),because rapamycin extends life span in mammalsby targeting cellular senescence and autophagy(Wilkinson et al., 2012; Lamming et al., 2013). Indeed,a recent clinical study suggests that a rapalog ever-olimus is able to reduce immunosenescence in elderlypeople (Mannick et al., 2014). Results in cancer therapyhave been disappointing (Chiarini et al., 2015), and thismay reflect the fact that blocking mTOR, which reducesS6K activation, results in blocking of a feedback in-hibitory loop that results in PI3K-Akt activation inmalignant cells. The reduction in autophagy by rapa-mycin may also work against the killing of malignantcells. Dual inhibitors of mTORC1 and 2, such asAZD2014 [3-(2,4-bis((S)-3-methylmorpholino)pyrido[2,3-d]pyrimidin-7-yl)-N-methylbenzamide], may bemore effective because this blocks the feedback loopwhere mTORC2 activates Akt (Dienstmann et al.,2014). This has suggested that dual inhibitors of PI3Kand mTOR may be more beneficial, because this avoidsthe PI3K-Akt feedback loop. There is close structuralhomology between these kinases, and several dualinhibitors have been developed and are now in clinicaltrials (Chiarini et al., 2015). None of these treatmentshas so far been tested in airway disease. However,rapamycin is very effective in inhibiting mTOR activa-tion in COPD lung and PBMC, which results in reducedcorticosteroids resistance (Mitani et al., 2015). In addi-tion, rapamycin is effective in suppressing allergicinflammation in sensitized mice and inhibits eosinophildifferentiation (Mushaben et al., 2013; Hua et al., 2015).Interestingly, rapamycin switches the phenotypeof lymphocytes from Th2 to regulatory T cells (Foxp3+)(Kim et al., 2010).

3. Activating Endogenous Inhibitors. Adenosinemonophosphate-activated kinase (AMPK) is an impor-tant regulator of PI3K signaling through the inhibition

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of mTORC1 via phosphorylation of the tumor suppres-sor complex TSC1/2 and plays an important role in theregulation of metabolism, cell energetics, andmitochon-drial function, but may also have antiaging effectsthrough increasing sirtuin-1 and anti-inflammatoryeffects (Steinberg and Kemp, 2009). This has prompteda search for AMPK activators, which should have valuein asthma and COPD (Scarpulla, 2011; Salt andPalmer, 2012). Metformin, a biguanide, is commonlyused to treat type 2 diabetes and works partly throughactivation of AMPK via changes in nucleotide levels.Experimentally it has been shown to extend life span inmice (Martin-Montalvo et al., 2013). It also has poten-tial as an anti-inflammatory drug (Salt and Palmer,2012). Metformin and the more specific AMPK activator59-aminoimidazole-4-carboxamide-1-b-4-ribofuranosidereduce allergic inflammation in a chronic allergic mousemodel, and this is reproduced in AMPK-deficient(AMPKa12/2) mice (Park et al., 2012). 59-Aminoimida-zole-4-carboxamide-1-b-4-ribofuranoside inhibits ciga-rette smoke-induced release of CXCL8 from A549 cells,and AMPKa12/2mice have reduced emphysema aftercigarette smoke exposure, indicating that activation ofthis pathway has therapeutic potential in asthma andCOPD (Lee et al., 2015). Flavones, such as quercitin,and resveratrol also activate AMPK. Resveratrol hasanti-inflammatory effects in COPD airway epithelialcells, although this may be mediated through severalmechanisms (Donnelly et al., 2004). More direct activa-tors of AMPK are now in development, such as thecompound A-769662, which is entering clinical trials fordiabetes (Goransson et al., 2007).Another approach is through activation of the phos-

phatases PTEN and SHIP, which inhibit PI3K signal-ing. It has been difficult to identify selective activatorsof PTEN, which may reflect the complexity of its regu-lation and may interactions (Song et al., 2012). SHIP ismore amenable to activation and a small moleculeactivator of SHIP-1, AQX-1125, and is effective inreducing p-Akt and reducing mediator release, mastcell activation, and leukocyte chemotaxis in vitro(Stenton et al., 2013b) and inflammation in allergen-induced and cigarette smoke-induced pulmonary in-flammation in mice in vivo (Stenton et al., 2013a). Arecent clinical trial of oral AQX-1125 in patients withmild asthma showed a significant reduction in laterresponse after allergen challenge and a (nonsignificant)reduction in inflammatory biomarkers (Leaker et al.,2014). The drug is well tolerated, suggesting that itmight be a potential treatment of asthma and COPD.Another downstream inhibitor of PI3K signaling

pathways is glycogen synthase-3b (GSK3b), which isconstitutively active but inhibited by phosphorylationby Akt and ERK1/2 as a result of oxidative stress(Takahashi-Yanaga, 2013). Inactivated GSK3b(p-GSKb-Ser9) is increased in PBMC, macrophages,and airway epithelial cells of patients with COPD,

especially those with severe disease (Ngkelo et al.,2015). This is associated with corticosteroid resistanceas a result of reduced HDAC2 in these cells, which ismimicked by GSK3b knockdown and a GSK3b inhibitorCT99021 [6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile]. This suggests that GSK3bactivators may be beneficial in reversing corticosteroidresistance, but it has been difficult to identify suchcompounds. In addition, GSK3b regulates many differ-ent signaling pathways, including NF-kB, and itsactivation might amplify inflammation.

