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Interdicting Gq Activation in Airway Disease by Receptor-Dependent and Receptor-Independent Mechanisms

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Interdicting Gq Activation in Airway Disease by Receptor-Dependent and Receptor-Independent Mechanisms 1521-0111/89/1/94–104$25.00 http://dx.doi.org/10.1124/mol.115.100339 MOLECULAR PHARMACOLOGY Mol Pharmacol 89:94–104, January 2016 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics Interdicting Gq Activation in Airway Disease by Receptor-Dependent and Receptor-Independent Mechanisms Richard Carr III, Cynthia Koziol-White, Jie Zhang, Hong Lam, Steven S. An, Gregory G. Tall, Reynold A. Panettieri, Jr., and Jeffrey L. Benovic Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (R.C., J.L.B.); Department of Medicine, Pulmonary, Allergy, and Critical Care Division, Airways Biology Initiative, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania (C.K.W., J.Z., R.A.P.); Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland (H.L., S.S.A.); and Department of Pharmacology and Downloaded from Physiology, University of Rochester Medical Center, Rochester, New York (G.G.T.) Received June 9, 2015; accepted October 9, 2015 ABSTRACT Ga bg heterotrimer (G ), an important mediator in the pathol- inhibits all G protein coupling to several G -coupled receptors, q q q molpharm.aspetjournals.org ogy of airway disease, plays a central role in bronchoconstric- including protease activated receptor 1, muscarinic acetylcho- tion and airway remodeling, including airway smooth muscle line M3, and histamine H1 receptors, while demonstrating no growth and inflammation. Current therapeutic strategies to direct effect on Gq. We also evaluated the ability of FR900359, treat airway disease include the use of muscarinic and leuko- also known as UBO-QIC, to directly inhibit Gq activation. triene receptor antagonists; however, these pharmaceuticals FR900359 inhibited spontaneous Gaq nucleotide exchange, demonstrate a limited clinical efficacy as multiple Gq-coupled while having little effect on Gasbg,Gaibg,orGa12/13bg hetero- receptor subtypes contribute to these pathologies. Thus, trimer activity. Both P4pal-10 and FR900359 inhibited Gq- broadly inhibiting the activation of Gq may be an advanta- mediated intracellular signaling and primary human airway geous therapeutic approach. Here, we investigated the effects smooth muscle growth, whereas only FR900359 effectively of broadly inhibiting Gq activation in vitro and ex vivo using interdicted agonist-promoted airway contraction in human pre- receptor-dependent and receptor-independent strategies. cision cut lung slices. These studies serve as a proof of concept at ASPET Journals on September 25, 2021 P4pal-10 is a protease activated receptor 4–derived pepducin that the broad-based inhibition of Gq activation may be a useful that exhibits efficacy toward multiple Gq-coupled receptors. therapeutic approach to treat multiple common pathologies of Mechanistic studies demonstrated that P4pal-10 selectively airway disease. Introduction mediate the parasympathetic and hormonal signaling regulating bronchomotor tone (Bel, 2013). G protein– Asthma manifests as a complex respiratory syndrome coupled receptor (GPCR) antagonists have a somewhat of airway hyperesponsiveness and inflammation. Airway limited efficacy in patients as the activation of multiple smooth muscle (ASM) shortening evokes an airway obstruc- Gq-coupled receptors contribute to the pathology of the tion that clinically manifests as a significant decrease in disease (Scadding and Scadding, 2010; Moulton and Fryer, peak airflow (Deshpande and Penn, 2006). Common thera- 2011). It is plausible that inhibiting Gq activation at the peutic interventions include the use of glucocorticoids to receptor or G protein level would be an advantageous asthma curb airway inflammation and are often combined with a therapeutic as Gq-mediated ASM shortening is a primary long-acting b-agonist. Other therapies include leukotriene contributor to bronchotone (Borchers et al., 2003; Pelaia et al., and muscarinic receptor antagonists that inhibit the acti- 2008). vation of Gaqbg heterotrimer (Gq)-coupled receptors that P4pal-10 is a pepducin antagonist derived from intracellu- lar loop 3 of protease-activated receptor (PAR) 4 (Covic et al., This research was supported by the National Institutes of Health National 2002). In the initial report, P4pal-10 demonstrated efficacy in Institute of General Medical Sciences [Grants R37-GM047417, R01-GM068857, R01-GM088242, and T32-GM100836], National Institutes of Health National preventing PAR4-mediated platelet activation ex vivo and Heart, Lung, and Blood Institute [Grants P01-HL114471 and R01-HL107361] exhibited antithrombotic activities in vivo. Interestingly, and National Institutes of Health