Soluble guanylate cyclase as an alternative target for PNAS PLUS bronchodilator therapy in asthma Arnab Ghosha, Cynthia J. Koziol-Whiteb, Kewal Asosingha, Georgina Chenga, Lisa Ruplea, Dieter Gronebergc, Andreas Friebec, Suzy A. A. Comhaira, Johannes-Peter Staschd, Reynold A. Panettieri Jr.b, Mark A. Aronicaa,e, Serpil C. Erzuruma,e,1, and Dennis J. Stuehra,1 aDepartment of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195; bRutgers Institute for Translational Medicine & Science, Rutgers University, New Brunswick, NJ 08901; cInstitute of Vegetative Physiology, Universität Würzburg, Wuerzburg 97070, Germany; dPharma Research Centre, Bayer Pharma AG, D-42096 Wuppertal, Germany; and eRespiratory Institute, Cleveland Clinic, Cleveland, OH 44195 Edited by Louis J. Ignarro, University of California, Los Angeles School of Medicine, Beverly Hills, CA, and approved March 11, 2016 (received for review December 10, 2015) Asthma is defined by airway inflammation and hyperresponsive- S1). Graded doses of the slow-release NO donor DETA/NO ness, and contributes to morbidity and mortality worldwide. Although [3,3-Bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene] produced bron- bronchodilation is a cornerstone of treatment, current bronchodilators chodilation in human PCLS similar to what was induced by a become ineffective with worsening asthma severity. We investigated standard β-agonist bronchodilator, Formoterol (Fig. 1B). In ad- an alternative pathway that involves activating the airway smooth dition, having a NO donor (sodium nitroprusside, SNP) present at a muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its subeffective concentration made the preconstricted human small natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists airways more responsive to lower doses of the β-agonist broncho- – – BAY 41 2272 and BAY 60 2770, triggered bronchodilation in normal dilator Isoproterenol (Fig. 1C). This finding shows that triggering the human lung slices and in mouse airways. Both BAY 41–2272 and BAY NO-sGC-cGMP pathway bronchodilated the preconstricted human 60–2770 reversed airway hyperresponsiveness in mice with allergic lung, and could also synergize with β-adrenergic-based bronchodi- asthma and restored normal lung function. The sGC from mouse asth- lators in doing so. We confirmed a similar role for the NO-sGC- matic lungs displayed three hallmarks of oxidative damage that render SCIENCES it NO-insensitive, and identical changes to sGC occurred in human lung cGMP pathway in bronchodilating the mouse airway, using tracheal sGC-β1−/− slices or in human airway smooth muscle cells when given chronic NO rings that we isolated from normal or mice (Fig. S1). APPLIED BIOLOGICAL exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based broncho- Direct-Acting Pharmacologic sGC Agonists Bronchodilate Human dilation, and reveal that pharmacologic sGC agonists can achieve bron- Lung. The pharmacologic agent Riociguat is an NO-independent chodilation despite this loss. sGC stimulator (10) that was recently approved to treat patients with different forms of pulmonary hypertension. Riociguat, and bronchodilation | bronchoconstriction | S-nitrosylation | nitric oxide | it’s structural analog BAY 41–2272 (BAY 41), only stimulate the heme protein NO-responsive, heme-containing form of sGC (9), whereas Cinaciguat or its structural analog BAY 60–2770 (BAY 60) only sthma is an inflammatory disease that causes airway hyper- stimulate the oxidatively damaged, heme-free and NO-insensitive Areactivity (AHR) and bronchoconstriction, which impedes forms of sGC (11), which may accumulate in cells and tissues under daily life activities and, when severe, can cause death. It is the most inflammatory oxidative stress (10, 12). We found that BAY 41–2272, common chronic disease of childhood, accounts for one in three and to a lesser extent BAY 60–2770, bronchodilated the pre- emergency department visits daily, and asthma diagnoses are in- constricted human PCLS obtained from healthy donors (Fig. 1D). creasing worldwide (1). The leading treatment for relief and acute care is bronchodilation, which relies heavily on the β-adrenergic Significance receptor-cAMP pathway. Nearly 70% of patients, however, de- β velop resistance or tachyphylaxis to the existing -agonist therapy Asthmatics depend on β-agonist bronchodilator drugs, but a (2), underscoring a need for new bronchodilators that can act majority develop resistance to these drugs in their lifetime, and through a different pharmacologic principle. new ways to bronchodilate are needed. We show that bro- The nitric oxide-soluble guanylate cyclase-cGMP pathway chodilation can be triggered in normal human and asthmatic (NO-sGC-cGMP) is the primary signal transduction pathway for mouse airways through an alternative signaling pathway, using relaxing vascular smooth muscle (3). In contrast, a role for the new pharmacologic agents that directly activate the soluble NO-sGC-cGMP pathway in relaxing airway smooth