Current Pharmaceutical Design - Volume 18, Issue 2, 2012

The cells need to respond rapidly and with high fidelity to their changing environment, having in their disposition a low abundance of signaling components in cell membranes. To achieve this task, plasticity is needed. Growing evidence has shown that structural plasticity provides the cell with the required diversity. Seven-transmembrane receptors (7TMRs), also called G protein-coupled receptors (GPCRs) are the most diverse family of receptors in the human genome and the most frequent targets for prescribed therapeutics [1]. In recent years there has been a radical reconceptualization of the components and the organization of the 7TMR signaling machinery and the way they transduce signals to their effectors, with profound implications for drug design and development strategies. The original concepts of rigidity and stability have been replaced by the concept of structural plasticity of the 7TMR signaling apparatus, as the basis for understanding the 7TMR functional complexity [2]. The topics selected for the present issue illustrate the major advances and the revisions, that have been made in the biological and pharmacological conceptualization of 7TMRs, focusing in particular on biased agonism. As long as receptors were viewed as bimodal switches, which oscillate only between two alternative conformations, related to the active (R*) versus inactive (R) functional states, differences in ligand efficacy were attributed to the accumulation of the stabilized single active state [1, 3]. However, when analyzed in a variety of readouts, ligands for a given 7TM receptor have been found to display distinct efficacy profiles, which cannot be accounted for by different amounts of a single active receptor conformation [4]. Data from fluorescence and spectroscopy studies have demonstrated that different ligands induce or stabilize different conformations of the receptor and modify receptor's interaction with G protein subunits [5, 6]. According to an alternative of the two-state model, heptahelical receptors may adopt several slightly different conformations, each with potentially different biochemical characteristics. Ligand stabilization of distinct receptor conformations, with different signaling properties, may allow the selective triggering of only a subset of the signaling capacity associated with the receptor activated by the endogenous related agonist [7]. This direction of the stimulus generated to a particular signaling pathway is called “stimulus trafficking” or “biased agonism” or “functional selectivity” [3, 8, 9]. The essential question is how the information contained in the ligand is conveyed, through receptor conformation change, to selective Gprotein coupling and effector signaling. Liapakis G et al. in their article provide the answer to this question by conceptualizing class A 7TMRs as allosteric proteins linked to intracellular components in a two way path. In one view, ligand binding to the receptor's pocket, stabilizes distinct active receptor conformations, which drive preferential recruitment and activation of certain effectors over others. In an alternative view, receptors in the membrane exist as preassembled complexes with G proteins and signaling partners [10]. Ligands selectively recognize specific receptor conformations present within signaling complexes of different composition [4]. In the inactive receptor conformation the transmembrane domains are arranged through a network of constraining intramolecular interactions, notably the “ionic lock”, which links the cytoplasmatic portions of transmembrane domains 3 (TM3) and 6 (TM6). Active states are achieved through disruption of ionic lock and subsequent separation of TM6 away from TM3, opening a G protein contact site in the cytoplasmic side of the 7TMR [11] and through a global toggle switch activation, with a vertical sew-saw inward movement of TM6 towards TM3 into the ligand binding pocket and outward movement of the intracellular end of TM6 opening for G-protein binding [11, 12]. In addition, interactions involve molecular microswitches, highly conserved residues with different interactions in the active vs. inactive states [12]. Novel methodologies, such as bioluminescence (BRET) or fluorescence (FRET) resonance energy transfer assays, based on labelling interacting signaling partners with energy donors or acceptors and measuring energy transfer between these intermolecular labels, have allowed the inference of spatial proximity between signalling components. Such approaches have permitted the monitoring of conformational changes in the components of signaling complexes and the study of spatiotemporal dynamics of the 7TMR signaling machinery in real time as analyzed elegantly by Denis C et al. in this issue....

G protein receptor kinase 2 (GRK2) has been for years mainly considered the negative regulator of the cardiac β adrenergic signaling. However GRK2 is a ubiquitous molecule and its kinase activity and scaffold properties brought to several investigations which have evidenced its involvement in pathophysiology of extra-cardiac diseases. Later discoveries, moreover, indicated that this molecule is also able to influence other pathways such as insulin signaling by an inhibitory role similar to what described years before on βAR signaling. The importance of this novel function is in particular related to the possibility that this molecule can regulate the cellular metabolism, modifying the ability of cells to utilize different substrates. This hypothesis has been recently investigated in animal model of Heart Failure, evidencing that upregulation of GRK2 leads to alterations of cardiac glucose metabolism in the early stages of the disease. However GRK2 shows increased level also in the early stages of others chronic disease such as Alzheimer's Disease, indicating that these findings could be possibly applied to others cellular system and supporting the emerging idea of GRK2 as master regulator of cellular metabolism.

