The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery

G protein-coupled receptors (GPCRs) are one of the most important target classes in the central nervous system (CNS) drug discovery, however the fact they are integral membrane proteins and are unstable when purified out of the cell precludes them from a wide range of structural and biophysical techniques that are used for soluble proteins. In this study we demonstrate how protein engineering methods can be used to identify mutations which can both increase the thermostability of receptors, when purified in detergent, as well as biasing the receptor towards a specific physiologically relevant conformational state. We demonstrate this method for the adenosine A_2A receptor and muscarinic M₁ receptor. The resultant stabilised receptors (known as StaRs) have a pharmacological profile consistent with the inverse agonist conformation. The stabilised receptors can be purified in large quantities, whilst retaining correct folding, thus generating reagents suitable for a broad range of structural and biophysical studies. In the case of the A_2A-StaR we demonstrate that surface plasmon resonance can be used to profile the association and dissociation rates of a range of antagonists, a technique that can be used to improve the in vivo efficacy of receptor antagonists.     

Allosteric modulation of G protein-coupled receptors: A pharmacological perspective

G protein-coupled receptor (GPCR)-based drug discovery has traditionally focused on targeting the orthosteric site for the endogenous agonist. However, many GPCRs possess allosteric sites that offer enormous potential for greater selectivity in drug action. The complex behaviors ascribed to allosteric ligands also present challenges to those interested in preclinical lead discovery. These challenges include the need to detect and quantify various phenomena when screening for allosteric ligands, such as saturability of effect, probe dependence, differential effects on orthosteric ligand affinity vs. efficacy, system-dependent allosteric agonism, stimulus-bias (functional selectivity), and the potential existence of bitopic (hybrid orthosteric/allosteric) ligands. These issues are also critical when interpreting structure-function studies of allosteric GPCR modulators because mutations in receptor structure, either engineered or naturally occurring, can differentially affect not only modulator affinity, but also the nature, magnitude and direction of the allosteric effect on orthosteric ligand function. The ever-expanding array of allosteric modulators arising from both academic and industrial research also highlights the need for the development of a uniform approach to nomenclature of such compounds.     

针对G蛋白偶联受体的药物筛选新方法

G蛋白偶联受体（GPCR）为具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质家族,其为极重要的药物靶点。本文针对GPCR的固有激活和变构效应的药物筛选模型开发新进展和高内涵药物筛选新技术进行综述。     

Advances in pharmacogenomics and individualized drug therapy: exciting challenges that lie ahead

Between the 1930s and 1990s, several dozen predominantly monogenic, high-penetrance disorders involving pharmacogenetics were described, fueling the crusade that gene–drug interactions are quite simple. Then, in 1990, the Human Genome Project was established; in 1995, the term pharmacogenomics was introduced; finally, the complexities of determining an unequivocal phenotype, as well as an unequivocal genotype, have recently become apparent. Since 1965, more than 1000 reviews on this topic have painted an overly optimistic picture—suggesting that the advent of individualized drug therapy used by the practicing physician is fast approaching. For many reasons listed here, however, we emphasize that these high expectations must be tempered. We now realize that the nucleotide sequence of the genome represents only a starting point from which we must proceed to a more difficult stage: knowledge of the function encoded and how this affects the phenotype. To achieve individualized drug therapy, a high level of accuracy and precision is required of any clinical test proposed in human patients. Finally, we suggest that metabonomics, perhaps in combination with proteomics, might complement genomics in eventually helping us to achieve individualized drug therapy.     

G protein-coupled receptors: novel targets for drug discovery in cancer

G protein-coupled receptors (GPCRs) belong to a superfamily of cell surface signalling proteins that have a pivotal role in many physiological functions and in multiple diseases, including the development of cancer and cancer metastasis. Current drugs that target GPCRs - many of which have excellent therapeutic benefits - are directed towards only a few GPCR members. Therefore, huge efforts are currently underway to develop new GPCR-based drugs, particularly for cancer. We review recent findings that present unexpected opportunities to interfere with major tumorigenic signals by manipulating GPCR-mediated pathways. We also discuss current data regarding novel GPCR targets that may provide promising opportunities for drug discovery in cancer prevention and treatment.     

PharmGKB: Understanding the Effects of Individual Genetic Variants

The Pharmacogenetics and Pharmacogenomics Knowledge Base (PharmGKB: http://www.pharmgkb.org) is devoted to disseminating primary data and knowledge in pharmacogenetics and pharmacogenomics. We are annotating the genes that are most important for drug response and present this information in the form of Very Important Pharmacogene (VIP) summaries, pathway diagrams, and curated literature. The PharmGKB currently contains information on over 500 drugs, 500 diseases, and 700 genes with genotyped variants. New features focus on capturing the phenotypic consequences of individual genetic variants. These features link variant genotypes to phenotypes, increase the breadth of pharmacogenomics literature curated, and visualize single-nucleotide polymorphisms on a gene's three-dimensional protein structure.     