VII. Janus-Activated Kinase Inhibition

The Janus kinase (JAK)-signal transducer of activa-tors of transcription (STAT) signaling pathway regu-lates the expression of multiple inflammatory andimmune proteins and has been implicated in manyinflammatory diseases (O’Shea et al., 2015). This hasled to the development of JAK inhibitors (Jakinibs),which are a new class of anti-inflammatory drug nowused for the treatment of inflammatory diseases, suchas rheumatoid arthritis, autoimmune diseases, hema-topoietic diseases, and cancer, with several inhibitors inclinical development and some already in clinical use(Kontzias et al., 2012; Menet et al., 2013). Afteroccupation of the cytokine receptor, the associated JAKsbecome activated and both phosphorylate each otherand the intracellular tail of their receptors, thuscreating docking sites for cytoplasmic STATs, which inturn directly bind as dimers to DNA and regulate geneexpression. There are four JAKs (JAK1-3) and Tyk2,which selectively bind different receptor chains (Fig. 4).The selective usage of JAKs by different receptorsexplains their distinct in vivo roles. There are sevenSTATs (STAT1, 2, 3, 4, 5a, 5b, 6) that are activated bydifferent combination of JAKs, but JAKs may alsodirectly affect genes through histone phosphorylation,which changes chromatin structure to influence geneexpression. Various polymorphisms of STAT-encodinggenes have been described in cancer and inflammatoryand immune diseases.

A. Role in Asthma and Chronic Obstructive Pulmo-nary Disease. There is recent evidence that JAKs areinvolved in asthma and COPD. Many of the cytokinesinvolved in asthma, including the Th2 cytokines (IL-4,IL-5, IL-9, and IL-13), GM-CSF, and thermic stromallymphopoietin, activate JAK-STAT signaling (Pernisand Rothman, 2002; Ghoreschi et al., 2009). Thereceptors for these cytokines involve activation of allof the JAKs, suggesting that a broad spectrum inhibitoris more likely to be useful. JAK3 expression is restrictedto hematopoietic cells and colocalizes with JAK1, trans-mitting signals through the common g-chain of thecytokine receptors for IL-2, IL-4, IL-7, IL-9, IL-15, andIL-21. Tofacitinib and pyridone-6, which are pan-JAK


inhibitors, are effective and reduce AHR in a murinemodel of allergic inflammation (Kudlacz et al., 2008;Matsunaga et al., 2011). There has been concern thatblocking JAK2 may have inhibitory effects on hemato-poiesis, so there has been a search for more selectiveinhibitors. JAKs play a key role in differentiation of Th2cells and a JAK1/3 inhibitor (R256) blocked the differ-entiation of Th2 cells and the phosphorylation of STAT5and STAT6, but not of Th1 or Th17 cells in vitro (Ashinoet al., 2014). In vivo R256 inhibited allergic inflamma-tion, AHR, and mucus hypersecretion in a mouse modelwithout any changes in Th2 cytokine concentrations.JAKs are also involved in the mechanisms of COPD

and several cytokines implicated in COPD activateJAK-STAT signaling, including IL-6, IL-12, andinterferon(IFN)-g, which activate JAK1, JAK2, andTyk2. In mice with hyperreactivity of the receptor forIL-6 (gp130), there was an increase in STAT3 andpulmonary inflammation that were prevented inSTAT3 knockout mice (Ruwanpura et al., 2012). Fur-thermore STAT3 was increased in the lungs of COPDpatients and correlated with inflammation but not withairway obstruction. STAT1 and STAT3 are phosphory-lated in the lungs of severe COPD patients to a greaterextent than in smokers and nonsmokers, indicatingactivation of several JAKs (Yew-Booth et al., 2015).STAT4 is also increased in the airway of COPDpatients, presumably in response to IL-12 or IL-23 (DiStefano et al., 2004). The chemokines CXCL9, CXCL10,and CXCL11 are all increased in the airways of COPDpatients and target a common receptor, CXCR3, whichis involved in the recruitment of T-cells (particularlyCD8+ cells) into the lung (Costa et al., 2008). These

chemokines are induced by IFNg through the activa-tion of JAKs and phosphorylation of predominantlySTAT1 (Tudhope et al., 2007). Pan-JAK inhibitorsare effective in reducing IFNg-induced secretion ofCXCR3-activating chemokines from human airwayepithelial cells, demonstrating their potential as anti-inflammatory treatments in COPD (Fenwick et al.,2015).

B. Jakinibs. There is a good rationale for the use ofJAK inhibitors in both asthma and COPD, because theymediate the effects of several mediators involved inthese diseases. Several oral JAK inhibitors (jakinibs)are now approved and in clinical trial for the treatmentof other inflammatory diseases, including rheumatoidarthritis, inflammatory bowel disease, and psoriasis(Kontzias et al., 2012; Menet et al., 2013; Schwartzet al., 2016). Over 20 pan-JAK inhibitors and isoform-selective inhibitors have now been developed. Tofaciti-nib is JAK1/3 selective and indicated for rheumatoidarthritis and psoriasis, whereas JAK2-selective inhibi-tors are indicated for hematologic malignancies andmyeloproliferative disease. Ruxolitinib is JAK1/2 selec-tive and indicated for rheumatoid arthritis and myelo-proliferative disease. However, the selective JAKinhibitors are not specific so tend to affect other JAKs,increasing the risk of side effects. Thus pan-JAKinhibitors may cause anemia, neutropenia, and thom-bocytopenia through inhibition of JAK2, which medi-ates the effects of erythropoietin, thrombopoietin, andGM-CSF. Side effects of jakinibs after systemic admin-istration are dose limiting and include increased infec-tions, diarrhea, increased cholesterol levels, andlymphomas, so a strategy to reduce these adverse

Fig. 4. JAK-STAT signaling pathways. Janus-activated kinases (JAK1-3, Tyk2) are activated by several extracellular stimuli and phosphorylateseveral signal transducer of activators of transcription (STAT) proteins, which translocate to the nucleus and activate target genes resulting in cellsurvival, activation of antiviral mechanisms, inflammation, and Th1 and Th2 immunity.

