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.

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 -arrestin-biased agonists in-adrenergic receptor (2AR), serotonin receptors 5-HT1B and 5-HT2B and for G-protein-biased agonists in the -opioid receptor. Using this computational method, we also performed a blind prediction followed by experimental testing and showed that the agonist carmoterol is-arrestin-biased in 2AR. Additionally, we have identified amino acid residues in the biased agonist binding site in both2AR and -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.

␤ 2 -Adrenoceptor (␤ 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 ␤ 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 ␤ 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/␤ 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 ␤ 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 ␤ 2 -AR that, with a dissociation half-life Ͼ17 h, might indeed be a rationale for the 24-h duration of action of olodaterol.

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. nonsmoker) 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. Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide and its prevalence continues to rise 1. The main risk factor to develop COPD is cigarette smoke 2,3. Smoking induces epithelial injury and this repeated injury of the epithelium triggers a pathophysiologic response, which leads to tissue remodeling of the airways that is characteristic for COPD 4,5. These changes of the small airway epithelium in COPD include: Goblet cell metaplasia 6-8 , reduced cilia function 9-13 , reduced club cell numbers 7,14,15 , basal membrane thickening 16,17 , epithelial barrier dysfunction 18-20 and squamous metaplasia 8,21-23. Furthermore, the epithelial defense mechanisms against inhaled particles and pathogens are impaired enabling sub-epithelial penetration of pathogens that increases the risk of COPD patients suffering from bacterial and viral infections and subsequent exacerbations 24-26. To address cigarette smoke (CS)-induced damage on epithelial cells in vitro, previous studies used primary epithelial cells or cell lines that were mostly exposed to cigarette smoke extract (CSE) or to whole CS. These studies demonstrate smoke effects e.g. on epithelial barrier integrity, mucus production and cilia toxicity 27-34. The majority of these studies focus on the pathophysiology of large airways, i.e. bronchial or tracheal epithelial cells.