Structure-based drug design strategies have been used to elucidate specific ligand recognition determinants and ultimately lead to the design of new small molecules for a specific target. The structure of the target receptor protein is the first requirement in structure-based drug design approaches. In the absence of a high resolution structure of a given G protein-coupled receptor (GPCR), computational techniques like homology modeling can be used to build a 3D model. Briefly, the best structural template is chosen from the Protein Database (PDB) mainly considering sequence identity and similarity with the target receptor and the quality of the crystallographic structure (e.g. resolution). The sequence of the target receptor is aligned to the template structure using highly conserved residues and the known shared structural features to guide the automated or semi-automated alignment. The sequence alignment and the template structures are the input for the homology modeling. Energy minimization or molecular dynamics can be used to further refine and optimize the resulting 3D models. The ligand-receptor interactions can be identified by means of structure-based approaches, e.g. molecular docking. The information contained in the ligand-receptor complexes from the docking can clarify structural elements for molecular recognition and lead to a further optimization of the compounds and the design of new derivatives. The binding site of a given GPCR can be mapped for each class of small molecule ligands. Also, virtual searching of chemically diverse databases for novel chemotypes to bind to a given GPCR structure has been productive using both structure-based and ligand-based strategies. We have applied mutagenesis and homology modeling to the study of GPCR families for extracellular purines and pyrimidines and used the structural insights gained to assist in the design of novel ligands. These families consist of the adenosine receptors (ARs) and the P2Y (nucleotide) receptors. The structures of the human A2AAR were recently reported in the antagonist-bound state and in the agonist-bound state. These structures can reliably serve as modeling templates, with some adjustment, for other ARs due to the relatively high sequence identity between ARs (average 47% between human subtypes). We achieved the structure-function analysis of P2YRs by indirect means, using mutagenesis and homology modeling based on a template of the high-resolution structure of similar GPCRs, such as the CXCR4 chemokine receptor. We have collaborated with one of the premier centers for X-ray crystallography of membrane-bound proteins, i.e. the lab of Prof. Ray Stevens (Scripps Research Inst.), to report the first X-ray structure of an agonist-bound A2AAR. Automatic docking of known potent nucleosides to the agonist-bound A2AAR crystallographic structure, and to homology models of other subtypes, was performed, resulting in new predictions of stabilizing interactions and a structural basis for previous empirical structure activity relationships. We predicted binding of novel C2 terminal and 5'derivatives of adenosine and used the models to interpret effects on measured binding affinity and efficacy of newly-synthesized agonists. Structures of G protein-coupled receptors (GPCRs) have a proven utility in the discovery of new antagonists and inverse agonists, which may modulate signaling of this important family of clinical targets. However, applicability of active-state GPCR structures to virtual screening and rational optimization of agonists, remains to be assessed. We have studied adenosine 5′derivatives and evaluated the performance of an agonist-bound A2A adenosine receptor (AR) structure in retrieval of known agonists, and then employed the structure to screenfor new fragments optimally fitting the corresponding subpocket. Biochemical and functional assays demonstrate high affinity of new derivatives that include polar heterocycles. The binding models also explain a modest selectivity gain for some substituents toward the closely related A1AR subtype and the modified agonist efficacy of some of these ligands. The study suggested further applicability of in silico fragment screening to rational lead optimization for GPCRs in general. In order to investigate the usability of homology models and the inherent selectivity of a particular model in relation to close homologs, we constructed multiple homology models for the A1 adenosine receptor (A1AR) and docked, 2.2 M lead-like compounds. High-ranking molecules were tested on the A1AR as well as the close homologs A2AAR and A3AR. While the screen yielded numerous potent and novel ligands (hit rate 21% and highest affinity of 400 nM), it delivered few selective compounds. Moreover, most compounds appeared in the top ranks of only one model. The structure−activity relationship (SAR) for a novel class of 1,2,4- triazole antagonists of the human A2A adenosine receptor (hA2A AR) was explored. Thirty-three analogs of a ligand that was discovered in a structure-based virtual screen against the hA2A AR were tested in AR radioligand binding assays and in functional assays for the A2B AR subtype. As a series of closely related analogs of the initial lead did not display improved binding affinity or selectivity, molecular docking was used to guide the selection of more distantly related molecules. This resulted in the discovery of novel AR antagonists (Ki ≥200 nM) with high ligand efficiency. In light of the SAR for the 1,2,4-triazole scaffold, we also investigated the binding mode of these compounds based on docking to several A2AAR crystal structures The P2Y1 receptor (P2Y1R) is a G protein-coupled receptor naturally activated by extracellular ADP. Its stimulation is an essential requirement of ADP-induced platelet aggregation, thus making antagonists highly sought compounds for the development of antithrombotic agents. Here, through a virtual screening campaign based on a pharmacophoric representation of the common characteristics of known P2Y1R ligands and the putative shape and size of the receptor binding pocket, we have identified novel antagonist hits of microM affinity derived from a N,N-bis-arylurea chemotype. Unlike the vast majority of known P2Y1R antagonists, these drug-like compounds do not have a nucleotidic scaffold or highly negatively charged phosphate groups. Hence, our compounds may provide a direction for the development of receptor probes with altered physicochemical properties. The P2Y12R is an ADP-activated GPCR that is well validated clinically as an important target for antithrombotic drugs. Three homology models of P2Y12R were compared based on different GPCR structural templates: bovine rhodopsin, human A2AAR and CXCR4R. The CXCR4-based model appeared to be the most consistent with known characteristics of P2Y12R. This conclusion was based on various criteria: sequence analysis, deviation from helicity in the second transmembrane helix (TM2), poses of docked ligands highlighting the role of key residues, the accessibility of a conserved disulfide bridge that is reactive toward irreversibly-binding antagonists, and the presence of a shared disulfide bridge between the third extracellular loop (EL3) and the N-terminus. Thus, ligand docking to the CXCR4-based model of the P2Y12R predicted poses of both reversibly and irreversibly-binding small molecules, consistent with observed pharmacology and mutagenesis studies. Farnesyl pyrophosphate was recently identified as an insurmountable antagonist of ADP-induced platelet aggregation mediated by the P2Y12R. Docking of farnesyl pyrophosphate in a P2Y12R model revealed molecular similarities with ADP and a good fit into the binding pocket for ADP.

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