Cigarette Smoke Specifically Affects Small Airway Epithelial Cell Populations and Triggers the Expansion of Inflammatory and Squamous Differentiation Associated Basal Cells.

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air-liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure. Smoke triggers detoxification and inflammatory responses and aberrantly activates and alters basal cell differentiation. This results in an increase of inflammatory basal-to-secretory cell intermediates and, particularly after chronic smoke exposure, a massive expansion of a rare inflammatory and squamous metaplasia associated KRT6A+ basal cell state and an altered secretory cell landscape. ALI cultures originating from healthy non-smokers and COPD smokers show similar responses to cigarette smoke exposure, although an increased pro-inflammatory profile is conserved in the latter. Taken together, the in vitro models provide high-resolution insights into the smoke-induced remodeling of the small airways resembling the pathological processes in COPD airways. The data may also help to better understand other lung diseases including COVID-19, as the data reflect the smoke-dependent variable induction of SARS-CoV-2 entry factors across SAEC populations.

Identifying Functional Hotspot Residues for Biased Ligand Design in G-Protein-Coupled Receptors.

G-protein-coupled receptors (GPCRs) mediate multiple signaling pathways in the cell, depending on the agonist that activates the receptor and multiple cellular factors. Agonists that show higher potency to specific signaling pathways over others are known as "biased agonists" and have been shown to have better therapeutic index. Although biased agonists are desirable, their design poses several challenges to date. The number of assays to identify biased agonists seems expensive and tedious. Therefore, computational methods that can reliably calculate the possible bias of various ligands ahead of experiments and provide guidance, will be both cost and time effective. In this work, using the mechanism of allosteric communication from the extracellular region to the intracellular transducer protein coupling region in GPCRs, we have developed a computational method to calculate ligand bias ahead of experiments. We have validated the method for several ß-arrestin-biased agonists in ß2-adrenergic receptor (ß2AR), serotonin receptors 5-HT1B and 5-HT2B and for G-protein-biased agonists in the κ-opioid receptor. Using this computational method, we also performed a blind prediction followed by experimental testing and showed that the agonist carmoterol is ß-arrestin-biased in ß2AR. Additionally, we have identified amino acid residues in the biased agonist binding site in both ß2AR and κ-opioid receptors that are involved in potentiating the ligand bias. We call these residues functional hotspots, and they can be used to derive pharmacophores to design biased agonists in GPCRs.

Functional and biochemical rationales for the 24-hour-long duration of action of olodaterol.

ß(2)-Adrenoceptor (ß(2)-AR) agonists are powerful bronchodilators and play a pivotal role in the management of pulmonary obstructive diseases, such as asthma and chronic obstructive pulmonary disease. Although these agents first were used many years ago, progress in drug development has resulted in better tolerated, long-acting ß(2)-AR agonists (LABAs), such as formoterol and salmeterol. Although LABAs have been on the market for several years, relatively little is known on the rationale(s) behind their long duration of action. In this study, we focused on olodaterol (previously known as BI1744CL), a novel inhaled LABA, which provides a bronchodilating effect lasting 24 h and is currently in Phase III clinical trials. To understand the rationale behind its long duration of action, different aspects of olodaterol were analyzed (i.e., its lipophilicity and propensity to accumulate in the lipid bilayer as well as its tight binding to the ß(2)-AR). In line with its physicochemical properties, olodaterol associated moderately with lipid bilayers. Instead, kinetic as well as equilibrium binding studies indicated the presence of a stable [(3)H]olodaterol/ß(2)-AR complex with a dissociation half-life of 17.8 h due to ternary complex formation. The tight binding of olodaterol to the human ß(2)-AR and stabilization of the ternary complex were confirmed in functional experiments monitoring adenylyl cyclase activity after extensive washout. Taken together, binding, kinetic, and functional data support the existence of a stable complex with the ß(2)-AR that, with a dissociation half-life >17 h, might indeed be a rationale for the 24-h duration of action of olodaterol.

The constitutive activity of the human muscarinic M3 receptor unmasks differences in the pharmacology of anticholinergics.

An activator protein 1-driven luciferase reporter assay was developed to monitor the activation of the human muscarinic M3 receptor (hM3-R) and evaluate functional potencies of different anticholinergics in Chinese hamster ovary cells. This assay proved to be superior to previously used functional assays [i.e., inositol phosphate accumulation (J Pharmacol Exp Ther 330:660-668, 2009)], thanks to the longer incubation times that allow reaching of pseudoequilibrium for ligands with slower dissociation kinetics, the long-acting muscarinic antagonists. Interestingly, within this system the hM3-R efficiently signaled in an agonist-independent manner. All the antagonists tested were able to inhibit the hM3-R constitutive activity in a concentration-dependent fashion, behaving as full inverse agonists. Curiously, significant differences in potency as antagonists (against carbachol) and inverse agonists were seen for some compounds (N-methyl scopolamine and tiotropium). Given the potential for inverse agonists to cause receptor up-regulation, the effect of chronic exposure to anticholinergics on the expression levels of hM3-R was also tested. Again, significant differences were seen, with some ligands (e.g., tiotropium) producing less than half of the receptor up-regulation caused by other anticholinergics. This study shows that anticholinergics can exhibit differential behaviors, which depend on the pathway investigated, and therefore provides evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation. In addition, differences in the potential of anticholinergics to induce hM3-R up-regulation might have clinical relevance, because many are on the market or in clinical trials as chronic treatment for chronic obstructive pulmonary disease, for example.

