Abstract
Despite intense interest in designing positive allosteric modulators (PAMs) as selective drugs of the adenosine A1 receptor (A1AR), structural binding modes of the receptor PAMs remain unknown. Using the first X-ray structure of the A1AR, we have performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) technique to determine binding modes of the A1AR allosteric drug leads. Two prototypical PAMs, PD81723 and VCP171, were selected. Each PAM was initially placed at least 20 Å away from the receptor. Extensive GaMD simulations using the AMBER and NAMD simulation packages at different acceleration levels captured spontaneous binding of PAMs to the A1AR. The simulations allowed us to identify low-energy binding modes of the PAMs at an allosteric site formed by the receptor extracellular loop 2 (ECL2), which are highly consistent with mutagenesis experimental data. Furthermore, the PAMs stabilized agonist binding in the receptor. In the absence of PAMs at the ECL2 allosteric site, the agonist sampled a significantly larger conformational space and even dissociated from the A1AR alone. In summary, the GaMD simulations elucidated structural binding modes of the PAMs and provided important insights into allostery in the A1AR, which will greatly facilitate the receptor structure-based drug design.
Highlights
The first A1AR positive allosteric modulators (PAMs), PD81723, was identified by Bruns and coworkers in 199011,12
Mutagenesis and molecular modeling studies have suggested that the A1AR allosteric site may reside within the second extracellular loop (ECL2)[26,27], the precise location of the allosteric site and the molecular mechanisms underlying the allosteric modulation of PD81723 and other PAMs remain unclear
The antagonist was removed from the X-ray structure and the agonist 5′-N-ethylcarboxamidoadenosine (NECA) placed in the receptor with atomic coordinates extracted from the A2AAR X-ray structure (PDB: 2YDV), after aligning the two receptor transmembrane domains
Summary
The first A1AR PAM, PD81723, was identified by Bruns and coworkers in 199011,12. Since a number of research groups have performed extensive structure-activity relationship (SAR) studies with the aim to improve the compound pharmacology and chemical properties[11,13,14,15,16,17,18,19,20,21]. Using the specialized supercomputer Anton, Dror et al performed microsecond-timescale MD simulations on the β1 and β2 adrenergic receptors (β1AR and β2AR)[34] These simulations showed that antagonist and agonist ligands entered the receptor orthosteric site through an opening between ECL2-ECL3, which was suggested to be a dominant binding pathway of GPCR drugs. The extracellular allosteric binding mode was confirmed by mutation experiments and later by the X-ray structure of the active M2 receptor that is recognized by a PAM36. Despite these successes, direct MD simulations are computationally expensive for studying protein-ligand binding. GaMD is well suited for investigating ligand binding of large biomolecules such as GPCRs
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