Cell surface G protein-coupled receptors (GPCRs) drive numerous signaling pathways involved in the regulation of a broad range of physiologic processes. Today, they represent the largest target for modern drugs development with potential application in all clinical fields. Recently, the concept of “ligand-directed trafficking” has led to a conceptual revolution in pharmacological theory, thus opening new avenues for drug discovery. Accordingly, GPCRs do not function as simple on-off switch but rather as filters capable of selecting the activation of specific signals and thus generating texture responses to ligands, a phenomenon often referred to as ligand-biased signaling. Also, one challenging task today remains optimization of pharmacological assays with increased sensitivity so to better appreciate the inherent texture of ligands. However, considering that a single receptor has pleiotropic signaling properties and that each signal can crosstalk at different levels, biased activity remains thus difficult to evaluate. One strategy to overcome these limitations would be examining the initial steps following receptor activation. Even, if some G protein independent functions have been recently described, heterotrimeric G protein activation remains a general hallmark for all GPCRs families and the first cellular event subsequent to agonist binding to the receptor. Herein, we review the different methodologies classically used or recently developed to monitor G protein activation and discussed them in the context of G protein biased-ligands

Components of 7TMR signaling machinery once considered as rigid, fixed and inflexible entities, operating in a onedimensional way, homogeneous spatially and temporally, are now proved to be structurally plastic, flexible and dynamic in space and time. 7TMRs are thought to exist as ensembles of multiple, inter-convertible, pre-existing conformations and this conformational diversity provides a structural plasticity and the molecular mechanism for the functional diversity of 7TMRs. Furthermore, 7TMRs appear to function not as monomers, but rather as higher order structures, within which allosteric mechanisms affect ligand binding, G protein selection and receptor mobility and signalling of GPCR protomers. Moreover, their function is regulated through compartmentalization of G protein receptor and effector molecules into specialized membrane microdomains, such as lipid rafts and caveolae. Different permutations of receptor localization and translocation in membrane microdomains and internalization patterns, employed by 7TMRs regulate their signalling. Finally, the previous clear distinction between cell signalling and endocytic membrane trafficking has been blurred by evidence that these processes are intertwined and bidirectionally linked. 7TMRs normally signaling from the plasma membrane can elicit signaling cascades from an intracellular location, with distinct biochemical outputs. All these developments have moved the search for selective drugs beyond the mere design of receptor subtype-selective ligands targeting their orthosteric site. Even the notion of ligand has been expanded, so as to include “superagonists” ”, acting as “super” 7TMR agonists to confer sustained endosomal signaling and biased agonists targeting GPCRs signalling from intracellular sites, as well as pharmacochaperones restoring insufficiently folded or misfolded receptors. The plasticity of the 7TMR signaling machinery and its dynamics in space and time will most probably impact further on the search for 7TMR drug selectivity.

The Protease-Activated Receptors ( PARs ) are G-protein-coupled receptors ( GPCRs ) characterized by a unique mechanism of activation. They carry built in their extended N-terminal structure their own activating agonist, in the form of a cryptic tethered ligand , unmasked by an irreversible proteolytic cleavage. Besides, PARs display several other particular properties, that converge and create interacting and intertwined layers of molecular processes regulating receptor's selective signaling with important biological and pharmacological consequences. These include the operation of multiple proteases, co-factors and protease inhibitors expressed in many types of cells and tissues, creating a dynamic balance between activators and inhibitors of PAR function in a tissue specific way. Membrane microdomain compartmentalization and allosteric modulation through intermolecular interactions between PARs adds further complexity to the receptor signaling and desensitization. Furthermore, molecular components interacting with thrombin and PARs take on new roles. In particular, activated protein C (APC) forms a significant negative feedback loop for thrombin with anticoagulant properties. In addition, APC exerts anti-inflammatory and direct neuroprotective effects in vivo and in vitro. This has informed the pharmacological dissection of anticoagulant from the anti-inflammatory and neuroprotective actions of APC and the generation of engineered APC mutations with diminished risk of serious bleeding, while preserving the cytoprotective effects of APC on cells. Even more important, these advances have made possible a paradigm shift , away from a “neurocentric” and towards a “vasculo-neuronal-inflammatory model of action”, which supports novel pharmacological strategies targeting multiple disease mechanisms.

