Current Pharmaceutical Design - Volume 12, Issue 14, 2006

G protein-coupled receptors (GPCRs) represent the largest known single gene family in the human genome. This superfamily of cell-surface proteins also constitutes the major target for approximately half the medicines on the market today. This issue of Current Pharmaceutical Design focuses on different aspects of the biology and the study of these receptors, with a broad emphasis on how these studies are influencing the modern drug discovery process. In her article, Evi Kostenis [2] presents an overview of functional assay systems for GPCRs that take advantage of the best-known signal transduction system associated with these receptors, namely, that of the G proteins themselves. The ability to translate ligand dependent and independent modulation of GPCRs into a measurable functional response in a manner that is as portable across GPCR systems as possible remains an important priority, and challenge, to GPCR-based drug discovery. The article describes how the manipulation of G protein alpha subunits, in particular, has been used to address this issue with respect to high throughput screening of GPCRs. Ethan Burstein and colleagues [3] focus on the emergence of chemical genomics, which combines genomic information, combinatorial chemistry and functional high throughput screening to accelerate drug discovery. In particular, the utility of functional assay platforms that allow a common screening approach against the widest possible array of genomic targets are discussed. The role of GPCR constitutive activity, intracellular signalling promiscuity, and G protein independent signaling are discussed within the context of general strategies for creating homogeneous assay platforms. Jen Estall and Daniel Drucker [4] focus on a specific subset of GPCRs, those targeted by glucagon and glucagonlike peptides, and discuss the potential of these receptors as drug targets. These receptors play essential roles in energy intake, absorption and disposal, and are currently hot targets with respect to diabetes, the regulation of food intake and related metabolic conditions both in the central nervous system and the periphery. The current state of knowledge with respect to selective agonists and antagonists, as well as specific aspects of the regulation of this system, are discussed. In their article, Spiro Pavlopoulos and colleagues [5] focus on another specific GPCR family that has also emerged as a very promising therapeutic target in recent years, namely the cannabinoid CB1 and CB2 receptors. Although there has been anectodal evidence for medicinal properties of cannabis and cannabis-related compounds for centuries, it is only in the modern molecular era that significant attempts have been made to delineate the biology of the receptors for these compounds and to design compounds that maintain therapeutic efficacy while minimizing and/or eliminating unwanted psychoactive effects. The discovery of endogenous cannabinoid compounds has greatly facilitated this process, and the article focuses on essential pharmacophoric elements that can be used to lead to selective signalling at cannabinoid receptors. Lei Shi and Jonathan Javitch [6] turn their attention to the area of informatics as applied to the study of GPCRs. Specifically, they highlight the key role for informatic approaches utilizing sequence information, mutagenesis data and the vast published literature to facilitate studies of GPCRs in the current climate characterized by a dearth of atomic resolution models. General approaches for integrating the vast amounts of information already available in databases and the literature, and the application to structure-funciton studies of GPCRs, are presented. Bryan Roth and Wesley Kroeze [7] draw attention to the wealth of therapeutic targets potentially available in the "receptorome", i.e., that portion of the human genome that encodes all receptors. By using a series of illuminating case studies, they demonstrate how massively parallel screening of the receptorome is a powerful drug discovery platform that can validate drug targets and yield novel information not only on mechanisms of drug action, but also on previously poorly understood mechanisms underlying drug side-effects. In addition, an overview of useful GPCR-related databases is also provided. Finally, Ignacio Torrecilla and Andrew Tobin [8] highlight the importance of delineating mechanisms that regulate GPCRs beyond the acute effects mediated by drugs that either activate or inhibit these receptors. In particular, their article focuses on the dynamic mechanisms that underlie covalent modifications of these receptors mediated by the processes of phosphorylation, palmitoylation and ubiquitination; it is envisaged that these pathways can provide alternative targets to the GPCRs themselves in the drug discovery process.We are extremely grateful to all our contributors for their efforts and patience during the preparation of this issue, and believe that it will serve as a timely overview of much of the state of play in GPCR-based drug discovery. References [1] Werry TD, Christopoulos A, Sexton PM. Mechanisms of ERK1/2 Regulation by Seven-Transmembrane- Domain Receptors. Curr Pham Design 2006; 12(14): 1683-1702. [2] Kostenis E. G Proteins in Drug Screening: From Analysis of Receptor-G Protein Specificity to Manipulation of GPCR-Mediated Signalling Pathways. Curr Pham Design 2006; 12(14): 1703-1715. [3] Burstein ES, Piu F, Ma J-N, Weissman JT, Currier EA, Nash NR, Weiner DM, Spalding TA, Schiffer HH, Tredici ALD, Brann MR. Integrative Functional Assays, Chemical Genomics and High Throughput Screening: Harnessing Signal Transduction Pathways to a Common HTS Readout. Curr Pham Design 2006; 12(14): 1717-1729. [4] Estall JL, Drucker DJ. Glucagon and Glucagon-Like Peptide Receptors as Drug Targets. Curr Pham Design 2006; 12(14): 1731-1750. [5] Pavlopoulos S, Thakur GA, Nikas SP, Makriyannis A. Cannabinoid Receptors as Therapeutic Targets. Curr Pham Design 2006; 12(14): 1751-1769. [6] Shi L, Javitch JA. A Role for Information Collection, Management, and Integration in Structure-Function Studies of G-Protein Coupled Receptors. Curr Pham Design 2006; 12(14): 1771-1783. [7] Roth BL, Kroeze WK. Screening the Receptorome Yields Validated Molecular Targets for Drug Discovery. Curr Pham Design 2006; 12(14): 1785-1795. [8] Torrecilla I, Tobin AB. Co-Ordinated Covalent Modification of G-Protein Coupled Receptors. Curr Pham Design 2006; 12(14): 1797-1808.

