G-protein coupled receptors: Structure, function and design of novel chemical modulators
G Protein-coupled Receptors (GPCRs) are transmembranal proteins in charge of signal transduction across cellular membranes. The mature stage of the research in this field have been recognized with the Nobel prize in Chemistry (2012), and the biomedical relevance is clear with 40% of the marketed drugs target a GPCR. Our research group has a longstanding experience on the structural biology and ligand design of GPCRs. We are currently running several projects, alone or in collaboration with experimental groups, to understand the structural basis of GPCR structure-function relationship and to assist in the ligand design process for several receptors. In the present proposal, we want to expand our computational exploration of GPCRs (project code SNIC 2015/1-304) as follows: 1- MD refinement and free energy simulations of GPCR-ligand complexes. Our webserver for the 3D structural modeling of GPCRs GPCR-ModSim (http://gpcr-modsim.org), includes a module called PyMemDyn for the membrane insertion and MD equilibration of GPCR structures and GPCR-ligand complexes. This module is installed as a standalone python wrapper for the GROMACS MD package in Triolith and has been recently updated as described in Esguerra et al, Nucl Acid Res, 2016, 44:W455. In this period we estimate to use this protocol to systematically setup several GPCR-ligand complexes arising from our multidisciplinary projects in drug design, followed by free energy calculations with the Linear Interaction Energy (LIE) method implemented in our MD software Q, in particular related to our collaboration with the medicinal chemistry lab of our collaborator Prof. Eddy Sotelo at the Santiago de Compostela University (see Rodriguez and Gutiérrez-de-Terán, Curr. Pharm. Des., 2013, 19:2216 and Gutiérrez-de-Terán et al., Methods Mol Biol, 2015, 1272:271 for a description of the flowchart and the project). This task is related with task 3C. 2- Design of peptidic agonists of the neuropeptide Y (NPY) type-2 (Y2) receptor. This project is executed in collaboration with Prof D. Larhammar (Uppsala University) and the pharmaceutical company Novo Nordisk (Denmark), and aims to identify the molecular determinants for enhanced agonistic activity of NPY and PYY family of peptides and assess the chemical modifications that enhance this activity. In the previous edition of this project (SNIC 2015/1-304) we performed long MD simulations to confirm and further refine our initial model of the Y2-PYY complex (Xu et al., Biochemistry, 2013, 52:7987), based on our PyMemDyn protocol (see previous point). As a result, we identified a series of pair interactions between receptor and peptide which were contrasted with the experimental data generated by our collaborators, based on site directed mutagenesis and chemical modifications of selected aminoacids. In the present edition we will use this result as starting point of a series of free energy perturbation (FEP) calculations performed on the positions identified, with our FEP as implemented in the Q-software installed in Triolith (Keränen, Chem. Commun., 2015, 51, 3522). With these calculations we will be able to interpret the experimental data referred above and further assess the design of new experiments for our collaborators. 3- Automation, testing and application of our MD protocol for free energy perturbations on amino acids. Our recently published tool for FEP calculations on amino acids (Keränen, Chem. Commun., 2015, 51, 3522) in combination with our GPCR-ModSim webserver (Esguerra, NAR, 2016, 44, 455) will be combined in a PyMol wrapper. This protocol will be used in several other projects with different collaborators. 3A) Benchmarking of the FEP protocol and GPCR-ModSim on crystalized GPCRs and homologous family member using collected mutagenesis data from Prof. C. de Graaf (Vrije Universiteit Amsterdam) and D. Gloriam (Copenhagen University). 3B) Characterization of energetic effect of mutations on endogenous ligand binding on adenosine receptors in cancer in collaboration with Prof. Ad IJzerman (Leiden University). 3C) Development of new ligands for the adenosine receptor with Prof. E. Sotelo (University of Santiago de Compostela), complementing the investigations described in the first point of this project. 4- Structure-based design of selective agonist ligands for the angiotensin II receptor type 2 (AT2). This program is running in collaboration with with Prof. Anders Hallberg (Medicinal Chemistry, Uppsala University). In a first stage, we used homology modeling and protein-ligand docking to understand how non-peptide antagonists designed by prof Hallberg interact with the angiotensin II receptors (AT1 and AT2, sharing 34% sequence homology). In the present proposal, we will use molecular dynamics simulations coupled to the linear interaction energy (LIE) method to assist on the design of chemical modifications of these ligand series. Furthermore, the role of key residues involved in ligand binding will be analyzed with our free energy perturbation (FEP) protocols (Keränen, Chem. Commun., 2015, 51, 3522) which will allow understanding of AT1/AT2 selectivity (NOTE: This part of the project was already proposed in our previous project SNIC 2015/1-304, but not executed due to a maternity leave of the postdoc associated to it, thus we will resume activities in this edition of the project).