In the course of this fiscal year, we have worked on the GPCR systems described in the following paragraphs. Some of these systems are very well characterized in the literature, where a wealth of information, including experimentally derived structures, can be found. Thus, they constitute an ideal platform for the development of computational methodologies subsequently applicable to the whole superfamily. Other systems, instead, are less well characterized but constitute attractive targets for the development of pharmaceutical agents. Rhodopsin. Rhodopsin is a GPCR activated by light, which causes the isomerization of the covalently bound 11-cis-retinal to all-trans-retinal, consequently triggering the activation of the receptor. Beta-adrenergic receptors. The beta-adrenergic receptors (beta-ARs) reside predominantly in smooth muscles and play crucial roles in the physiology of heart and airways. Antagonists of the beta-ARs are widely used for various indications, particularly the treatment of hypertension and cardiac arrhythmias. Agonists of the beta2-AR are clinically used in the treatment of asthma. Adenosine receptors. The adenosine receptors are widely expressed in several organs of the human body, and mediate important physiological functions in the heart, lungs, blood vessels, and platelets. Muscarinic receptors. The muscarinic receptors are a family of GPCRs stimulated by acetylcholine. Ligands of the muscarinic receptors are amply used for the treatment of a variety of conditions, including Parkinsons disease. P2Y receptors. P2Y receptors are GPCRs activated by extracellular nucleotides. Of note, antagonists of the P2Y12 receptor are amply used as antithrombotic agents. TRH-Rs. Thyrotropin-releasing hormone (THR) is a tripeptide hormone which stimulates the release of thyrotropin by activating specific GPCRs known as thyrotropin-releasing hormone receptors (TRH-Rs). In particular, during this fiscal year, we have conducted the research and accomplished the results described in the following paragraphs. 1) Rationalized the structural basis of the selectivity of the beta2-adrenergic receptors for fluorinated catecholamines, through molecular modeling-guided site directed mutagenesis experiments. Experimental collaborators: Jurgen Wess (NIDDK) and Kenneth Kirk (NIDDK). 2) Finalized and published a unique review article that covers, in a systematic manner, over a century of GPCR history. 3) Finalized and published a review describing the use of NMR spectroscopy to unravel the structure-function relationships of GPCRs. 4) Worked on controlled a posteriori virtual screening experiments for beta2-adrenergic receptors ligands. Notably, we devised a way of steering the screening towards the identification of agonists or blockers. Moreover, in collaboration with Claudio N. Cavasotto (University of Texas), we improved the performance of the screenings by incorporating the flexibility of the receptor into the virtual screening process. 5) Worked on a strategy to computationally classify ligands of the adrenergic receptors into agonists and blockers. Notably, the study furnished also an insightful view into the mechanism of agonist binding. 6) Reviewed the possibility of modeling G protein-coupled receptors. 7) Work on a chapter on virtual screening for GPCR ligands, to be published in a book edited by the Royal Chemical Society. 8) Conducted a controlled virtual screening for agonists of the TRH-R receptor. 9) Conducted a bioinformatics study, in collaboration with Carson C. Chow (NIDDK), intended to shed light onto the evolution of the GPCR superfamily. 10) Conducted computer-assisted design of analogs of the adenosine A2A receptor. Experimental collaborators: Kenneth A. Jacobson (NIDDK) and Giampiero Spalluto (University of Trieste, Italy). 11) Discovered compounds with enhanced selectivity for the P2Y6 receptor through the molecular modeling-guided chemical engineering of nucleotides. Experimental collaborators: Kenneth A. Jacobson (NIDDK). 12) Conducted computer-assisted design of analogs of novel antagonists of the P2Y1 receptor previously identified by us through virtual screening. Experimental collaborators: Kenneth A. Jacobson (NIDDK) and T. Kendall Harden (University of North Carolina). 13) Rationalized the mechanism of binding of functionalized congeners of P2Y1 agonists. Experimental collaborators: Kenneth A. Jacobson (NIDDK). 14) Generated a structural model of macromolecular complex formed by the muscarinic M3 receptor coupled to the Gq heterotrimer through molecular modeling guided by biochemical cross-linking experiments. Experimental collaborators: Jurgen Wess (NIDDK). 15) Conducted structure-based design of TRH-R ligands. Experimental collaborators: Marvin C. Gershengorn (NIDDK).

Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2010
Total Cost
$311,583
Indirect Cost
City
State
Country
Zip Code
Costanzi, Stefano; Santhosh Kumar, T; Balasubramanian, Ramachandran et al. (2012) Virtual screening leads to the discovery of novel non-nucleotide P2Y? receptor antagonists. Bioorg Med Chem 20:5254-61
Costanzi, Stefano (2012) Homology modeling of class a G protein-coupled receptors. Methods Mol Biol 857:259-79
Hu, Jianxin; Thor, Doreen; Zhou, Yaru et al. (2012) Structural aspects of Mýýý muscarinic acetylcholine receptor dimer formation and activation. FASEB J 26:604-16
Costanzi, Stefano; Vilar, Santiago (2012) In silico screening for agonists and blockers of the ?(2) adrenergic receptor: implications of inactive and activated state structures. J Comput Chem 33:561-72
Jacobson, Kenneth A; Costanzi, Stefano (2012) New insights for drug design from the X-ray crystallographic structures of G-protein-coupled receptors. Mol Pharmacol 82:361-71
Maruoka, Hiroshi; Jayasekara, M P Suresh; Barrett, Matthew O et al. (2011) Pyrimidine nucleotides with 4-alkyloxyimino and terminal tetraphosphate ?-ester modifications as selective agonists of the P2Y(4) receptor. J Med Chem 54:4018-33
McMillin, Sara M; Heusel, Moritz; Liu, Tong et al. (2011) Structural basis of M3 muscarinic receptor dimer/oligomer formation. J Biol Chem 286:28584-98
Vilar, Santiago; Ferino, Giulio; Phatak, Sharangdhar S et al. (2011) Docking-based virtual screening for ligands of G protein-coupled receptors: not only crystal structures but also in silico models. J Mol Graph Model 29:614-23
Wei, Qiang; Costanzi, Stefano; Liu, Qiu-Zhen et al. (2011) Activation of the P2Y1 receptor induces apoptosis and inhibits proliferation of prostate cancer cells. Biochem Pharmacol 82:418-25
Jacobson, Kenneth A; Deflorian, Francesca; Mishra, Shilpi et al. (2011) Pharmacochemistry of the platelet purinergic receptors. Purinergic Signal 7:305-24

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