Structural Insights and Thermodynamics of Membrane‐Facilitated Binding and Unbinding Mechanisms of Long‐Acting β2‐Adrenergic Agonists

Objective The β2‐adrenergic receptor (β2‐AR) is a member of the G protein‐coupled receptor family. β2‐AR agonists such as salbutamol, salmeterol, and formoterol constitute the frontline treatment for both asthma and chronic obstructive pulmonary disorder. These drugs differ significantly in their onset and duration of actions. The structural basis and mechanisms underlying these differences in pharmacology are unknown. Experimental studies suggest that a “microkinetic model” in which the plasma membrane acts as a depot enriching local drug concentration around the receptor adequately explains the long‐lasting effects of lipophilic salmeterol and formoterol. This membrane contribution was also shown to affect drugs’ efficacy by influencing their observed binding kinetics, especially the association rate constant k on . In addition, published mutagenesis studies have shown that transmembrane helices (TMH) 2 and 7 are essential for salmeterol’s affinity to β2‐AR. Our central hypothesis is that distinct structural features of these drugs resulting in specific lipid interactions cause differences in membrane‐involved receptor binding and unbinding events. Specifically, we hypothesize that differences in bilayer depths of ligands would affect their receptor access through the transmembrane helices, and thus the association and dissociation pathways of the long‐acting drugs are likely different from that of short‐acting salbutamol. Here, we present an extensive computational study elucidating thermodynamics and potential lipid‐mediated association and dissociation pathways of salmeterol and formoterol and compare them with that of salbutamol. Methods The membrane partitioning characteristics of the ligands in a model membrane (containing POPC lipid and cholesterol) were investigated by steered molecular dynamics and Umbrella Sampling simulations, using NAMD software. The dissociation and association pathways of each ligand were investigated by advanced simulation techniques using the crystal structure of β2‐AR bound to salmeterol (PDB ID 6MXT). The absolute binding free energies were estimated using GROMACS/PLUMED software. Results The association and dissociation paths of salmeterol and formoterol reveal potential membrane‐involved receptor access routes through TMHs 1 and 7. Salbutamol accesses the binding site through a unique aqueous path lined with polar residues. The calculated binding free energies and kinetic parameters are in good agreement with the published experimental values. The residue‐ligand interactions critical for receptor access and binding within the site provide structural basis for membrane‐involved binding mechanisms and agree with the published crystallographic and mutagenesis studies. Conclusions Our results illustrate the functional role of membrane lipids in influencing access and binding of ligands to β2‐AR. The distinct structural features of the ligands and their specific interactions with membrane lipids appear to influence their binding kinetics. The obtained structural insights can be used for rational design of ligands to exploit membrane interactions for optimal pharmacology. Support or Funding Information National Institutes of Health‐NIGMS R15 GM131293‐01Membrane‐involved ligand‐binding to beta2‐AR