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effects is to deliver the drugs topically. Thus, topicaltofacitinib and ruxolitinib have been developed for skinapplication for the treatment of psoriasis. Inhaled pan-JAK inhibitors, such as LAS194046 and R256, are nowin development for asthma and COPD and have shownefficacy in models of allergic asthma (Ramis et al.,2014). An inhaled pan-JAK inhibitor (VR560) markedlyreduced the allergic inflammatory response in micesensitized and exposed to chronic house dust mites,inhibited Th2 cytokines and IL-17, and inhibitedSTAT3 phosphorylation (Wiegman et al., 2015). Thissuggests that inhaled pan-JAK inhibitors may havegood potential in asthma and COPD and clinical trialsare now in progress.

VIII. Spleen Tyrosine Kinase Inhibition

Spleen tyrosine kinase (Syk) is a nonreceptor cyto-plasmic protein tyrosine kinase that is primarilyexpressed in hematopoietic cells (particularlyB lymphocytes) and links immune cell receptors tomultiple intracellular signaling pathways that regulatemany cellular responses, including cytokine release andcell survival (Mocsai et al., 2010; Riccaboni et al., 2010).Syk appears to have many diverse functions, includinginnate and adaptive immunity, cell adhesion, and organdevelopment and has been implicated in many autoim-mune, inflammatory, and malignant diseases. Immu-noreceptors include B-cell and T-cell receptors andFc-receptors, which associate with transmembraneproteins that have immunoreceptor-tyrosine-based ac-tivation motifs (ITAMs), which are short peptide

sequences containing two tyrosine residues 6–12 aminoacids apart. ITAM tyrosines are phosphorylated afterligand binding, resulting in the recruitment and acti-vation of Syk or the related enzyme z-activated proteinkinase of 70 kDa, which results in recruitment of otherkinases and the activation of downstream signalingpathways, includingNF-kBand PI3K pathways (Fig. 5).

A. Role in Asthma and Chronic Obstructive PulmonaryDisease. In a chronic allergic murine model of asthmathere was an increase in Syk activation in epithelial cells,and a Syk inhibitor [N-[4-[6-(cyclobutylamino)-9H-purin-2-ylamino]phenyl]-N-methylacetamide (NVP-QAB-205)]significantly reduceAHRafter allergen and the enhancedAHR after additional exposure to diesel particulates andozone, although there was no reduction in inflammatorycells (Penton et al., 2013). A Syk inhibitor [6-(5-fluoro-2-(3,4,5-trimethoxyphenylamino) pyrimidin-4-ylamino)-2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (R406)]also prevents activation of sensitized mast cells anddendritic cells from mice, with a reduction in AHR andallergic inflammation in vivo (Matsubara et al., 2006a,b).Similar effects are seen with an inhaled Syk antisense,indicating the specificity of the small molecule Sykinhibitors (Stenton et al., 2000, 2002). An oral inhibitor[2-(7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino)-nicotinamide HCl (Bay 61-3606)] is also effec-tive in reducing the allergic reaction to inhaled allergensin rats and also in preventing passive cutaneous anaphy-laxis and mast cell activation (Yamamoto et al., 2003).This Syk inhibitor also inhibits the activation of humanbasophils. The reduction in AHR seen after a Sykinhibitor may be due to an effect on airway smooth

Fig. 5. Spleen tyrosine kinase signaling. Syk is activated by immune receptors via phosphorylation by Lyn kinase and may activate PI3K orphospholipase Cg, resulting in activation of immune cells, such as mast cells, B-lymphocytes, and T-lymphocytes. Syk may also be activated byintegrins. Several Syk inhibitors have been identified (shown in green box).


muscle contractility, because Syk-deficient mice do notdevelop AHRafter allergens and the contractility of smallairways to spasmogens is reduced (Wang et al., 2015).This is mimicked by treatment with a Syk inhibitor (Moyet al., 2013). Syk is also activated by infection of humanairway epithelial cells after rhinovirus binding to ICAM-1and results in the activation of p38 MAPK and PI3Kactivation, with release of mediators such as CXCL8 andVEGF and so may play a role in acute exacerbations ofboth asthma and COPD (Lau et al., 2008). Syk alsoregulates internalization of rhinovirus via clathrin-mediated endocytosis, which involves ITAMs previouslyonly associated with immune cells (Lau et al., 2011).B. Spleen Tyrosine Kinase Inhibitors. Because of

the key role of Syk in mast cell and immune cellsignaling, the effects of Syk inhibition on the allergicinflammatory response, AHR, and the inhibition ofrhinovirus signaling there is a persuasive case fordevelopment of Syk inhibitors (Geahlen, 2014). Severalorally administered Syk inhibitors have been developedfor the treatment of rheumatoid arthritis and B cellleukemias (Norman, 2014b; Thorarensen and Kaila,2014) For example, fostamatinib (R788) has shownefficacy in rheumatoid arthritis patients (but not thoserefractory to biologics), and the most frequent adverseeffects are gastrointestinal symptoms, hypertension,and neutropenia, but no serious infections (Nijjaret al., 2013). To avoid side effects, topical formulationshave been developed. A nasal spray formulation of R112was weakly effective in patients with seasonal allergicrhinitis exposed to outdoor allergens (Meltzer et al.,2005) and transiently decreased the concentration ofPGD2 in nasal lavage after nasal allergen challenge buthad no effect on either histamine or mast cell tryptase(Guyer et al., 2006). A novel Syk inhibitor suitable forinhalation, LAS189386 [1-{2-[(1S,4S)-2,5-diazabicyclo[2.2.1]hept-2-yl]pyridin-4-yl}-N-pyrazin-2-yl-1H-indazol-3-amine], delivered to ovalbumin-sensitized BrownNorway rats showed good inhibition of the early re-sponse to allergen and of mast cell degranulation,without any effects on systemic mast cells (Ramiset al., 2015), and so is in clinical development forasthma.

