Structural Basis for Binding of Allosteric Drug Leads in the Adenosine A1 Receptor

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.

Figure 1

(A) X-ray structure of the DU172 antagonist-bound adenosine A₁ receptor (A₁AR) (PDB: 5UEN), (B) the structure of the two prototypical A₁AR positive allosteric modulators (PAMs), PD81723 and VCP171, used in this study and (C) computational model used for the simulations. The receptor was inserted into a POPC lipid bilayer and solvated in an aqueous medium of 0.15 M NaCl. After removal of antagonist from the A₁AR X-ray structure, NECA was placed in the orthosteric pocket with atomic coordinates copied from the A_2AAR X-ray structure (PDB: 2YDV) after aligning the two receptor transmembrane domains. Four molecules of each PAM were placed >20 Å away from the receptor.

Figure 2

GaMD simulations predicted the A₁AR PAM PD81723 recognized an allosteric site defined by extracellular loop 2 (ECL2): (A–C) Low-energy binding modes of PD81723 identified from (A) dual-boost GaMD simulations using AMBER, (B) dual-boost GaMD simulations using NAMD and (C) dihedral-boost GaMD simulations using NAMD. The receptor, orthosteric agonist NECA and PAM PD81723 are shown in ribbons, spheres and sticks, respectively. Residues found within 5 Å of the bound PD81723 are highlighted in balls-and-sticks. (D–G) A₁AR residues for which alanine substitution were shown in a previous structure-function study to significantly decrease (orange) or enhance (red) PD81723 affinity (D), binding cooperativity (E), efficacy (F) or functional cooperativity (G).

Figure 3

GaMD simulations predicted the A₁AR PAM VCP171 recognized an allosteric site defined by extracellular loop 2 (ECL2): (A) Low-energy binding mode of VCP171 identified from dual-boost GaMD simulations using AMBER. The receptor, orthosteric agonist NECA and PAM VCP171 are shown in ribbons, spheres and sticks, respectively. Residues found within 5 Å of the bound VCP171 are highlighted in balls-and-sticks. (B–E) A₁AR residues for which alanine substitution were shown in a previous structure-function study to significantly decrease (orange) or enhance (red) VCP171 affinity (B), binding cooperativity (C), efficacy (D) or functional cooperativity (E).

Figure 4

PD81723 stabilized NECA binding within the A₁AR orthosteric site: (A–C) structural clusters of NECA identified in simulations of the “A₁AR + NECA” system using (A) dual-boost GaMD with AMBER, (B) dual-boost GaMD with NAMD and (C) dihedral-boost GaMD with NAMD. (D–F) structural clusters of NECA identified in simulations of the “A₁AR + NECA + PD81723” system with no PD81723 bound at the ECL2 allosteric site using (D) dual-boost GaMD with AMBER, (E) dual-boost GaMD with NAMD and (F) dihedral-boost GaMD with NAMD. (G–I) structural clusters of NECA identified in simulations of the “A₁AR + NECA + PD81723” system with PD81723 bound at the ECL2 allosteric site using (G) dual-boost GaMD with AMBER, (H) dual-boost GaMD with NAMD and (I) dihedral-boost GaMD with NAMD. The receptor, orthosteric agonist (NECA) and PAM (PD81723) are shown in ribbons, sticks and spheres, respectively. NECA clusters are colored by the potential of mean force (PMF) in a green(0 kcal/mol)-white-red(8 kcal/mol) scale and the NECA conformation extracted from the 2YDV X-ray structure of the A_2AAR with two receptor transmembrane domains aligned is shown in orange for reference.

Figure 5

Structural clusters of NECA identified in dual-boost GaMD simulations using AMBER of the “A₁AR + NECA + VCP171” system: (A) binding of NECA in the A₁AR orthosteric site was stabilized upon VCP171 binding to the ECL2 allosteric site. (B) Dissociation of NECA was observed in the absence of VCP171 binding to the ECL2 allosteric site. The receptor, orthosteric agonist (NECA) and PAM (VCP171) are shown in ribbons, sticks and spheres, respectively. NECA clusters are colored by free energy in a green(0 kcal/mol)-white-red(8 kcal/mol) scale and the NECA conformation extracted from the 2YDV X-ray structure of the A_2AAR with two receptor transmembrane domains aligned is shown in orange for reference.

Figure 6

PAM binding closed a salt bridge E172^ECL2-K265^ECL3 in the A₁AR extracellular vestibule: (A) A 2D PMF profile of the E172^ECL2-K265^ECL3 distance and NECA RMSD relative to the starting bound conformation obtained from AMBER dual-boost GaMD simulation of the “A₁AR + NECA” system. The C_δ atom in E172 and N_ζ atom in K265 were used to calculate the distance. (B) Three low-energy states, “Open”, “Intermediate” and “Closed”, identified in (A) are shown using the X-ray structure of antagonist DU172-bound A₁AR (PDB: 5UEN), cryo-EM structure of adenosine-G_i-bound A₁AR (PDB: 6D9H) and GaMD predicted structure of the NECA and PAM PD81723 co-bound A₁AR. (C,D) 2D PMF profiles of the E172^ECL2-K265^ECL3 distance and PAM occupancy at the ECL2 allosteric site obtained from AMBER dual-boost GaMD simulations of the (C) “A₁AR + NECA + PD81723” and (D) “A₁AR + NECA + VCP171” systems. PAM binding biased conformation ensemble of the E172^ECL2-K265^ECL3 salt bridge towards the closed state, leading to stabilized agonist binding at the orthosteric site.