Quantifying the Kinetics of Signaling and Arrestin Recruitment by Nervous System G-Protein Coupled Receptors

Neurons integrate inputs over different time and space scales. Fast excitatory synapses at boutons (ms and μm), and slow modulation over entire dendritic arbors (seconds and mm) are all ultimately combined to produce behavior. Understanding the timing of signaling events mediated by G-protein-coupled receptors is necessary to elucidate the mechanism of action of therapeutics targeting the nervous system. Measuring signaling kinetics in live cells has been transformed by the adoption of fluorescent biosensors and dyes that convert biological signals into optical signals that are conveniently recorded by microscopic imaging or by fluorescence plate readers. Quantifying the timing of signaling has now become routine with the application of equations in familiar curve fitting software to estimate the rates of signaling from the waveform. Here we describe examples of the application of these methods, including (1) Kinetic analysis of opioid signaling dynamics and partial agonism measured using cAMP and arrestin biosensors; (2) Quantifying the signaling activity of illicit synthetic cannabinoid receptor agonists measured using a fluorescent membrane potential dye; (3) Demonstration of multiplicity of arrestin functions from analysis of biosensor waveforms and quantification of the rates of these processes. These examples show how temporal analysis provides additional dimensions to enhance the understanding of GPCR signaling and therapeutic mechanisms in the nervous system.

BitterMatch: Recommendation systems for matching molecules with bitter taste receptors

Bitterness is an aversive cue elicited by thousands of chemically diverse compounds. Bitter taste may prevent consumption of foods and jeopardize drug compliance. The G protein-coupled receptors for bitter taste, TAS2Rs, have species-dependent number of subtypes and varying expression levels in extraoral tissues. Molecular recognition by TAS2R subtypes is physiologically important, and presents a challenging case study for ligand-receptor matchmaking. Inspired by hybrid recommendation systems, we developed a new set of similarity features, and created the BitterMatch algorithm that predicts associations of ligands to receptors with ~80% precision at ~50% recall. Associations for several compounds were tested in-vitro, resulting in 80% precision and 42% recall. The encouraging performance was achieved by including receptor properties and integrating experimentally determined ligand-receptor associations with chemical ligand-to-ligand similarities. BitterMatch can predict off-targets for bitter drugs, identify novel ligands and guide flavor design. Inclusion of neighbor-informed similarities improves as experimental data mounts, and provides a generalizable framework for molecule-biotarget matching.

Novel Molecular Targets of Antidepressants

Antidepressants target a variety of proteins in the central nervous system (CNS), the most important belonging to the family of G-protein coupled receptors and the family of neurotransmitter transporters. The increasing number of crystallographic structures of these proteins have significantly contributed to the knowledge of their mechanism of action, as well as to the design of new drugs. Several computational approaches such as molecular docking, molecular dynamics, and virtual screening are useful for elucidating the mechanism of drug action and are important for drug design. This review is a survey of molecular targets for antidepressants in the CNS and computer based strategies to discover novel compounds with antidepressant activity.

Disrupting GPCR Complexes with Smart Drug-like Peptides

G protein-coupled receptors (GPCRs) are a superfamily of proteins classically described as monomeric transmembrane (TM) receptors. However, increasing evidence indicates that many GPCRs form higher-order assemblies made up of monomers pertaining to identical (homo) or to various (hetero) receptors. The formation and structure of these oligomers, their physiological role and possible therapeutic applications raise a variety of issues that are currently being actively explored. In this context, synthetic peptides derived from TM domains stand out as powerful tools that can be predictably targeted to disrupt GPCR oligomers, especially at the interface level, eventually impairing their action. However, despite such potential, TM-derived, GPCR-disrupting peptides often suffer from inadequate pharmacokinetic properties, such as low bioavailability, a short half-life or rapid clearance, which put into question their therapeutic relevance and promise. In this review, we provide a comprehensive overview of GPCR complexes, with an emphasis on current studies using GPCR-disrupting peptides mimicking TM domains involved in multimerization, and we also highlight recent strategies used to achieve drug-like versions of such TM peptide candidates for therapeutic application.

Development of OPLS-AA/M Parameters for Simulations of G Protein-Coupled Receptors and Other Membrane Proteins

G protein-coupled receptors (GPCRs) and other membrane proteins are valuable drug targets, and their dynamic nature makes them attractive systems for study with molecular dynamics simulations and free energy approaches. Here, we report the development, implementation, and validation of OPLS-AA/M force field parameters to enable simulations of these systems. These efforts include the introduction of post-translational modifications including lipidations and phosphorylation. We also modify previously reported parameters for lipids to be more consistent with the OPLS-AA force field standard and extend their coverage. These new parameters are validated on a variety of test systems, with the results compared to high-level quantum mechanics calculations, experimental data, and simulations with CHARMM36m where relevant. The results demonstrate that the new parameters reliably reproduce the behavior of membrane protein systems.