IX. Receptor Tyrosine Kinases

Growth factors are cytokines that induce structuralchanges, such as fibrosis and tissue remodeling, and acton high-affinity surface receptors that have tyrosinekinase activity. Of the 90 human protein tyrosinekinase genes, over half encode receptor tyrosine kinases(RTK) (Hubbard and Till, 2000). RTKs are important innormal tissue growth, development, and repair afterinjury and have been closely linked to cancers. MostRTKs function as single unit receptors, although some(e.g., insulin receptor) are multimeric. An N-terminalextracellular domain interacts with the protein ligand

(growth factor or hormone), and the C-terminalintracellular domain contains a relatively well-conserveddomain that catalyzes receptor autophosphorylation withreceptor dimerization and phosphorylation of RTK sub-strates, which include proteins with Src homology-2domains and phosphotyrosine domains (Lemmon andSchlessinger, 2010). There are several distinct familieswith RTKs, but those most relevant to airway disease arethe epidermal growth factor receptor (EGFR, ErbB)family, fibroblast growth factor receptor (FGFR) family,vascular-endothelial growth factor receptor (VEGFR)family, and platelet-derived growth factor receptor(PDGFR).


EGFR play an important role in mediating mucushypersecretion and mucus metaplasia, with goblet cellproliferation in both asthma and COPD (Nadel andBurgel, 2001). EGFR shows increased expression inairway epithelial cells of asthmatic airways and isassociated with CXCL8 release and neutrophilic in-flammation (Hamilton et al., 2003). EGFR plays apivotal role in mucus hypersecretion (chronic bronchi-tis) in COPD through increased expression of MUC5ACandmay be activated by ligand such as TGF-a that maybe generated by neutrophilic inflammation and throughnonligand mechanism such as oxidative stress(Takeyama et al., 2000; Takeyama et al., 2001; Shaoand Nadel, 2005). There is increased expression ofEGFR in airway epithelia cells of COPD airways(de Boer et al., 2006).

Basic fibroblast growth factor-2 (bFGF) also stimu-lates airway smooth muscle proliferation and acts inconcert with other growth factors (Bosse and Rola-Pleszczynski, 2008). Chronic allergen exposure in sensitizedmice induces bFGF expression in airway macrophages andis associated with airway remodeling, both of whichwere reduced after treatment with a corticosteroid(Yum et al., 2011).

Increased angiogenesis contributes to the structuralremodeling in asthma, with increased numbers ofbronchial vessels, which show increased leakiness(Paredi and Barnes, 2009). These effects are largelymediated by VEGF secreted from airway epithelial cellsand airway smooth muscle cells, T cells and eosinophils(Hoshino et al., 2001). There is increased expression ofVEGF, VEGF receptors, and angiogenin-1 in airways ofasthmatic patients that correlates with increased vas-cularity (Feltis et al., 2006). By contrast, VEGF levelsare reduced in COPD sputum and lung parenchyma(Taraseviciene-Stewart et al., 2006; Kanazawa et al.,2003) and may be linked to the development of emphy-sema through apoptosis of endothelial cells. VEGFlevels are reduced in the epithelia lining fluid ofperipheral airways of patients with COPD and normalsmokers (Kanazawa et al., 2014).

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The platelet-derived growth factor (PDGF) familyincludes the isoforms A-D, which may exist as homo- orheterodimers and activate the receptors PDGFR-a and -b,which dimerize. (PDGF)-BB is a potent stimulator ofairway smooth muscle proliferation (Hirst et al., 1996)and may act synergistically with other growth factorssuch as endothelin-1 (Yahiaoui et al., 2006). The pro-liferative effect of PDGF is mediated via activation ofSTAT-3 and the small GTPase Rac-1 (Simeone-Penneyet al., 2008). Overexpression of PDGF-BB in airwayepithelial cells induces airway smooth muscle (ASM)proliferation and airway hyperresponsiveness in amousemodel (Hirota et al., 2011). Chronic allergen exposureinduces secretion of PDGF into BAL fluid in this model.

B. Receptor Tyrosine Kinase Inhibitors

Several small molecule EGFR inhibitors, such asgefitinib and erlotinib, have been developed for thetreatment of solid cancers, including lung cancers,where mutations of EGFR are commonly seen. Exper-imental studies with EGFR reduces mucus secretion.An EGFR inhibitor AG1478 [N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinamine] is effective at inhibitingMUC5AC secretion induced by LPS, TNF-a, or cigarettesmoke from human airway epithelial cells in vitro andin cigarette smoke-induced MUC5AC secretion in ratsin vivo (Hegab et al., 2007; Takezawa et al., 2016).However, a potent and selective EGFR inhibitor BIBW2948 given by inhalation for 4 weeks in patients withCOPD failed to show any reduction in epithelial mucus,despite evidence for target engagement measured byEGFR internalization (Woodruff et al., 2010). In addi-tion, there was a reduction in lung function, and liverenzyme elevation was seen in some patients. VEGFRinhibitors, such as cediranib, have been developed forcertain solid cancers but have not been tested in asthmaand would be contraindicated in COPD because experi-mental VEGFR inhibitors have induced emphysema.Several drugs inhibit PDGFR and usually inhibit othertyrosine kinases. For example, masitinib targets PDGFRand c-Kit and tyrosine kinases, improves asthma controlin severe asthma patients, and reduces the requirementfor oral corticosteroids, although it was associated withskin rashes and edema (Humbert et al., 2009). Ninteda-nib inhibits PDGFR, but also VEGFRandFGFR, and hasbeen approved for pulmonary fibrosis but has not beenstudied in airway disease (Keating, 2015). Similarly,imatinib inhibits PDGFR and c-Kit, as well as ABL-kinase, the key target in chronic myeloid leukemia, buthas not been studied in asthma or COPD.

