Can molecular dynamics simulations improve the structural accuracy and virtual screening performance of GPCR models?

The determination of G protein-coupled receptor (GPCR) structures at atomic resolution has improved understanding of cellular signaling and will accelerate the development of new drug candidates. However, experimental structures still remain unavailable for a majority of the GPCR family. GPCR structures and their interactions with ligands can also be modelled computationally, but such predictions have limited accuracy. In this work, we explored if molecular dynamics (MD) simulations could be used to refine the accuracy of in silico models of receptor-ligand complexes that were submitted to a community-wide assessment of GPCR structure prediction (GPCR Dock). Two simulation protocols were used to refine 30 models of the D3 dopamine receptor (D3R) in complex with an antagonist. Close to 60 µs of simulation time was generated and the resulting MD refined models were compared to a D3R crystal structure. In the MD simulations, the receptor models generally drifted further away from the crystal structure conformation. However, MD refinement was able to improve the accuracy of the ligand binding mode. The best refinement protocol improved agreement with the experimentally observed ligand binding mode for a majority of the models. Receptor structures with improved virtual screening performance, which was assessed by molecular docking of ligands and decoys, could also be identified among the MD refined models. Application of weak restraints to the transmembrane helixes in the MD simulations further improved predictions of the ligand binding mode and second extracellular loop. These results provide guidelines for application of MD refinement in prediction of GPCR-ligand complexes and directions for further method development.

Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts.

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-ß-cyclodextrin (methyl-ß-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

GPCRmd uncovers the dynamics of the 3D-GPCRome.

G-protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new three-dimensional (3D) molecular structures of GPCRs (3D-GPCRome) over the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. Molecular dynamics (MD) simulations have become a widely established technique for exploring the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations require efficient storage resources and specialized software. Here we present GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyze and share GPCR MD data. GPCRmd originates from a community-driven effort to create an open, interactive and standardized database of GPCR MD simulations.

Publisher Correction: GPCRmd uncovers the dynamics of the 3D-GPCRome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Performance of virtual screening against GPCR homology models: Impact of template selection and treatment of binding site plasticity.

Rational drug design for G protein-coupled receptors (GPCRs) is limited by the small number of available atomic resolution structures. We assessed the use of homology modeling to predict the structures of two therapeutically relevant GPCRs and strategies to improve the performance of virtual screening against modeled binding sites. Homology models of the D2 dopamine (D2R) and serotonin 5-HT2A receptors (5-HT2AR) were generated based on crystal structures of 16 different GPCRs. Comparison of the homology models to D2R and 5-HT2AR crystal structures showed that accurate predictions could be obtained, but not necessarily using the most closely related template. Assessment of virtual screening performance was based on molecular docking of ligands and decoys. The results demonstrated that several templates and multiple models based on each of these must be evaluated to identify the optimal binding site structure. Models based on aminergic GPCRs showed substantial ligand enrichment and there was a trend toward improved virtual screening performance with increasing binding site accuracy. The best models even yielded ligand enrichment comparable to or better than that of the D2R and 5-HT2AR crystal structures. Methods to consider binding site plasticity were explored to further improve predictions. Molecular docking to ensembles of structures did not outperform the best individual binding site models, but could increase the diversity of hits from virtual screens and be advantageous for GPCR targets with few known ligands. Molecular dynamics refinement resulted in moderate improvements of structural accuracy and the virtual screening performance of snapshots was either comparable to or worse than that of the raw homology models. These results provide guidelines for successful application of structure-based ligand discovery using GPCR homology models.

Synthesis, molecular modelling studies and biological evaluation of new oxoeicosanoid receptor 1 agonists.

The oxoeicosanoid receptor 1 (OXER1) is a member of the G-protein coupled receptors (GPCR) family, and is involved in inflammatory processes and oncogenesis. As such it is an attractive target for pharmacological intervention. The present study aimed to shed light on the molecular fundaments of OXER1 modulation using chemical probes structurally related to the natural agonist 5-oxo-ETE. In a first step, 5-oxo-ETE and its closely related derivatives (5-oxo-EPE and 4-oxo-DHA) were obtained by conducting concise and high-yielding syntheses. The biological activity of obtained compounds was assessed in terms of potency (EC50) and efficacy (Emax) for arrestin recruitment. Finally, molecular modelling and simulation were used to explore binding characteristics of 5-oxo-ETE and derivatives with the aim to rationalize biological activity. Our data suggest that the tested 5-oxo-ETE derivatives (i) insert quickly into the membrane, (ii) access the receptor via transmembrane helices (TMs) 5 and 6 from the membrane side and (iii) drive potency and efficacy by differential interaction with TM5 and 7. Most importantly, we found that the methyl ester of 5-oxo-ETE (1a) showed even a higher maximum response than the natural agonist (1). In contrast, shifting the 5-oxo group into position 4 results in inactive compounds (4-oxo DHA compounds (3) and (3a)). All in all, our study provides relevant structural data that help understanding better OXER1 functionality and its modulation. The structural information presented herein will be useful for designing new lead compounds with desired signalling profiles.

