Membrane-Facilitated Receptor Access and Binding Mechanisms of Long-Acting β2-Adrenergic Receptor Agonists

Abstract

The drugs salmeterol, formoterol, and salbutamol constitute the frontline treatment for asthma and other chronic pulmonary diseases. These drugs activate the β2-adrenergic receptors (β2-AR), a class A G-protein-coupled receptor (GPCR) and differ significantly in their clinical onset and duration of actions. According to the "microkinetic model," the long duration of action of salmeterol and formoterol compared to salbutamol were attributed, at least in part, to their high lipophilicity and increased local concentrations in the membrane near the receptor. However, the structural and molecular bases of how the lipophilic drugs reach the binding site of the receptor from the surrounding membrane remain unknown. Using a variety of classical and enhanced molecular dynamics (MD) simulation techniques, we investigated the membrane partitioning characteristics, binding, and unbinding mechanisms of the ligands. The obtained results offer remarkable insight into the functional role of membrane lipids in the ligand association process. Strikingly, salmeterol entered the binding site from the bilayer through transmembrane helices 1 and 7. The entry was preceded by membrane-facilitated rearrangement and presentation of its phenyl-alkoxy-alkyl tail as a passkey to an access route gated by F193, a residue known critical for salmeterol's affinity. Formoterol's access is through the aqueous path shared by other β2-AR agents. We observed a novel secondary path for salbutamol that is distinct from its primary route. Our study offers a mechanistic description for the membrane-facilitated access and binding of ligands to a membrane protein and establishes a groundwork for recognizing membrane lipids as an integral component in the molecular recognition process.

Characterization of a novel positive allosteric modulator of the α1A-Adrenergic receptor

“…The high affinity Ki measurement we observed (0.13 pM) are obtained from algorithms and based upon shallow curves and may not be absolute. Allosteric modulators such as cmpd-3 can have additional allosteric effects depending upon the ligand used, the amount of receptor pre-coupled to G-protein (based upon the degree of overexpression), and with possible interactions with the lipid environment ( Szlenk et al., 2021 ) that can alter observed affinity.…”

Molecular basis for the recognition of 24-(S)-hydroxycholesterol by integrin αvβ3

“…WT-metaD technique has been successfully applied to predict the access paths and binding modes of many GPCR ligands. For example, in our recently published studies, we have successfully used the WT-metaD technique in recreating the experimentally determined binding modes of antiasthma agents such as salmeterol, formoterol, and salbutamol to the β2-adrenergic receptor and for a negative allosteric modulator, ORG27569, of the cannabinoid CB1 receptor 60 , 61 . Therefore, we believe that 24HC and 25HC likely recognize site II of integrin αVβ3 in the binding modes, as predicted in this study.…”

Membrane Lipids Are an Integral Part of Transmembrane Allosteric Sites in GPCRs: A Case Study of Cannabinoid CB1 Receptor Bound to a Negative Allosteric Modulator, ORG27569, and Analogs

“…We determined the membrane partitioning characteristics of ORG27569 using a combination of steered molecular dynamics (SMD) and umbrella sampling, as previously published. , The ligand was parameterized using the CHARMM-GUI ligand reader and modeler, and charges were assigned using CGenFF . The CHARMM-GUI membrane builder was used to generate a bilayer consisting of 65 lipids per leaflet, of which 59 of them were POPC lipids and 6 cholesterol molecules, adding up to a total of 130 lipid molecules.…”

“…Membrane lipids have been shown to actively participate in the access and binding of ligands to transmembrane orthosteric and allosteric sites of many integral membrane proteins. This topic has been extensively discussed in recent reviews. , Lipid pathways have been described for several ligands binding to various GPCRs such as cannabinoid, − sphingosine-1-phosphate, rhodopsin, protease-activated receptor-1, β2 adrenergic receptor, , dopamine D3, angiotensin II receptor type 1, and P2Y purinoceptor 1 indicating a crucial role of the membrane in enabling access to receptor binding sites. The plasma membrane can act as a “reservoir” or “depot” for lipophilic and amphiphilic drugs, such as salmeterol, a β2 adrenergic receptor agonist, which on dissociation, partitions back into the membrane, enabling rebinding and thereby prolonging the duration of action. , The lipid bilayer has also been shown to “preconfigure” molecules in the orientation and conformation favorable for binding by reducing the dimensionality of the ligands as it takes a two-dimensional lateral diffusion route to access the binding sites. ,, …”

“…The plasma membrane can act as a "reservoir" or "depot" for lipophilic and amphiphilic drugs, such as salmeterol, a β2 adrenergic receptor agonist, which on dissociation, partitions back into the membrane, enabling rebinding and thereby prolonging the duration of action. 26,30 The lipid bilayer has also been shown to "preconfigure" molecules in the orientation and conformation favorable for binding by reducing the dimensionality of the ligands as it takes a two-dimensional lateral diffusion route to access the binding sites. 29,31,32 Recently, a crystal structure of cannabinoid CB1 receptor (CB1R) was reported with an agonist, CP55940, bound to the orthosteric site and a negative allosteric modulator (NAM), ORG27569, bound to a lipid-facing extrahelical transmembrane site located near the inner leaflet of the membrane 33 (Figure 1 and Supporting Table S1).…”