C. c-Kit Inhibitors

c-Kit (also known as CD117 and SCFR) is a proto-oncogene, a tyrosine kinase receptor for stem cell factor,which is an essentialmast cell growth factor. It is encodedby the KIT gene and expressed on hematopoietic pro-genitor cells and mature mast cells (Lennartsson and

Ronnstrand, 2012). Activating mutations of KIT areassociated with mastocytosis, with an accumulation ofmast cells in tissues. Silencing of c-Kit with a smallinterference RNA reduces the inflammatory response toallergen in amurinemodel of asthma, with a reduction inTh2 cytokines and eosinophils (Wu et al., 2012). Imatinib,an ABL-kinase inhibitor for chronic myeloid leukemia, isalso an inhibitor of c-Kit tyrosine kinase activity and iseffective in a mouse model of asthma with hypersensitiv-ity to cockroach allergen (Berlin and Lukacs, 2005). Thissuggests that more selective c-Kit inhibitors may beuseful in the treatment of asthma (Reber et al., 2006).As discussed above, masitinib inhibits c-Kit and hasproduced benefits in asthma. Other receptor tyrosinekinases, such as sunitinib and nilotinib, have beendeveloped for cancer therapy and may also be effectivebut have dose-limiting side effects (von Mehren, 2006).

X. Transforming Growth Factor-bReceptor Kinase

Transforming growth factor(TGF)-b is a member of alarge superfamily, which includes bone morphogenicproteins, activins, and inhibins. It plays an importantrole in tissue repair, proliferation, and cell differentia-tion (Massague, 2012). It is implicated in several chroniclung diseases, including asthma and COPD, where it isinvolved in tissue remodeling (Aschner and Downey,2016). It is often associated with tissue fibrosis throughthe induction of the growth factor connective tissuegrowth factor (also known as CCN2) (Ihn, 2002). TGF-balso plays an important role in immunoregulation and issecreted by subtypes of regulatory T cells (Levings et al.,2002). There are three isoforms of TGF-b (TGF-b1, TGF-b2, TGF-b3), secreted noncovalently bound to latency-associated peptide, which dissociate to release activeTGF-b with acidification, oxidative stress, certain integ-rins, and the action of MMP-2 and MMP-9, so that TGFbis activated at sites of inflammation (Yu and Stamenkovic,2000). Activated TGF-b binds to activate TGF-b type I(T-bRI) via phosphorylation by T-bRII, which has Ser/Thr kinase activity. Phosphorylation of T-bRI thenrecruits Smad proteins, enabling Smad phosphorylationand nuclear translocation, resulting in activation ofvarious genes. There are eight Smad proteins in humans,including receptor-Smads (1, 2, 3, 5, 8), co-Smad4, andinhibitory Smads (6, 7). TGF-b signaling may also beactivated in a Smad-independent manner by MAPkinases, PI3K and Src-family kinases.


TGF-b plays a key role in the airway remodeling ofasthma (Aschner and Downey, 2016). TGF-b1 levels areincreased in asthmatic airways and there is increasedTGF-b in the submucosa of patients with asthma(Kokturk et al., 2003). TGF signaling may promote


ECM deposition, airway smooth muscle proliferation,and collagen deposition after allergen challenge. TGF-bmay be derived from eosinophils, mast cells, macro-phages, epithelial cells, or fibroblasts (Minshall et al.,1997; Halwani et al., 2011). It is increased particularlyin severe asthma (Balzar et al., 2005). TGF-b inducesproliferation of human airway smooth muscle cells viaactivation of multiple kinases, including PI3K, ERK,JNK and by NF-kB activation (Goldsmith et al., 2006).There is increased expression of TGF-b in epithelial

cells from small airways of COPD patients (Takizawaet al., 2001; Wang et al., 2008) and this may becompounded by downregulation of inhibitory Smads(Smad6, Smad7) as a result of exposure to cigarettesmoke (Springer et al., 2004). TGF-b is also expressed inmacrophages in the walls of small airways of patientswith COPD (de Boer et al., 1998). Oxidative stress is apotent stimulus to increased TGF-b activation andexpression (Koli et al., 2008) and TGF-b is activatedfrom its latent precursor byMMP-9, which is secreted inCOPD (Yu and Stamenkovic, 2000; Dallas et al., 2002).

B. T-bRI Kinase Inhibitors

Several small molecule inhibitors of T-bRI kinase havebeen developed, such as SB-431542 [4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide] andSB-505154 [2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride], whichinhibit TGF-b signaling and Smad activation (Lapinget al., 2002; DaCosta Byfield et al., 2004). For example,these drugs are effective in the bleomycin model of lungfibrosis (Koh et al., 2015). In a rat model of chronicallergen exposure, a T-bRI kinase inhibitor, SD-208 [2-(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-yl-amine],reduced airway hyperresponsiveness and collagen de-position, but there was no reduction in airway smoothmuscle proliferation (Leung et al., 2006). However, aT-bRI kinase inhibitor was effective in reducing humanASM proliferation in vitro (Xie et al., 2007). Clinicaldevelopment of these inhibitors has proven problematicbecause of adverse effects, so no clinical trials appear tohave been conducted. A T-bRI kinase inhibitor, galuni-sertib, was entered into clinical trials for cancers but hadmarked cardiac toxicity that has slowed further develop-ment (Herbertz et al., 2015). Inhibiting TGF-b signalingmay have unwanted effects through inhibiting regulatoryT cells activity, so that targeting fibrosis may be betterachieved by targeting the downstream target connectivetissue growth factor (Jun and Lau, 2011).

