GS-5759, a Bifunctionalβ₂-Adrenoceptor Agonist and Phosphodiesterase 4 Inhibitor for Chronic Obstructive Pulmonary Disease with a Unique Mode of Action: Effects on Gene Expression in Human Airway Epithelial Cells

Abstract

(R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl] carbamoyl}phenyl)sulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide trifluoroacetic acid (GS-5759) is a bifunctional ligand composed of a quinolinone-containing pharmacophore [β-adrenoceptor agonist orthostere (β2A)] found in several β-adrenoceptor agonists, including indacaterol, linked covalently to a phosphodiesterase 4 (PDE4) inhibitor related to 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) by a pent-1-yn-1-ylbenzene spacer. GS-5759 had a similar affinity for PDE4B1 and the native β-adrenoceptor expressed on BEAS-2B human airway epithelial cells. However, compared with the monofunctional parent compound, β2A, the K of GS-5759 for the β-adrenoceptor was 35-fold lower. Schild analysis determined that the affinities of the β-adrenoceptor antagonists, (2R,3R)-1-[(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl) amino]-2-butanol (ICI 118551) and propranolol, were agonist-dependent, being significantly lower for GS-5759 than β2A. Collectively, these data can be explained by "forced proximity," bivalent binding where the pharmacophore in GS-5759 responsible for PDE4 inhibition also interacts with a nonallosteric domain within the β-adrenoceptor that enhances the affinity of β2A for the orthosteric site. Microarray analyses revealed that, after 2-hour exposure, GS-5759 increased the expression of >3500 genes in BEAS-2B cells that were highly rank-order correlated with gene expression changes produced by indacaterol and GSK 256066 in combination (Ind/GSK). Moreover, the line of regression began close to the origin with a slope of 0.88, indicating that the magnitude of most gene expression changes produced by Ind/GSK was quantitatively replicated by GS-5759. Thus, GS-5759 is a novel compound exhibiting dual β-adrenoceptor agonism and PDE4 inhibition with potential to interact on target tissues in a synergistic manner. Such polypharmacological behavior may be particularly effective in chronic obstructive pulmonary disease and other complex disorders where multiple processes interact to promote disease pathogenesis and progression.

Analysis of the Indacaterol-Regulated Transcriptome in Human Airway Epithelial Cells Implicates Gene Expression Changes in the Adverse and Therapeutic Effects ofβ2-Adrenoceptor Agonists

“…Role of Canonical cAMP Signaling. Previous studies have established that SABA-and LABA-induced gene expression changes in BEAS-2B cells are mediated by the b 2 -adrenoceptor (Holden et al, 2014;Joshi et al, 2015Joshi et al, , 2017Kaur et al, 2008). The ability of PKIa to prevent the induction of several salmeterol-induced genes that are associated with enriched GO terms related to proinflammatory or therapeutic processes extends those findings and provides evidence that cAMP/PKA signaling is a major regulator of gene transcription.…”

Impact of Phosphodiesterase 4 Inhibition on the Operational Efficacy, Response Maxima, and Kinetics of Indacaterol-Induced Gene Expression Changes in BEAS-2B Airway Epithelial Cells: A Global Transcriptomic Analysis

“…Effect of PDE4 inhibition on gene expression changes produced by submaximal concentrations of indacaterol and formoterol. (A) BEAS-2B cells were pretreated (30 minutes) with either GSK 256066 (GSK; 10 nM) or roflumilast (Rof; 1 mM) and then exposed to either indacaterol (Ind; 1 nM) or concentration 3800Â greater than its K I for the inhibition of human PDE4B1 (Joshi et al, 2017), GSK 256066 did not affect gene expression even at a FDR ,10%. While these data suggested that genomic mechanisms play little role in the mechanism of action of PDE4 inhibitors, further analyses ascertained that GSK 256066 was a weak stimulus in BEAS-2B cells, and the small sample size used for the arrays lacked statistical power to detect small changes in gene expression.…”

“…Total RNA was extracted (RNeasy Mini Kit; Qiagen Inc., Mississauga, ON, Canada) and reverse transcribed using a qscript cDNA synthesis kit according to the manufacturer's instructions (Quanta Biosciences, Gaithersburg, MD). Real-time polymerase chain reaction (PCR) analysis of cDNA was performed using the primer sequences shown in Supplemental Table 1 as described previously (Joshi et al, 2017;Yan et al, 2018).…”

“…Recently, we reported that the LABAs indacaterol and salmeterol promoted significant, and potentially beneficial, gene expression changes in BEAS-2B airway epithelial cells and human primary bronchial epithelia by mechanisms that involve canonical, Gsa/adenylyl cyclase/cAMP-dependent signaling (Yan et al, 2018). Therefore, logic dictates that PDE4 inhibitors may also provide clinical benefit by modulating gene expression (Tannheimer et al, 2012;Moodley et al, 2013;Giembycz and Maurice, 2014;BinMahfouz et al, 2015;Joshi et al, 2017). A genomic, anti-inflammatory mechanism of action also accommodates the likelihood that airway smooth muscle is but one of several tissues that are therapeutic targets of orally active PDE4 inhibitors.…”

Inhaled Phosphodiesterase 4 (PDE4) Inhibitors for Inflammatory Respiratory Diseases

“…GS-5759 is a bifunctional, moderately potent PDE4 inhibitor (IC 50 = 5 nM) and β 2 adrenergic agonist (EC 50 = 8 nM) made by linking a GSK256066 structurally related PDE4 inhibitor to an indacaterol structurally related β 2 -agonist (Tannheimer et al, 2014;Joshi et al, 2017). Although it has shown preclinical efficacy in a variety of species (Salmon et al, 2014) this program is assumed to be discontinued as no development has been reported.…”