G-protein coupled receptors (GPCRs) comprise the largest family of proteins in our body, which have many important physiological functions and are implicated in the pathophysiology of many serious diseases. GPCRs therefore are significant targets in pharmaceutical research. GPCRs share the common architecture of seven plasma membrane-spanning segments connected to each other with three extracellular and three intracellular loops. In addition, GPCRs contain an extracellular N-terminal region and an intracellular C-terminal tail. GPCRs could stimulate different intracellular G-proteins (internal stimuli) and signaling pathways after their interaction with different ligands (external stimuli). The exceptional functional plasticity of GPCRs could be attributed to their inherent dynamic nature to adopt different active conformations, which are stabilized differentially by different stimuli as well as by several mutations. This review describes the structural changes of GPCRs associated with their activation. Understanding the dynamic nature of GPCRs could potentially contribute in the development of future structure-based approaches to design new receptor-specific, signaling-selective ligands, which will enrich the pharmaceutical armamentarium against various diseases

Despite significant advances in pharmacological and clinical treatment, heart failure (HF) remains the number one killer disease in the western world. HF is a chronic and progressive clinical syndrome mainly characterized by reduction in left ventricular ejection fraction and adverse remodeling of the myocardium. One of its hallmark molecular abnormalities is elevation of cardiac G protein-coupled receptor (GPCR) kinase (GRK)-2, originally termed beta-adrenergic receptor kinase-1 (βARK1), a member of the GRK family of serine/threonine protein kinases which phosphorylate and desensitize GPCRs. Up-regulated GRK2 in the heart underlies the diminished contractile responsiveness of the heart to positive inotropes, as it abrogates the pro-contractile signaling of various important cardiac receptors: mainly β-adrenergic receptors (βARs), but also angiotensin II type 1 receptors (AT1Rs), etc. Thus, cardiac-specific GRK2 inhibition via various transgenic approaches is postulated to combat chronic HF symptoms by increasing cardiac function, and even be salutary in some cases by increasing survival. This has been extensively documented over the past 15 years through a vast series of preclinical studies on animals of all sizes and shapes, from small mice up to large rabbits and pigs closely resembling human physiology, and genetically manipulated to have cardiac GRK2 inhibited or deleted, transiently or permanently. However, over the past several years, it has become increasingly clear that GRK2, like other members of the GRK family, exerts additional effects that can aggravate HF, in addition to merely blunt cardiac contractility by opposing cardiac βAR G protein-mediated signaling. One of these newly discovered cardiotoxic effects of GRK2, uncovered by our laboratory, is promotion by adrenal GRK2 of sympathetic hyperactivity of the failing heart, a significant morbidity factor in HF, targeted therapeutically nowadays by the use of beta-blockers in HF pharmacotherapy. Thus, new avenues for therapeutic exploitation of GRK2 inhibition in HF treatment might be possible in the near future. The present review gives first a brief account of what has already been documented about the benefits of cardiac GRK2 genetic manipulation in HF as a positive inotropic therapy for the disease, and then goes on to discuss in detail the intriguing new possibility that has emerged of lowering GRK2 activity in the adrenal gland, which could constitute a novel sympatholytic therapy for HF that helps relieve the devastatingly cardiotoxic sympathetic overload of the failing heart.

Heptahelical, G protein-coupled or seven transmembrane-spanning receptors, such as the β-adrenergic and the angiotensin II type 1 receptors, are the most diverse and therapeutically important family of receptors in the human genome, playing major roles in the physiology of various organs/tissues including the heart and blood vessels. Ligand binding activates heterotrimeric G proteins that transmit intracellular signals by regulating effector enzymes or ion channels. G protein signaling is terminated, in large part, by phosphorylation of the agonist-bound receptor by the G-protein coupled receptor kinases (GRKs), followed by βarrestin binding, which uncouples the phosphorylated receptor from the G protein and subsequently targets the receptor for internalization. As the receptor-βarrestin complex enters the cell, βarrestin-1 and -2, the two mammalian βarrestin isoforms, serve as ligand-regulated scaffolds that recruit a host of intracellular proteins and signal transducers, thus promoting their own wave of signal transduction independently of G-proteins. A constantly increasing number of studies over the past several years have begun to uncover specific roles played by these ubiquitously expressed receptor adapter proteins in signal transduction of several important heptahelical receptors regulating the physiology of various organs/ systems, including the cardiovascular (CV) system. Thus, βarrestin-dependent signaling has increasingly been implicated in CV physiology and pathology, presenting several exciting opportunities for therapeutic intervention in the treatment of CV disorders. Additionally, the discovery of this novel mode of heptahelical receptor signaling via βarrestins has prompted a revision of classical pharmacological concepts such as receptor agonism/antagonism, as well as introduction of new terms such as “biased signaling”, which refers to ligand-specific activation of selective signal transduction pathways by the very same receptor. The present review gives an overview of the current knowledge in the field of βarrestin-dependent signaling, with a specific focus on CV heptahelical receptor βarrestin-mediated signaling and on “biased” CV heptahelical receptor ligands that promote or inhibit it. Exciting new possibilities for cardiovascular therapeutics arising from the delineation of this βarrestin-dependent signaling are also discussed.