Control of cell growth and differentiation has long been a focus of intense research interest, particularly in the context of cancer therapeutics. The evolutionarily-conserved extracellular signal-regulated kinases 1 and 2 (ERK1/2) are serine-threonine kinases that respond to a wide range of mitogens and growth factors to initiate changes in cellular proliferation and differentiation, and are the most important members of the mitogen-activated protein kinase (MAPK) family in terms of seven transmembrane-domain receptor (7TMR)-mediated regulation of mitogenic processes. Regulation of the ERK1/2 signaling cascade by 7TMRs is highly complex and cell type-specific. Recent advances in our knowledge of this effector pathway have revealed that its regulation is at least partly independent of traditional G protein-mediated actions arising from the stimulation of 7TMRs. This review summarizes the current position of our knowledge of ERK1/2 regulation, and illustrates the wealth of potential targets available for the development of new strategies for the treatment of proliferative and other ERK-related disorders.

Seven transmembrane G protein coupled receptors (7TM GPCRs) represent one of the largest gene familes in the human genome. Because of the size of the GPCR family, their proven history of being valuable targets for small molecule drug design, the fact that the absolute number of GPCRs that are targets for current medicines represents only a small fraction of the total encoded by the human genome, and that ligands for GPCRs do not have to enter the cell to exert their function, it is very likely that GPCRs will remain major targets for the pharmaceutical industry in the foreseeable future. Despite recent evidence indicating that GPCRs can provide information to cells, that does not require activation of G proteins ("signaling at zero G"), most of the GPCRs known to date function via interaction with and activation of heterotrimeric (αβγ) G proteins. Thus, assay systems translating ligand modulation of GPCRs into G protein-dependent intracellular responses are a key component of both basic research and the drug discovery process. This article will review the current knowledge and recent progress in understanding molecular aspects of specific receptor-G protein recognition. It will also highlight how the knowledge generated by such studies can be transformed into assay systems for GPCR drug discovery.

Chemical genomics is a drug discovery strategy that relies heavily on high-throughput screening (HTS) and therefore benefits from functional assay platforms that allow HTS against all relevant genomic targets. Receptor Selection and Amplification Technology (R-SAT™) is a cell-based, high-throughput functional assay where the receptor stimulus is translated into a measurable cellular response through an extensive signaling cascade occurring over several days. The large biological and chronological separation of stimulus from response provides numerous opportunities for enabling assays and increasing assay sensitivity. Here we review strategies for building homogeneous assay platforms across large gene families by redirecting and/or amplifying signal transduction pathways.