XI. Protein Kinase C Inhibition

Protein kinase C (PKC) is a family of protein kinasesthat regulate a variety of downstream proteins via Ser/Thr phosphorylation. PKCs are activated by diacylgly-cerol, generated via phospholipase C activation, byphospholipids, or by calcium ions to regulate signal

propagation within cells (Rosse et al., 2010). There are15 subtypes of PKC, organized into three main families,determined by the method of activation. On activation,PKCs translocate from the cytoplasm to the cell mem-brane, where they remain activated for a long time andmediate myriad functions, including cell proliferation,receptor activation, and inhibition and contraction andproliferation of smooth muscle.


PKCs have multiple signaling roles relevant toairway disease (Dempsey et al., 2007). PKC isoformsare involved in bronchoconstriction, as well as pro-liferation of airway smoothmuscle cells. PKC activationby the phorbol ester phorbol 12,13-dibutyrate causes aslowly developing and sustained contraction of humanairway smooth muscle that is blocked by the PKCinhibitor staurosporine (Rossetti et al., 1995). SeveralPKC isoforms are detectable in ASM (Webb et al., 1997;Donnelly et al., 1995). PKC-z plays a role in theproliferation of ASM cells in response to PDGF (Carlinet al., 2000). PKC activation in response to phorbolesters and acetylcholine results in uncoupling ofb2-receptors in ASM, because diacylglycerol mediatesphosphorylation of the b2-receptor and the stimulatoryG-protein Gs (Grandordy et al., 1994). PKC is involvedin the activation of murine Th2 cells through thephosphorylation and activation of voltage-gated cal-cium channels (CaV1.1-1.4) and PKC inhibition inhibitscytokine release from Th2 cells (Robert et al., 2014).PKC-d is known to activate mTOR and suppression ofPKC-d by the inhibitor rottlerin inhibits PI3K-mTORsignaling after allergen exposure in a murine model ofasthma (Choi et al., 2013). PKC-d is involved in theactivation of mast cells via IgE-receptors and regulatesthe synthesis and secretion of cysteinyl-leukotrienes(Cho et al., 2004a,b). PKC-u is involved in activation ofT-cells and IL-2 secretion so its inhibition causesimmunosuppression (Chand et al., 2012). PKC-d andPKC-z are involved in eosinophil migration (Langloiset al., 2009). Allergen challenge in patients with asthmaresults in long-term activation of PKC-z in eosinophils(Evans et al., 1999). PKC is also involved in the mucussecretion response and expression of MUC5AC afterexposure to human neutrophil elastase (Shao andNadel, 2005).

B. Protein Kinase C Inhibitors

The multiple roles of PKC are bronchoconstriction,inflammatory cell activation, mucus secretion, andremodeling and suggest that PKC inhibitors may havetherapeutic potential in themanagement of asthma andCOPD. Nonselective inhibitors, such as staurosporine,are too toxic for human administration, but severalisoform-selective PKC inhibitors have been developedand entered clinical trials in various diseases, including

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cancer, cardiovascular disease, diabetes, transplanta-tion, and neuropsychiatric diseases (Mochly-Rosenet al., 2012). Most clinical trials have been negativeand there have been several unexpected adverse effects.No clinical trials have been reported in asthma andCOPD, and there are no PKC drugs currently approvedfor human therapy.

XII. Other Kinase Targets

Several other kinases may be potential therapeutictargets because they are involved in chronic inflamma-tion or have effects on ASM.

A. Rho-Associated Protein Kinase Inhibition

Rho-associated protein kinases (ROCK1 and ROCK2)are Ser/Thr kinases that are mainly involved in regulat-ing cytoskeletal function and thus the shape and move-ment of cells (Riento and Ridley, 2003). ROCK istherefore important in migration of inflammatory cellsto sites of inflammation. ROCK1 is the major down-stream target of the small GTPase RhoA. ROCK1 andROCK2 phosphorylate actin and result in myosin lightchain phosphorylation, resulting in contraction and in-creased contractility of smooth muscle. Thus ROCKplays a key role in determining contractility in bovineASM (Gosens et al., 2004). ROCKs are thought tomediate some of the pleiotropic effects of statins.The ROCK inhibitor Y-27632 administered by inhala-

tion inhibits eosinophilic inflammation, AHR, and air-way remodeling after chronic allergen exposure insensitized guinea pigs (Schaafsma et al., 2008a; Possaet al., 2012; Righetti et al., 2014). This drug inhibitsAHR, bronchoconstriction, and eosinophilic inflamma-tion in a mouse model of acute allergic inflammation(Henry et al., 2005). Another ROCK inhibitor, fasudil,reduces airway inflammation and AHR induced by ozoneexposure inmice (Kasahara et al., 2015). There is growingevidence that ROCK inhibitors inhibit fibrosis and somayreduce airway remodeling in airway diseases (Knipe et al.,2015). This suggests that ROCK inhibitors might beuseful as bronchodilators and anti-inflammatory andantifibrotic therapy, but clinical development hasbeen slow (Fernandes et al., 2007; Schaafsma et al.,2008b). Y-27632 [(R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] is notvery selective for ROCK and may inhibit other kinases. Ithas not been developed clinically because of toxicity.Fasudil is a more selective ROCK inhibitor that has beenapproved in Japan for the treatment of cerebral vaso-spasm. There are no reported clinical studies in asthma orCOPD. It is possible that there may be a positive in-teraction between a ROCK inhibitor and existing bron-chodilator therapy, but there are no studies exploring this.