Development of a miniaturized 96-Transwell air-liquid interface human small airway epithelial model.

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo, miniaturization of respiratory drug discovery assays is of pivotal importance. Thus, a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture, a pseudostratified epithelium containing basal, club, goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition, ciliary beating frequency, and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements, together with dextran permeability measurements, confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor ß1 (TGF-ß1) and tumor necrosis factor α (TNF-α) in a concentration dependent manner. Thus, the miniaturized cellular model system enables the recapitulation of a physiologically responsive, differentiated small airway epithelium, and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery, for instance, in respect of fibrotic distal airway/lung diseases.

Intermittent exposure to whole cigarette smoke alters the differentiation of primary small airway epithelial cells in the air-liquid interface culture.

Cigarette smoke (CS) is the leading risk factor to develop COPD. Therefore, the pathologic effects of whole CS on the differentiation of primary small airway epithelial cells (SAEC) were investigated, using cells from three healthy donors and three COPD patients, cultured under ALI (air-liquid interface) conditions. The analysis of the epithelial physiology demonstrated that CS impaired barrier formation and reduced cilia beat activity. Although, COPD-derived ALI cultures preserved some features known from COPD patients, CS-induced effects were similarly pronounced in ALI cultures from patients compared to healthy controls. RNA sequencing analyses revealed the deregulation of marker genes for basal and secretory cells upon CS exposure. The comparison between gene signatures obtained from the in vitro model (CS vs. air) with a published data set from human epithelial brushes (smoker vs. non-smoker) revealed a high degree of similarity between deregulated genes and pathways induced by CS. Taken together, whole cigarette smoke alters the differentiation of small airway basal cells in vitro. The established model showed a good translatability to the situation in vivo. Thus, the model can help to identify and test novel therapeutic approaches to restore the impaired epithelial repair mechanisms in COPD, which is still a high medical need.

Molecular basis for the long duration of action and kinetic selectivity of tiotropium for the muscarinic M3 receptor.

Antagonizing the human M3 muscarinic receptor (hM3R) over a long time is a key feature of modern bronchodilating COPD drugs aiming at symptom relief. The long duration of action of the antimuscarinic drug tiotropium and its kinetic subtype selectivity over hM2R are investigated by kinetic mapping of the binding site and the exit channel of hM3R. Hence, dissociation experiments have been performed with a set of molecular matched pairs of tiotropium on a large variety of mutated variants of hM3R. The exceedingly long half-life of tiotropium (of more than 24 h) is attributed to interactions in the binding site; particularly a highly directed interaction of the ligands' hydroxy group with an asparagine (N508(6.52)) prevents rapid dissociation via a snap-lock mechanism. The kinetic selectivity over hM2R, however, is caused by differences in the electrostatics and in the flexibility of the extracellular vestibule. Extensive molecular dynamics simulations (several microseconds) support experimental results.

Differential inverse agonism at the human muscarinic M3 receptor.

Human muscarinic M3 receptors (hM3Rs) induce smooth muscle contraction and mucus gland secretion in response to parasympathetic stimulation. As a consequence of hM3R function, muscarinic antagonists have wide therapeutic use to treat overactive bladder, abdominal pain (irritable bowel syndrome), and chronic obstructive pulmonary disease (COPD). In this chapter, we describe the set up and results obtained with different in vitro assays to monitor hM3R activation (agonist-dependent and constitutive) and evaluate functional potencies of different anticholinergics in CHO cells. Given the G(q) coupling of hM3R, assays measuring the second messengers inositol phosphates (InsP) and an AP-1-driven reporter luciferase were developed. In our hands, the reporter gene assay shows advantages: firstly, thanks to the longer incubation times, it allows reaching of pseudo-equilibrium also for ligands with slower receptor dissociation kinetics (e.g., tiotropium). Secondly, the AP-1-driven luciferase detects significant constitutive activity of the hM3R, which allows characterizing the different anticholinergics for their inverse agonist properties. Given the potential for inverse agonists to cause changes in receptor expression, monitoring hM3R upregulation is another important pharmacological parameter. Here, we describe how to measure the effect of chronic exposure to anticholinergics on the expression levels of hM3R, with particular attention to ensure full antagonist removal from receptor pool before hM3R quantification. Taken together, our results indicate that anticholinergics exhibit differential pharmacological behaviors, which are dependent on the pathway investigated, and therefore provide evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation.