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. The crystal structure of the A2A subtype of the adenosine receptor has been recently crystallized in complex with the potent antagonist ZM241385. In coordination with the solution of the A2A structure, a blind assessment of GPCR structure modeling and docking has been organized by the same authors, who asked modelers from all around the world to submit their prediction of the A2A receptor in complex with ZM241385. Among all those submitted, my models resulted to be the most accurate, with very low RMSDs between experimental and predicted structures for both protein and ligand. In line with what was published by me last year, this experiment demonstrated that homology modeling and molecular docking can be effectively used to study the interactions of GPCRs with their ligands. The results have recently been published in Nature Reviews Drug Discovery. 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. With rhodopsin, the beta-ARs are the most studied GPCRs and constitute an ideal platform for the development of computational methodologies subsequently applicable to the whole superfamily. In the course of this fiscal year, we published the development of a series of regression models that combine ligand-based and structure-based techniques for the prediction of ligand-receptor affinities, applicable to virtual screenings aimed at identifying new active molecules and to the design of more potent analogs. We are currently running a series of controlled, a posteriori, virtual screenings using the crystal structure as well as homology models of the beta2-AR. The results indicate that, molecular docking is very effective in prioritizing true binders versus non-binders, not only with the crystal structure, but also when using homology models. We are currently studying the possibility of incorporating the flexibility of the receptor into the virtual screening process. 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. As stated, rhodopsin, together with the adrenergic receptors, is the most well characterized GPCR. In the course of this fiscal year, we worked on a review entitled """"""""Rhodopsin and the others: a historical perspective on structural studies of G protein-coupled receptors"""""""". This unique article covers in a systematic manner over a century of history, illustrating the amount of structural information that, since the late eighteen hundreds, has been accumulated for rhodopsin, providing a platform for the study of the structure-function relationships of the whole superfamily. Moreover, we continued our molecular dynamics simulations intended to uncover the structure-function relationships of rhodopsin, this time concentrating on the role of water molecules in the activation process. Preliminary results suggest a functional role of water molecules in the conformational changes that lead from the inactive to the active state of the receptor. P2Y receptors. P2Y receptors are GPCRs activated by extracellular nucleotides. Of particular interest are P2Y1 antagonists, which have shown the potential of being developed into novel potent antithrombotic agents. In collaboration with the laboratories of Drs. Kenneth Jacobson (NIDDK) and Kendall Harden (University of North Carolina), for the past few years I have been conducting bioinformatics and molecular modeling studies intended to shed light onto the structure-function relationships of the P2Y receptors. In the course of this fiscal year, as a result of a virtual screening conducted by us, we identified novel diverse ligands of the P2Y1 receptor. We are currently designing analogs of these hits, with the aim of improving their affinity of the receptor. Moreover, molecular docking of novel analogs, combined with NMR experiments, allowed us to further understand the process of molecular recognition for this receptor, in particular with regard to the conformational requirements. Work in this direction is still in progress. FFAR1 (a.k.a. GPR40). FFAR1 (a.k.a. GPR40) is a GPCR activated by long chain free fatty acids (FFAs), which is involved in the regulation of metabolic processes and glucose homeostasis. In particular, FFAR1 is considered an appealing target for the treatment of type two diabetes. Our research, in collaboration with the laboratory of Dr. Marvin Gershengorn (NIDDK), is intended for the elucidation of the structure-function relationships of FFAR1 and for the discovery of novel ligands. In the course of this fiscal year, we published the identification of two potential ionic locks between the second extracellular loop and the transmembrane domain, the disruption of which possibly triggers the activation of the receptor. These results are very much in line with the above mentioned observations concerning the requirement of freedom of motion of the second extracellular loop for the activation of the adrenergic receptors, suggesting that a common activation mechanism might be shared by several members of the GPCR superfamily. 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). Two different TRH-R subtypes have been identified in rodents. The goal of our research, in collaboration with the laboratory of Dr. Marvin Gershengorn (NIDDK), is the elucidation of the structure-function relationships of the two subtypes and the identification of novel ligands able to discriminate against them. Combining molecular modeling with site-directed mutagenesis experiments, we are currently investigating the functional role of the C-terminal domain of the receptor.
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