Orphan G protein-coupled receptors: from DNA to drug targets

Although G protein-coupled receptors (GPCRs) are the most numerous therapeutic targets to date, ligands remain unknown for approximately two-thirds of these receptors. The challenge in the post-genomic era is to evaluate the role of these 'orphan' GPCRs in normal physiology and disease, and to develop new therapeutics based on this information. A prevalent strategy to determine the function of these orphan GPCRs is to identify specific ligands that conditionally modulate their function. These ligands can then be used to explore the role of the receptor-ligand pair in relevant models of disease. Some promise is emerging from this infant field of functional genomics as several recently deorphanized GPCRs may be potential drug targets for many diseases, including obesity, cardiovascular disease, inflammation and cancer.     

Heterodimerisation of G protein-coupled receptors: implications for drug design and ligand screening

Importance of the field: In recent times many G protein-coupled receptors (GPCRs) have been shown to dimerise/oligomerise and, in some cases, such structural organization has been found to be essential for receptor function or to play a modulatory role in living cells. The fact that these complexes may display differential pharmacology through, for example, the formation of a new binding pocket or signalling properties, as well as different functions or regulation in physiological tissues, offers novel opportunities for drug discovery. As a consequence, it seems necessary to develop new approaches suitable for GPCR heterodimer identification and selective ligand screening. Areas covered in this review: This review gives an overview of new strategies that have been developed in an effort to incorporate the possibilities added by GPCR hetero-oligomerisation on the screening of compounds as drug candidates. What the reader will gain: The reader will gain a wider knowledge about how the current understanding of GPCR oligomeric structure and function has mandated that hetero-oligomeric receptors must be considered as novel targets in the identification of future lead compounds. Take home message: For the improvement of novel drug discovery, more structural and functional information on the process of receptor oligomerisation is needed, and the realisation that the function of GPCRs can be greatly influenced by other interacting receptors or proteins also demands consideration in the lead-compound developing process in order to achieve better therapeutic agents.     

与类风湿关节炎相关的G蛋白偶联受体及治疗药物作用靶点

很多胞外信号直接或间接通过G蛋白偶联受体向胞内传输。多种G蛋白偶联受体包括趋化因子受体、前列腺素(prostaglandins,PGs)受体、β2-肾上腺素受体(β2-adrener-gicreceptor)、致炎肽P物质(proinflammatorypeptidesub-stanceP,SP)受体、蛋白酶活化受体2(protease-activatedre-ceptor2,PAR-2)等在免疫应答调节中起至关重要的作用。该文综述了与类风湿关节炎相关的G蛋白偶联受体(Gpro-tein-coupledreceptors,GPCRs)信号转导的一些蛋白作用,包括G蛋白偶联受体激酶、视紫红质抑制蛋白(arrestin)、G蛋白信号转导调节因子、G蛋白偶联致炎受体等。作用于这些信号或其转导过程的药物正成为类风湿关节炎治疗的新策略之一。     

Virally encoded G protein-coupled receptors: targets for potentially innovative anti-viral drug development

Various herpes- and poxviruses contain DNA sequences encoding proteins with homology to cellular chemokine receptors, which belong to the family of G protein-coupled receptors (GPCRs). Since GPCRs play a crucial role in cellular communication and chemokine receptors play a prominent role in the immune system, the virally encoded GPCRs may be crucial determinants of viral action. The Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8), implicated in the pathogenesis of Kaposi's sarcoma (KS), a highly vascularized tumor, encodes a GPCR, referred to as ORF74. This virally encoded receptor was found to induce tumorigenesis and transgenic expression of ORF74 induces an angioproliferative disease resembling KS. Cytomegalovirus (CMV), suggested to play a role in atherosclerosis, encodes four GPCRs, among which US28. This virally encoded GPCR is able to induce migration of smooth muscle cells, a feature essential for the development of atherosclerosis. Remarkably, the KSHV and some CMV-encoded GPCRs display constitutive activity, while their cellular homologs do not. It remains to be determined whether this phenomenon contributes to the pathogenesis of viral action. Also, the family of poxviruses encodes GPCRs of which the function is not clear yet. In this review we will give an overview of the different virally encoded GPCRs, and discuss their putative role in viral action and potential as drug target.     

Calcium signalling by G protein-coupled sphingolipid receptors in bovine aortic endothelial cells

Besides its role as a putative second messenger releasing Ca²⁺ from intracellular stores, sphingosine-1-phosphate (SPP) has recently been identified as an extracellularly acting ligand activating a high affinity G protein-coupled membrane receptor in various cell types. Since SPP can be released from activated platelets, we examined in the present study whether endothelial cells express receptors for SPP and related sphingolipids. In bovine aortic endothelial cells loaded with fura-2, addition of SPP caused a rapid and transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), amounting to maximally about 230 nM. Removal of extracellular Ca²⁺ revealed that SPP-induced [Ca²⁺]_i elevations were due to both release of Ca²⁺ from intracellular stores and influx of extracellular Ca²⁺. Pretreatment of the cells with pertussis toxin inhibited the SPP-induced increase in [Ca²⁺]_i by 83%, in line with the previously reported involvement of G proteins of the G_i/o family in SPP signalling in other cell types. In contrast to other [Ca²⁺]_i-elevating agonists, e.g., ATP and bradykinin, SPP did not activate phospholipase C in bovine aortic endothelial cells, suggesting the involvement of a novel, unidentified signalling pathway in SPP-induced release of intracellular Ca²⁺. Furthermore, SPP also did not cause activation of either phospholipase D or A₂. Out of various related sphingolipids studied, only sphingosylphosphorylcholine (SPPC) induced a similar maximal increase in [Ca²⁺ _i as SPP, and its effect was also fully pertussis toxin-sensitive. However, the potencies of the two sphingolipids to increase [Ca²⁺]_i differed by more than two orders of magnitude, with the EC₅₀ values being 0.8 nM and 260 nM for SPP and SPPC, respectively. These results identify SPP and SPPC as novel and potent endothelial agonists, inducing calcium signalling by activation of a G_i/o protein-coupled receptor(s). Given the recently reported release of SPP from thrombin-activated platelets, SPP may represent a novel mediator of platelet-endothelial cell interactions.     

Structural insights into RAMP modification of secretin family G protein-coupled receptors: implications for drug development

Secretin family G protein-coupled receptors (GPCRs) are important therapeutic targets for migraine, diabetes, bone disorders, inflammatory disorders and cardiovascular disease. They possess a large N-terminal extracellular domain (ECD) known to be the primary ligand-binding determinant. Structural determination of several secretin family GPCR ECDs in complex with peptide ligands has been achieved recently, providing insight into the molecular determinants of hormone binding. Some secretin family GPCRs associate with receptor activity-modifying proteins (RAMPs), resulting in changes to receptor pharmacology. Recently, the first crystal structure of a RAMP ECD in complex with a secretin family GPCR was solved, revealing the elegant mechanism governing receptor selectivity of small molecule antagonists of the calcitonin gene-related peptide (CGRP) receptor. Here we review the structural basis of ligand binding to secretin family GPCRs, concentrating on recent progress made on the structural basis of RAMP-modified GPCR pharmacology and its implications for rational drug design.     

GPCR-Gα融合蛋白及其在oGPCRs配基筛选中的应用

GPCRGα融合蛋白是近几年用于受体研究的新颖手段之一,它的表达确保了受体与G蛋白之间1∶1的化学计量关系、空间位置上的邻近性及适宜于高通量的配基筛选,使其为孤儿G蛋白偶联受体提供了一种新的研究策略,将在孤儿受体的配基筛选中发挥重要作用,对研发以oGPCR为作用靶点的新药产生积极意义。     

Internal PDZ ligands: novel endocytic recycling motifs for G protein-coupled receptors

Internalization, recycling and lysosomal sorting are key processes that regulate the temporal and spatial signaling of G protein-coupled receptors (GPCRs). Interactions between GPCR intracytosolic sorting signals and adaptor proteins facilitate trafficking through the endocytic pathway. To date only a few sorting signals and molecules that regulate GPCR trafficking have been identified. A study reported in the May 2005 issue of Molecular Pharmacology has now identified an internal PDZ ligand motif that seems to regulate efficient recycling of the ET(A) endothelin receptor. This finding now expands the diversity of GPCR sorting motifs to include internal and C-terminal PDZ ligands, tyrosine-based motifs, and lysine residues capable of being ubiquitinated.     

药物基因组学与心律失常疾病的治疗

抗心律失常药物的治疗剂量和中毒剂量非常接近,而个体的差异易引起药物的不良反应。为此研究抗心律失常药物的药物基因组学来正确地引导药物的使用,以期达到理想的治疗效果并减少毒副作用。本文就近年来抗心律失常药物基因组学的研究进展作一综述。