Molecular Dynamics Simulations Reveal a Possible Membrane‐Involved Microkinetic Mechanism for Long‐Lasting Action of Salmeterol, a β2‐Adrenergic Receptor Agonist
Authors: Szlenk Christopher T. , Jeevan GC B. , Natesan Senthil 
Published: June 2021

Objective

The exact mechanisms underlying the longer duration of action and capacity to “reassert” airway smooth muscle relaxation in vitro despite repeated washing, of several long‐acting β2‐adrenergic receptor (β2‐AR) agonists such as salmeterol remain unresolved. Recent experimental studies suggest that the “microkinetic model” in which membrane acts as a depot enriching local drug concentration around the receptor adequately explains the long‐lasting effects. Specifically, the 700‐fold higher potency of salmeterol to β2‐AR in comparison to the structurally related agonists was found to be majorly due to its interactions with the membrane. The ‘reduction of dimensionality’ effect whereby ligands move on or within the membrane by 2D lateral diffusion could likely produce further enhancement of drug association rates and corresponding affinity values. These studies also suggested that the “exosite” described before probably reflects the phospholipid membrane rather than specific residues on the receptor. Most recently, a crystal structure of salmeterol bound to the active β2‐AR revealed an exosite for its aryloxyalkyl tail at the extracellular vestibule of the receptor. However, the association and dissociation pathways through which salmeterol access and leave the binding site is unknown. In addition, the possibility of lipophilic salmeterol reaching the binding site through transmembrane helices from the surrounding membrane environment is compelling and warrants thorough investigation, especially with the availability of the bound crystal structure. Here, we present an extensive computational study investigating the plausible lipid‐mediated association and dissociation pathways of salmeterol.

Methods

The most favorable bilayer location, orientation, and conformation of salmeterol were investigated using a 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) model membrane with 10% cholesterol by steered molecular dynamics (MD) and Umbrella Sampling simulations, using NAMD2.12 software. The dissociation and association pathways of salmeterol from the bound β2‐AR crystal structure (PDB ID 6CSY) were investigated by: 1) steered MD, 2) temperature accelerated MD, and 3) funnel metadynamics simulations. The protein, POPC lipids, and salmeterol were parameterized using the CHARMM36 and CGenFF force fields respectively.

Results

The results thus far, reveal that the dissociation of salmeterol from its bound form proceeds through the extracellular side of the receptor, with a transient stay in the aqueous bulk before finally partitioning into the membrane (near the β2‐AR) in its energetically favorable bilayer depth (~15 Å from the bilayer center) determined by our partitioning studies. The specific interactions of the ligand with the receptor residues along the binding pathways and associated free energies were analyzed.

Conclusions

Computational results suggest that the ‘microkinetic” mechanism attributed to the high membrane affinity of salmeterol may play a concerted role with its exosite binding towards the unique pharmacological properties.

Support or Funding Information

NIH – R15GM131293

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.