Glucagon and the glucagon-like peptides are derived from a common proglucagon precursor, and regulate energy homeostasis through interaction with a family of distinct G protein coupled receptors. Three proglucagon-derived peptides, glucagon, GLP-1, and GLP-2, play important roles in energy intake, absorption, and disposal, as elucidated through studies utilizing peptide antagonists and receptor knockout mice. The essential role of glucagon in the control of hepatic glucose production, taken together with data from studies employing glucagon antagonists, glucagon receptor antisense oligonucleotides, and glucagon receptor knockout mice, suggest that reducing glucagon action may be a useful strategy for the treatment of type 2 diabetes. GLP-1 secreted from gut endocrine cells controls glucose homeostasis through glucose-dependent enhancement of β-cell function and reduction of glucagon secretion and gastric emptying. GLP-1 administration is also associated with reduction of food intake, prevention of weight gain, and expansion of β-cell mass through stimulation of β-cell proliferation, and prevention of apoptosis. GLP-1R agonists, as well as enzyme inhibitors that prevent GLP-1 degradation, are in late stage clinical trials for the treatment of type 2 diabetes. Exenatide (Exendin-4) has been approved for the treatment of type 2 diabetes in the United States in April 2005. GLP-2 promotes energy absorption, inhibits gastric acid secretion and gut motility, and preserves mucosal epithelial integrity through enhancement of crypt cell proliferation and reduction of epithelial apoptosis. A GLP-2R agonist is being evaluated in clinical trials for the treatment of inflammatory bowel disease and short bowel syndrome. Taken together, the separate receptors for glucagon, GLP-1, and GLP-2 represent important targets for developing novel therapeutic agents for the treatment of disorders of energy homeostasis.

The cannabinoid receptors CB1 and CB2 are family A, G-protein Coupled Receptors that mediate the effects of cannabinoids, a class of compounds that are so named because the first members were isolates of the cannabis plant. In recent history, there has been much anecdotal evidence that the potent and diverse physiological responses produced by these compounds can be turned to therapeutic benefit for a wide variety of maladies. The remarkable abundance of cannabinoid receptors and the discovery of several endogenous ligands along with enzyme and transporter proteins for which they are substrates, suggests that an endogenous cannabinoid neuromodulatory system is an important mediator of biological function. For these reasons CB1 and CB2 receptors are attractive targets for the design of therapeutic ligands. The action of these receptors, however, may also be modulated by manipulating the enzymes and membrane transporters that regulate the endogenous ligands. Despite the range of physiological processes and activities that are mediated by cannabinoid receptors, it is clear that it is possible to produce ligands that result in differential responses. In this paper, we review the pharmacophoric elements that lead to these differential responses and in order to discuss them in context we present an overview of structural aspects governing cannabinoid receptor function, the cannabinergic system and its physiological functions.

Elucidation of protein function is greatly facilitated by the availability of an atomic resolution structure or a reliable molecular model. The difficulty of obtaining atomic resolution structures of membrane proteins in general, and of G-protein coupled receptors (GPCRs) in particular, has made the information available from sequence analysis, mutagenesis, and the literature on related GPCRs exceptionally important. Here, we review previous studies of GPCR structurefunction from the perspectives of sequence analysis, management of mutagenesis and ligand binding data, and literature data mining. The knowledge derived from these information resources not only constitutes the prerequisites for reliable molecular modeling, but also can provide other insights into GPCR functions. Finally, we review approaches for information integration and applying knowledge discovery techniques to structure-function studies of GPCRs, including molecular modeling itself.

With the recently completed sequencing and annotation of the human genome, it has become clear that a significant portion of the genome encodes signal-transducing molecules including receptors, protein kinases, ion channels, transporters and coupling proteins. This review focuses on membrane-localized receptors, which represent the largest single group of signal-transducing molecules. Indeed, one can estimate that nearly 10% of the human genome encodes membrane- localized receptors (e.g. G-protein coupled receptors, ligand-gated ion channels and transporters). We have defined that portion of the human genome that encodes 'receptors' the receptorome. In this article, we will demonstrate how the massively parallel screening of the receptorome provides a facile and under-utilized screening platform for drug discovery. Using case studies, we will show how receptorome-based screening elucidates the mechanisms responsible for serious side-effects of both approved and investigational medications. Additionally, we will provide evidence that receptorome- based screening provides insights into novel therapeutic indications of approved medications and serves to validate targets for therapeutic drug discovery.

The G-protein coupled receptor (GPCR) gene family represents one of the largest families in the mammalian genome. The flexibility of signalling and widespread tissue distribution of these receptors has allowed GPCRs to be employed in the physiological regulation of nearly all biological functions. This, coupled with the fact that it is possible to chemically produce highly specific ligands to these receptors have made GPCRs attractive targets for pharmacological intervention in a wide variety of disease states. When targeting GPCRs in therapeutic drug design it is traditional, and eminently sensible, to focus on ligands that will provide agonism, antagonism or allosteric modulation. However, as more is understood of the mechanisms that regulate GPCRs, and in particular the dynamic covalent modifications that might endow tissue specific functions, then these regulatory processes may provide alternative targets for GPCR drug discovery. In this review we consider three of the covalent modifications which are considered to regulate the function of GPCRs namely; receptor phosphorylation, palmitoylation and ubiquitination. In particular, we will describe the mechanisms of modification, the functional consequences and the relationship between these three covalent modification events