Molecular Biophysics of Class A G Protein Coupled Receptors-Lipids Interactome at a Glance-Highlights from the A2A Adenosine Receptor.

G protein-coupled receptors (GPCRs) are embedded in phospholipid membrane bilayers with cholesterol representing 34% of the total lipid content in mammalian plasma membranes. Membrane lipids interact with GPCRs structures and modulate their function and drug-stimulated signaling through conformational selection. It has been shown that anionic phospholipids form strong interactions between positively charged residues in the G protein and the TM5-TM6-TM 7 cytoplasmic interface of class A GPCRs stabilizing the signaling GPCR-G complex. Cholesterol with a high content in plasma membranes can be identified in more specific sites in the transmembrane region of GPCRs, such as the Cholesterol Consensus Motif (CCM) and Cholesterol Recognition Amino Acid Consensus (CRAC) motifs and other receptor dependent and receptor state dependent sites. Experimental biophysical methods, atomistic (AA) MD simulations and coarse-grained (CG) molecular dynamics simulations have been applied to investigate these interactions. We emphasized here the impact of phosphatidyl inositol-4,5-bisphosphate (PtdIns(4,5)P2 or PIP2), a minor phospholipid component and of cholesterol on the function-related conformational equilibria of the human A2A adenosine receptor (A2AR), a representative receptor in class A GPCR. Several GPCRs of class A interacted with PIP2 and cholesterol and in many cases the mechanism of the modulation of their function remains unknown. This review provides a helpful comprehensive overview for biophysics that enter the field of GPCRs-lipid systems.

Comparative Study of Receptor-, Receptor State-, and Membrane-Dependent Cholesterol Binding Sites in A2A and A1 Adenosine Receptors Using Coarse-Grained Molecular Dynamics Simulations.

We used coarse-grained molecular dynamics (CG MD) simulations to study protein-cholesterol interactions for different activation states of the A2A adenosine receptor (A2AR) and the A1 adenosine receptor (A1R) and predict new cholesterol binding sites indicating amino acid residues with a high residence time in three biologically relevant membranes. Compared to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-cholesterol and POPC-phosphatidylinositol-bisphosphate (PIP2)-cholesterol, the plasma mimetic membrane best described the cholesterol binding sites previously detected for the inactive state of A2AR and revealed the binding sites with long-lasting amino acid residues. We observed that using the plasma mimetic membrane and plotting residues with cholesterol residence time ≥2 µs, our CG MD simulations captured most obviously the cholesterol-protein interactions. For the inactive A2AR, we identified one more binding site in which cholesterol is bound to residues with a long residence time compared to the previously detected, for the active A1R, three binding sites, and for the inactive A1R, two binding sites. We calculated that for the active states, cholesterol binds to residues with a much longer residence time compared to the inactive state for both A2AR and A1R. The stability of the identified binding sites to A1R or A2AR with CG MD simulations was additionally investigated with potential of mean force calculations using umbrella sampling. We observed that the binding sites with residues to which cholesterol has a long residence time in A2AR have shallow binding free energy minima compared to the related binding sites in A1R, suggesting a stronger binding for cholesterol to A1R. The differences in binding sites in which cholesterol is stabilized and interacts with residues with a long residence time between active and inactive states of A1R and A2AR can be important for differences in functional activity and orthosteric agonist or antagonist affinity and can be used for the design of allosteric modulators, which can bind through lipid pathways. We observed a stronger binding for cholesterol to A1R (i.e., generally higher association rates) compared to A2AR, which remains to be demonstrated. For the active states, cholesterol binds to residues with much longer residence times compared to the inactive state for both A2AR and A1R. Taken together, binding sites of active A1R may be considered as promising allosteric targets.

Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure-Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations.

Drugs targeting adenosine receptors (AR) can provide treatment for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]pyridines L2-L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]pyridine (A17) displaying the highest affinity at both receptors with a long residence time of binding, as determined using a NanoBRET-based assay. Two binding orientations of A17 produce stable complexes inside the orthosteric binding area of A1R in molecular dynamics (MD) simulations, and we selected the most plausible orientation based on the agreement with alanine mutagenesis supported by affinity experiments. Interestingly, for drug design purposes, the mutation of L2506.51 to alanine increased the binding affinity of A17 at A1R. We explored the structure-activity relationships against A1R using alchemical binding free energy calculations with the thermodynamic integration coupled with the MD simulation (TI/MD) method, applied on the whole G-protein-coupled receptor-membrane system, which showed a good agreement (r = 0.73) between calculated and experimental relative binding free energies.

Investigation of Tumor Cells and Receptor-Ligand Simulation Models for the Development of PET Imaging Probes Targeting PSMA and GRPR and a Possible Crosstalk between the Two Receptors.

Prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) have both been used in nuclear medicine as targets for molecular imaging and therapy of prostate (PCa) and breast cancer (BCa). Three bioconjugate probes, the PSMA specific: [68Ga]Ga-1, ((HBED-CC)-Ahx-Lys-NH-CO-NH Glu or PSMA-11), the GRPR specific: [68Ga]Ga-2, ((HBED-CC)-4-amino-1-carboxymethyl piperidine-[D-Phe6, Sta13]BN(6-14), a bombesin (BN) analogue), and 3 (the BN analogue: 4-amino-1-carboxymethyl piperidine-[(R)-Phe6, Sta13]BN(6-14) connected with the fluorescent dye, BDP-FL), were synthesized and tested in vitro with PCa and BCa cell lines, more specifically, with PCa cells, PC-3 and LNCaP, with BCa cells, T47D, MDA-MB-231, and with the in-house created PSMA-overexpressing PC-3(PSMA), T47D(PSMA), and MDA-MB-231(PSMA). In addition, biomolecular simulations were conducted on the association of 1 and 2 with PSMA and GRPR. The PSMA overexpression resulted in an increase of cell-bound radioligand [68Ga]Ga-1 (PSMA) for PCa and BCa cells and also of [68Ga]Ga-2 (GRPR), especially in those cell lines already expressing GRPR. The results were confirmed by fluorescence-activated cell sorting with a PE-labeled PSMA-specific antibody and the fluorescence tracer 3. The docking calculations and molecular dynamics simulations showed how 1 enters the PSMA funnel region and how pharmacophore Glu-urea-Lys interacts with the arginine patch, the S1', and S1 subpockets by forming hydrogen and van der Waals bonds. The chelating moiety of 1, that is, HBED-CC, forms additional stabilizing hydrogen bonding and van der Waals interactions in the arene-binding site. Ligand 2 is diving into the GRPR transmembrane (TM) helical cavity, thereby forming hydrogen bonds through its amidated end, water-mediated hydrogen bonds, and π-π interactions. Our results provide valuable information regarding the molecular mechanisms involved in the interactions of 1 and 2 with PSMA and GRPR, which might be useful for the diagnostic imaging and therapy of PCa and BCa.