muscle is less guanylate cyclase (sGC) enzyme. Because an sGC-based drug clear (4, 5), and bronchodilation was instead suggested to depend was recently approved to treat pulmonary arterial hyperten- on glutathione nitrosothiol levels in the lung (6, 7). However, re- sion, our findings imply that such drugs could become new cent studies have shown that inflammation can desensitize sGC bronchodilators in asthma. Our work also provides insight on toward its natural activator, NO (8), and new drugs have become how the sGC signaling enzyme becomes desensitized toward available that directly activate sGC, independent of NO (9). These NO in inflammatory asthma, and thus helps to explain why NO developments encouraged us to re-examine the NO-sGC-cGMP is an ineffective bronchodilator in this disease. pathway regarding its role in bronchodilation, its becoming dam- aged in inflammatory asthma, and its potential for alternative Author contributions: A.G., K.A., A.F., R.A.P., M.A.A., S.C.E., and D.J.S. designed research; A.G., C.J.K.-W., G.C., L.R., and D.G. performed research; S.A.A.C. and J.-P.S. contributed bronchodilator development under this circumstance. new reagents/analytic tools; A.G., C.J.K.-W., S.C.E., and D.J.S. analyzed data; and A.G. and D.J.S. wrote the paper. Results The authors declare no conflict of interest. The NO-sGC-cGMP Pathway Bronchodilates Human Lung. We first This article is a PNAS Direct Submission. tested if stimulating the NO-sGC-cGMP pathway would dilate 1To whom correspondence may be addressed. Email: [email protected] or [email protected]. preconstricted small airways in human precision-cut lung slices This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (PCLS) obtained from healthy donor lungs (Fig. 1A and Table 1073/pnas.1524398113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1524398113 PNAS | Published online April 11, 2016 | E2355–E2362 Downloaded by guest on September 26, 2021 Fig. 1. NO donors and sGC stimulators and activators bronchodilate human lung slices. (A) Small airways in human PCLS were contracted with CCh followed by addition of the indicated compounds and image collection and processing to determine bronchiole lumen area, expressed as percent compared with baseline. (B) NO donor (DETA/NO) caused a dose-dependent bronchodilation in a manner similar to the β-agonist Formoterol. (C) Coadministering a subthreshold dose of NO donor (SNP, 1 μM) enhanced Isoproterenol (ISO) dilation of PCLS. (D) Differing capacity of the BAY sGC activators to dilate PCLS relative to Formoterol, with the final sGC-α1andβ1 expression levels in the slices compared below. For A, n = 3; for B–D, n = 2; mean ± SD; four to seven slices per condition. Thus, pharmacologic agents that act directly on sGC can broncho- β-agonist drugs, it implies that sGC-targeted drugs could be ef- dilate constricted airways in human lung. fective for inducing bronchodilation in asthmatics. sGC Agonist Drugs Eliminate Airway Hyperresponsiveness in Mouse Biomarkers of sGC Damage Manifest in the Mouse Asthmatic Lung Models of Allergic Asthma. We next tested if the sGC stimulator and Correlate with an Altered sGC Drug-Response Profile. To explore BAY 41–2272 and sGC activator BAY 60–2770 would act as this mechanism, we analyzed lung tissues that were harvested bronchodilators in vivo in two different mouse models of asthma, from the naïve and OVA or HDME asthmatic mice. The lung one in which the mice are sensitized and challenged to ovalbumin sGC from both the OVA and HDME mice had a decreased protein (OVA), and another in which mice are exposed to house catalytic response (cGMP production) toward NO and BAY 41– dust mite allergen extract (HDME) (Fig. S2). OVA is a standard 2272 and an increased catalytic response toward BAY 60–2770, experimental model of mouse allergic airway inflammation, whereas relative to lung sGC from naïve mice (Fig. 3A). This response HDME more closely mimics human sensitization via airway pattern matches what we saw in the live asthmatic mice regarding epithelial cells, and dust mites are a common worldwide asth- their nearly equivalent airway responses toward BAY 41–2272 matic allergen (13). The allergen sensitizations and subsequent and BAY 60–2770 (Fig. 2), and implies that the allergic in- 7-d exposure caused an expected asthma-like inflammation to flammation caused a significant portion of the lung sGC to ac- develop in the mouse lungs for both models, as judged by an cumulate in an oxidatively damaged and NO-unresponsive—but increase in eosinophil numbers and an increased expression of BAY 60–2270-responsive—form. Indeed, the sGC-β1 from the the NO-generating enzyme, inducible NO synthase (iNOS) (Fig. asthmatic lungs exhibited three biochemical hallmarks of being S3). The inflammation only partly diminished (OVA) or did not damaged and NO-unresponsive (14, 15): an increased level of alter (HDME) the sGC protein expression levels in mouse lungs cysteine S-nitrosylation (Fig. 3 B and C), a diminished association (Fig.