B. Interleukin-1 Receptor-AssociatedKinase Inhibition

IL-1 receptor-associated kinases (IRAKs) are impor-tant enzymes in the signaling pathways from IL-1 andIL-18 receptors and from TLRs and are thereforeupstream of NF-kB and MAPK activation. Of the fourIRAKs identified, IRAK-4 appears to be the mostimportant in inflammation and is therefore a potentialtarget for anti-inflammatory therapy (Wang et al.,2009). In humans, mutations in IRAK-4 have beenassociated with susceptibility to bacterial infections.Small molecule IRAK-4 inhibitors are in clinical devel-opment and have been facilitated by the discovery of thecrystal structure of IRAK-4 (Chaudhary et al., 2015). Anaturally occurring compound lancemaside C inhibitsmacrophage activation induced by LPS via TLRthrough inhibition of IRAK-4 (Joh and Kim, 2010). AnIRAK-4 inhibitor is effective in suppressing release ofchemokines form airway epithelial cells of patients withCOPD in vitro (Fenwick et al., 2013). To date no clinicalstudies with IRAK-4 inhibitors have been reported.

C. Src Family Kinase Inhibition

Proto-oncogene tyrosine protein kinase Src is a non-receptor tyrosine kinase linked to cell proliferation andmalignancy but also involved in smoothmuscle functionand inflammation and may be activated by growthfactors, adhesion molecules, and oxidative stress(Roskoski, 2015). Src family kinases play a key role insensitizing airway smoothmuscle andmay play a role inAHR via activation of ROCK and is reversed by the Srcinhibitor PP2 (Shaifta et al., 2015). TNF-a disruptsairway epithelial barrier function and this is mediatedvia Src activation and prevented by a Src inhibitorSU6656 [2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide] (Hardyman et al., 2013). The Src inhibitorPP2 and siRNA knockdown of Src prevents rhinovirus-induced release of CXCL8 from human airway epithelialcells (Bentley et al., 2007). Analysis of an asthma proteininteractome suggests that Src plays a central role andmay be a good target to inhibit (Randhawa and Bagler,2012). Several Src inhibitors, such as dasatinib, havebeen developed for the treatment of hematologic malig-nancies but inhibit other kinases and have frequent sideeffects and have not been tested in airway disease.

D. Hematopoietic Cell Kinase Inhibition

Hematopoietic cell kinase (Hck), a Src family kinase, isa myeloid-specific nonreceptor tyrosine kinase involvedin neutrophil production, adhesion and cytokine pro-duction. Hck overexpression in mice induces chronicneutrophilic inflammation and emphysema (Ernst,2002). Hck is involved in neutrophil and myeloid cellrecruitment into the lung, because Hck knockout micefail to recruit these cells in response to TNF-a and LPS


(Mazzi et al., 2015). There is increased expression of Hckin circulating neutrophils from patients with COPD(Yanagisawa et al., 2009), suggesting that this kinasemay be a good target for inhibition. Several drugs thatinhibit Hck, including PP2 and dasatinib, are available,and new drugs are in development (Poh et al., 2015).Inhaled drugs that have inhibitory effects onHck are alsoin clinical development for the treatment of severeasthma and COPD (Norman, 2014a; Onions et al., 2016).

E. Bruton’s Tyrosine Kinase

Bruton’s tyrosine kinase (BTK) pays a critical role inB-lymphocyte development and in mast cell activationthrough high-affinity IgE receptors (Crofford et al., 2016).Mutations of BTK are associated with X-linked agamma-globulinemia, which causes immunodeficiency. SelectiveBTK inhibitors, such as ibrutinib, have been developed fortreatment of B cell malignancies, but so far have not beentested in airway disease (Foluso et al., 2016).

XIII. Future Developments

Although many hundreds of kinases have beenimplicated in chronic inflammatory diseases, relativelyfew have been successfully targeted therapeutically.Major problems involve specificity and adverse effects,often due to off-target effects and loss of efficacy overtime. Although over 30 kinase inhibitors have beenapproved for the treatment of various forms of malig-nancy, relatively few are approved for the treatment of

chronic inflammatory diseases and none so far forasthma and COPD therapy (Wu et al., 2015c).

A. Improving Selectivity and Safety

Most kinase inhibitors target the ATP-bindingpocket, where they compete with ATP in high concen-trations, and so need to be of at least nanomolar potencyto be effective. Because ATP binding is common to allkinases, it is difficult to develop kinases with specificityfor a particular kinase because of the great similarity inbinding site between different kinases. The off-targeteffects often account for sometimes unexpected adverseeffects that have severely limited the clinical develop-ment of these drugs. Although this is relatively lessimportant for treatment of life-threatening malignan-cies, it is unacceptable in long-term treatment of chronicinflammatory diseases, such as asthma and COPD.Understanding the cocrystal structure of the ligand-ATP-binding site of particular kinases may aid thediscovery of more selective inhibitors (Norman et al.,2012b). For example, this approach has been used todevelop selective inhibitors of FGFR tyrosine kinase(Norman et al., 2012a). Targeting allosteric bindingsites that lie outside the highly conserved ATP-bindingsite offers hope for greater potency and specificity andmay also protect against the development of drugresistance by mutation of the ATP-binding site (Wuet al., 2015b). Binding may be to an allosteric site closeor distant from the ATP-binding pocket. Over 10 allo-steric kinase inhibitors are now in clinical development