Regulation of PDF Receptor signaling controlling daily locomotor rhythms in Drosophila

G protein-coupled receptors (GPCRs) trigger second messenger signaling cascades following activation by cognate ligands. GPCR signaling ceases following receptor desensitization or uncoupling from G proteins. Each day and in conjunction with ambient daylight conditions, neuropeptide PDF regulates the phase and amplitude of locomotor activity rhythms in Drosophila through its receptor, a Family B GPCR. Its time of action – when it starts signaling and when it stops – must change every day to following changing day lengths. We studied the process by which PDF Receptor (PDFR) signaling turns off in vivo, by modifying as many as half of the 28 potential sites of phosphorylation in its C terminal tail. We report that many such sites are conserved evolutionarily, and that in general their conversion to a non-phosphorylatable residue (alanine) creates a specific behavioral syndrome opposite to loss of function phenotypes previously described for pdfr. Such “gain of function” pdfr phenotypes include increases in the amplitudes of both Morning and Evening behavioral peaks as well as multi-hour delays of their phases. Such effects were most clearly associated with a few specific serine residues, and were seen following alanine-conversion of as few as one or two residues. The behavioral phenotypes produced by these PDFR sequence variants are not a consequence of changes to the pharmacological properties or of changes in their surface expression, as measured in vitro. We conclude that the mechanisms underlying termination of PDFR signaling are complex and central to an understanding of how this critical neuropeptide modulates daily rhythmic behavior.

The Two β-Arrestins Regulate Distinct Metabolic Processes: Studies with Novel Mutant Mouse Models

The two β-arrestins (β-arrestin-1 and -2; alternative names: arrestin-2 and -3, respectively) are well known for their ability to inhibit signaling via G protein-coupled receptors. However, β-arrestins can also act as signaling molecules in their own right. Although the two proteins share a high degree of sequence and structural homology, early studies with cultured cells indicated that β-arrestin-1 and -2 are not functionally redundant. Recently, the in vivo metabolic roles of the two β-arrestins have been studied using mutant mice selectively lacking either β-arrestin-1 or -2 in cell types that are of particular relevance for regulating glucose and energy homeostasis. These studies demonstrated that the β-arrestin-1 and -2 mutant mice displayed distinct metabolic phenotypes in vivo, providing further evidence for the functional heterogeneity of these two highly versatile signaling proteins.

Transient Receptor Potential (TRP) and Thermoregulation in Animals: Structural Biology and Neurophysiological Aspects

This review presents and analyzes recent scientific findings on the structure, physiology, and neurotransmission mechanisms of transient receptor potential (TRP) and their function in the thermoregulation of mammals. The aim is to better understand the functionality of these receptors and their role in maintaining the temperature of animals, or those susceptible to thermal stress. The majority of peripheral receptors are TRP cation channels formed from transmembrane proteins that function as transductors through changes in the membrane potential. TRP are classified into seven families and two groups. The data gathered for this review include controversial aspects because we do not fully know the mechanisms that operate the opening and closing of the TRP gates. Deductions, however, suggest the intervention of mechanisms related to G protein-coupled receptors, dephosphorylation, and ligands. Several questions emerge from the review as well. For example, the future uses of these data for controlling thermoregulatory disorders and the invitation to researchers to conduct more extensive studies to broaden our understanding of these mechanisms and achieve substantial advances in controlling fever, hyperthermia, and hypothermia.

Binding site identification of G protein-coupled receptors through a 3D Zernike polynomials-based method: application to C. elegans olfactory receptors

Studying the binding processes of G protein-coupled receptors (GPCRs) proteins is of particular interest both to better understand the molecular mechanisms that regulate the signaling between the extracellular and intracellular environment and for drug design purposes. In this study, we propose a new computational approach for the identification of the binding site for a specific ligand on a GPCR. The method is based on the Zernike polynomials and performs the ligand-GPCR association through a shape complementarity analysis of the local molecular surfaces. The method is parameter-free and it can distinguish, working on hundreds of experimentally GPCR-ligand complexes, binding pockets from randomly sampled regions on the receptor surface, obtaining an Area Under ROC curve of 0.77. Given its importance both as a model organism and in terms of applications, we thus investigated the olfactory receptors of the C. elegans, building a list of associations between 21 GPCRs belonging to its olfactory neurons and a set of possible ligands. Thus, we can not only carry out rapid and efficient screenings of drugs proposed for GPCRs, key targets in many pathologies, but also we laid the groundwork for computational mutagenesis processes, aimed at increasing or decreasing the binding affinity between ligands and receptors.