National Institute of Environmental Health Sciences [Grant P30-ES013508]. P4pal-10 inhibited both PAR1- and PAR4-mediated platelet dx.doi.org/10.1124/mol.115.100339. aggregation and it was suggested this was due to disruption of ABBREVIATIONS: AP, agonist peptide; b2AR, b2-adrenergic receptor; ASM, airway smooth muscle; BSA, bovine serum albumin; CXCR, CXC chemokine receptor; DMSO, dimethylsulfoxide; DPBS, Dulbecco’s phosphate-buffered saline; DTT, dithiothreitol; EGF, epidermal growth factor; Fura2-AM, Fura-2 acetoxymethyl ester; G12/13,Ga12/13bg heterotrimer; Gi,Gaibg heterotrimer; Gq,Gaqbg heterotrimer; Gs,Gasbg heterotrimer; GPCR, G protein–coupled receptor; GST, glutathione S-transferase; GTPgS, guanosine 59-O-[gamma-thio]triphosphate; HBSS, Hank’s balanced salt solution; HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis; PAR, protease activated receptor; P4pal-10 Scr, scrambled P4pal-10; RBD, Rhotekin Rho-binding domain; SDS, sodium dodecyl sulfate; TBST, Tris-buffered saline with Tween 20; TXA2R, thromboxane A2 receptor. 94 Interdicting Gq Activation in Airway Disease 95 the activity of PAR1/PAR4 heterodimers and loss of respon- bovine serum, 25 mM HEPES, pH 7.2, 1.7 mM CaCl2,2mM siveness from both receptor subtypes. Moreover, although L-glutamine, 0.2 units/ml of penicillin, and 100 mg/ml streptomycin). Covic et al. reported that P4pal-10 had little effect on The use of human ASM cells does not constitute human subjects research as all donor tissue is harvested anonymously and deidenti- thromboxane A2 receptor (TXA2R) function in platelets (Covic et al., 2002), other investigators reported that TXA R fied. All cells were maintained at 37°C in a humidified 5% CO2 2 incubator. Cells were untransfected unless specifically noted. was sensitive to P4pal-10 in human platelet aggregation at Calcium Mobilization. Calcium mobilization was performed as concentrations comparable to those used to inhibit PAR1 and previously described, with slight modifications (Luo et al., 2008). PAR4 (Stampfuss et al., 2003). Here, we further investigated HEK293 cells or primary human ASM cells were grown to confluence the putative multiple efficacies of P4pal-10 and applied in 15-cm dishes. Cells were treated with CellStripper (CellGro), its unique properties to understand the mechanisms and washed, and concentrated to 5 Â 106 cells/ml in Hank’s balanced salt application of broad-based Gq inhibition in the treatment solution (HBSS) (CellGro) with 0.025% BSA. Fura-2 acetoxymethyl of airway disease. ester (Fura2-AM) (Life Technologies, Carlsbad, MA) dye was added to 2 mM, gently rocked, and incubated for 45 minutes at 37°C. Cells The relative therapeutic advantage of broad-based Gq inhibition was also studied using a small molecule inhibi- were pelleted by centrifugation, and the supernatant was replaced tor of G activation, FR900359 (also known as UBO-QIC). with 5 ml of HBSS and incubated at room temperature for 15 minutes q to allow Fura2-AM processing. Cells were pelleted by centrifugation, FR900359 is a cyclic depsipeptide isolated from the roots of 6 washed, and resuspended at 30 Â 10 cells/ml in ice-cold HBSS with Downloaded from Ardisia crenatasims that is nearly structurally identical to 0.025% BSA. To measure calcium mobilization, 50,000 cells were the well studied Gq inhibitor YM-254890 (Fujioka et al., incubated in HBSS without calcium and magnesium (CellGro) with 1988; Taniguchi et al., 2003; Takasaki et al., 2004; Bernard 0.025% bovine serum albumin (BSA) in the presence or absence of et al., 2014). Although not as well characterized as YM-254890, various concentrations of P4pal-10 for 1 minute at 37°C. Cells FR900359 likely retains the same properties and mode of treated with FR900359 were incubated on ice for 10 minutes before operation. Structural studies of a YM-254890/Gaq complex addition to the assay cuvette and 1-minute incubation at 37°C before revealed that the compound interacts with the hinge region the addition of agonists (final concentrations: 100 mM PAR1-AP, molpharm.aspetjournals.org 100 mM carbachol, 100 nM bradykinin, 100 mM histamine, and 10 mM between the a-helical domain and GTPase domain of Gaq (Nishimura et al., 2010). These contacts may inhibit the ATP; concentrations that promoted maximal calcium mobilization were used). Fura2-AM fluorescence was monitored using excitation opening of the nucleotide-bindingpocketthatisrequisitefor at 340 and 380 nm and emission at 510 nm over a time course (LS55; GDP/GTP exchange and subsequent G protein activation. PerkinElmer, Waltham, MA). Percent responsiveness was calcu- Recent studies using FR900359 demonstrate efficacy in lated as the fraction of peak response of the inhibitor-treated cells as inhibiting Gq-mediated signaling in human platelets, with compared with the response of the cognate
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