TABLE 1Kinase inhibitors in clinical development

Kinase Inhibitors Development Stage Disease Indication Comments

IKK (NF-kB) IMD-0354 Phase 2? COPD DiscontinuedBay 65-1942 Preclinical COPD Oral

p38 MAPK Dilmapimod Phase 2 COPD OralLosmapimod Phase 2 COPD OralPH-797804 Phase 2 COPD OralPF-03715455 Phase 2 Asthma, COPD InhaledAZD7624 Phase 2 COPD InhaledRV-568 Phase 2 COPD Inhaled

JNK SP600125 Preclinical Asthma, COPD? OralMEK1/ERK Trametinib Phase 3 Cancer OralASK-1 GS-4997 Phase 2 Diabetes OralPI3K Idelisib Approved B cell malignancy Oral PI3Kd-selective

TG100-115 Preclinical Asthma, COPD InhaledPI3Kg/d-selectiveGSK2269557 Phase 2 COPD Inhaled PI3Kd-selectiveRV-1729 Phase 2 Asthma, COPD Inhaled PI3Kg/d-selective

JAK LAS194046 Preclinical Asthma, COPD InhaledR256 Preclinical Asthma, COPD InhaledVR560 Preclinical Asthma, COPD Inhaled

SYK Fostamatinib Phase 3 Arthritis, lymphoma OralR112 Phase 2 Rhinitis Nasal, discontinuedR343 Phase 2 Asthma Inhaled, discontinuedLAS189386 Preclinical Asthma Inhaled

RTK BIBW 2948 Phase 2 COPD Inhaled EGFRMasitinib Phase 2 Severe asthma Oral, PDGFR, c-Kit

ROCK Fasudil Approved Cerebral vasospasm Oral

ASK, apoptosis signal-regulatory kinase; EGFR, epidermal growth factor receptor; ERK, extracellular regulatingkinase; IKK, inhibitor of kB kinase; JAK, Janus-activated kinase; JNK, Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; NF-kB, nuclear factor-kappa B; PDGFR, platelet-derived growth factor receptor; PI3K,phosphoinositide-3-kinase; ROCK, Rho-associated protein kinase; RTK, receptor tyrosine kinase; Syk, spleen tyrosinekinase.

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and somemay be combinedwith conventional inhibitorsof the same kinase.Another way to limit side effects is to deliver kinase

inhibitors topically and, in the case of airway disease, byinhalation, which should greatly reduce systemic expo-sure. This requires the development of drugs with highpotency that have low oral bioavailability and topicalactivity. It has so far proven difficult to develop suchdrugs, but inhaled p38 MAPK inhibitors are currentlyin clinical development for severe asthma and COPD(Millan, 2011; Norman, 2015). Inhaled PI3Kg/d inhibi-tors are also in development for severe asthma andCOPD because they may overcome the corticosteroidsresistance in these diseases (Norman, 2014a; Onionset al., 2016).

B. Targeting Several Kinases

Inhibitingmore than one kinasemay increase efficacybecause theremay be additive or synergistic effects seenby inhibiting more than one kinase. In addition, it maydecrease the risks of developing resistance by blockingpathways that may reduce the effect of monokinaseinhibition (Morphy, 2010). Several multikinase inhibi-tors have been developed for the treatment of malig-nancies (Garuti et al., 2015). Sorafenib targets severaltyrosine kinases (VEGFR, PDGFR, c-Raf, c-Kit), suni-tinib (VEGFR, PDGFR), vandetanib (VEGFR, EGFR,RET), and pazopanib (VEGFR, PDGFR, c-Kit). Thesedrugs are indicated for advanced malignancies, andadverse effects are common. Nintedanib inhibits sev-eral growth factor receptors (VEGFR, PDGFR, FGFR)and is approved for the treatment of idiopathic pulmo-nary fibrosis but has significant side effects (Keating,2015). There is a case for the development of inhaledmultikinase inhibitors for severe airway disease. Sev-eral narrow spectrum kinase inhibitors suitable forinhaled delivery have been developed for the treat-ment of corticosteroid-resistant inflammation in severeasthma and COPD (Onions et al., 2016). These drugsinhibit p38a, p38g and Hck and are more effective thanp38a inhibitors in suppressing the release of inflamma-tory cytokines from macrophages in vitro and in treat-ing inflammation in mice exposed to cigarette smoke,which is resistant to corticosteroids.

C. Overcoming Kinase Inhibitor Resistance

A major limitation to the use of kinase inhibitors inthe treatment of malignancy is mutation of the ATPbinding site, which overcomes the inhibitory effects ofkinases. This is addressed by the development of multi-kinase inhibitors and allosteric kinase inhibitors, whichmake development of resistance less likely. Covalentkinase inhibitors are another approach, and irrevers-ible inhibitors are now in clinical trials for malignantdisease. Loss of effect of kinase incubators is also seen inthe treatment of chronic inflammatory disease, such asrheumatoid arthritis and inflammatory bowel disease,

and tends to develop over several weeks of treatment(Barouch-Bentov and Sauer, 2011). This may arise ascompensatory pathways are upregulated to circumventthe pathway blocked by the kinase inhibitor and may beovercome by also blocking the compensatory pathway,for example with narrow spectrum kinase inhibitors(Onions et al., 2016).

D. Conclusions

Although this review highlighted the involvement ofmany different kinases in both asthma and COPD, sofar no kinase inhibitors have been approved for clinicaluse and have not even yet reached Phase 3 studies.Table 1 summarizes the clinical status of many of thedrugs that are discussed in the review. Many preclinicalstudies and early clinical trials are underway, sohopefully the field will advance. A major challenge hasbeen the development of potent inhaled kinase inhibi-tors to avoid systemic exposure and the lack of suchcompounds has slowed development in this area. Inview of the pressing unmet need for new treatments insevere asthma and COPD, it is hoped that there will besignificant investment for the pharmaceutical industryin this area in the future.

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Kinases as Novel Therapeutic Targets in Asthma...

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