Membrane cholesterol effect on the 5-HT2A receptor: Insights into the lipid-induced modulation of an antipsychotic drug target.

The serotonin 5-hydroxytryptamine 2A (5-HT2A ) receptor is a G-protein-coupled receptor (GPCR) relevant for the treatment of CNS disorders. In this regard, neuronal membrane composition in the brain plays a crucial role in the modulation of the receptor functioning. Since cholesterol is an essential component of neuronal membranes, we have studied its effect on the 5-HT2A receptor dynamics through all-atom MD simulations. We find that the presence of cholesterol in the membrane increases receptor conformational variability in most receptor segments. Importantly, detailed structural analysis indicates that conformational variability goes along with the destabilization of hydrogen bonding networks not only within the receptor but also between receptor and lipids. In addition to increased conformational variability, we also find receptor segments with reduced variability. Our analysis suggests that this increased stabilization is the result of stabilizing effects of tightly bound cholesterol molecules to the receptor surface. Our finding contributes to a better understanding of membrane-induced alterations of receptor dynamics and points to cholesterol-induced stabilizing and destabilizing effects on the conformational variability of GPCRs.

Challenges and Opportunities in Drug Discovery of Biased Ligands.

The observation of biased agonism in G protein-coupled receptors (GPCRs) has provided new approaches for the development of more efficacious and safer drugs. However, in order to rationally design biased drugs, one must understand the molecular basis of this phenomenon. Computational approaches can help in exploring the conformational universe of GPCRs and detecting conformational states with relevance for distinct functional outcomes. This information is extremely valuable for the development of new therapeutic agents that promote desired conformational receptor states and responses while avoiding the ones leading to undesired side-effects.This book chapter intends to introduce the reader to powerful computational approaches for sampling the conformational space of these receptors, focusing first on molecular dynamics and the analysis of the produced data through methods such as dimensionality reduction, Markov State Models and adaptive sampling. Then, we show how to seek for compounds that target distinct conformational states via docking and virtual screening. In addition, we describe how to detect receptor-ligand interactions that drive signaling bias and comment current challenges and opportunities of presented methods.

Membrane cholesterol access into a G-protein-coupled receptor.

Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.

In silico Exploration of the Conformational Universe of GPCRs.

The structural plasticity of G protein coupled receptors (GPCRs) leads to a conformational universe going from inactive to active receptor states with several intermediate states. Many of them have not been captured yet and their role for GPCR activation is not well understood. The study of this conformational space and the transition dynamics between different receptor populations is a major challenge in molecular biophysics. The rational design of effector molecules that target such receptor populations allows fine-tuning receptor signalling with higher specificity to produce drugs with safer therapeutic profiles. In this minireview, we outline highly conserved receptor regions which are considered determinant for the establishment of distinct receptor states. We then discuss in-silico approaches such as dimensionality reduction methods and Markov State Models to explore the GPCR conformational universe and exploit the obtained conformations through structure-based drug design.

MEMBPLUGIN: studying membrane complexity in VMD.

SUMMARY: Computer simulations are giving way to more complex and accurate studies of biological membranes by molecular dynamics (MD) simulations. The analysis of MD trajectories comprises the biophysical characterization of membrane properties or the study of protein-lipid interactions and dynamics. However, there is a lack of automated tools to analyse MD simulations of complex membrane or membrane-protein systems. Here we present MEMBPLUGIN, a plugin for the Visual Molecular Dynamics package that provides algorithms to measure a host of essential biophysical properties in simulated membranes. MEMBPLUGIN features are accessible both through a user-friendly graphical interface and as command-line procedures to be invoked in analysis scripts. AVAILABILITY AND IMPLEMENTATION: MEMBPLUGIN is a VMD extension written in Tcl. Multi-platform source code, documentation and tutorials are freely available at http://membplugin.sourceforge.net. CONTACT: toni.giorgino@isib.cnr.it or jana.selent@upf.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.