It is with great sadness that we record here the premature death of Herve Paris, who died at his home in Toulouse, in January 23, 2010. With his death, molecular pharmacology research of GPCRs has lost one of its creative, productive and most consistent supporters. He studied in the University of Paul Sabatier, Toulouse III, France and at the University of Miami, USA. With this basic background he served at INSERM (Institut National de la Recherche Medicale, France, Toulouse) as researcher, Charge de Recherche and as Director of research, a position he held until his death. His main field of research was alpha2-adrenergic receptor pharmacology, mechanisms of signal transduction and regulation of expression. Among others, he showed in 1985, that the human colon adenocarcinoma cell-line HT 29 expresses functional alpha 2-adrenergic receptors and used it as a model cell system to characterize pharmacological properties of a2A-adrenergic receptors. He also conclusively proved that binding of the radioligand [3H]RX821002 is a valuable tool for labeling α2A-adrenoceptors. These tools were instrumental in later advances in the molecular pharmacology of alpha2-adrenergic receptor subtypes by his research group. As a colleague, I have deeply appreciated his intelligence and his exceptional scientific qualifications and as a friend I was touched by his kindness, enduring solidarity and his consistency, all these years I had the chance to know him. We dedicate this issue on biased agonism at 7TMRs to his memory. He will be deeply missed.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the degeneration of dopamine neurons in the substantia nigra pars compacta. To date, although a bulk of evidences suggest that the etiology of PD is multifactorial, none of the mechanisms yet proposed have been considered conclusive. Activated glia seem to play a critical role in the degeneration of nigral dopaminergic neurons in PD, by secreting a complex array of cytokines, chemokines, proteolytic enzymes, reactive oxygen/nitrogen species and complement proteins that may have deleterious effects on the dopaminergic system. Recently, it has been reported that microglia activation and immunity are key factors contributing to disease progression. Here, we review studies on the role of inflammation mediated by the innate and adaptive immune systems involved in the pathogenesis of PD and highlight some of the important areas for future investigation.

Recent years have broadened the spectrum of therapeutic strategies and specific agents for treatment of multiple sclerosis (MS). While immune-modulating drugs remain the first-line agents for MS predominantly due to their benign safety profile, our growing understanding of key processes in initiation and progression of MS has pioneered development of new agents with specific targets. One concept of these novel drugs is to hamper migration of immune cells towards the affected central nervous system (CNS). The first oral drug approved for MS therapy, fingolimod inhibits egress of lymphocytes from lymph nodes; the monoclonal antibody natalizumab prevents inflammatory CNS infiltration by blocking required adhesion molecules. The second concept is to deplete T cells and/or B cells from the peripheral circulation using highly specific monoclonal antibodies such as alemtuzumab (anti-CD52) or rituximab/ocrelizumab (anti-CD20). All of these novel, highly effective agents are a substantial improvement in our therapeutic armamentarium; however, they have in common to potentially lower the abundance of immune cells within the CNS, thereby collaterally affecting immune surveillance within this well-controlled compartment. In this review, we aim to critically evaluate the risk/benefit ratio of therapeutic strategies in treatment of MS with a specific focus on infectious neurological side effects.

Enkephalins play a great role in management of pain, blood pressure, hypertension and cardiovascular diseases. Enkephalins are short-lived molecules being rapidly hydrolyzed following their synaptic release by enkephalin degrading enzymes. The inhibitors of enkephalin degrading enzymes are able to prolong the duration of action of enkephalins. This review will focus on the inhibitors of enkephalin degrading enzymes as a novel therapeutic approach for cancer itself and also in cancer and neuropathic pain management with discussion on the present status and future directions for a new class of drugs.