Alternative drug discovery approaches for orphan GPCRs

G protein-coupled receptors (GPCRs) are well-known drug targets. However, a question mark remains for the more than 100 orphan GPCRs as current deorphanisation strategies failed to identify specific ligands for these receptors. Recent advances have shown that orphan GPCRs may have important functions that are ligand-independent. Orphan GPCRs can modulate the function of well-defined drug targets such as GPCRs with identified ligands and neurotransmitter transporters though physical association with those molecules. Thus, compounds that bind to orphan GPCRs and allosterically regulate the function of the interacting partner or even disrupt the interaction with the latter could become new drugs.     

Novel G-protein-coupled receptor genes expressed in the brain: continued discovery of important therapeutic targets

The rhodopsin family of G-protein-coupled receptors (GPCRs) is the largest known group of cell-surface mediators of signal transduction. The vast majority of these receptors were discovered by methods based upon shared sequence homologies found throughout this family. While such efforts identified a multitude of receptor subtypes for previously known ligands, numerous receptors have been discovered for which endogenous ligands were unknown. These receptors are commonly referred to as orphan receptors. One of the most important tasks of modern pharmacology lies in elucidating the functions of these receptors. Of particular interest are receptors with recognised expression in the central nervous system, given that many psychiatric and neurodegenerative disorders are mediated by unknown mechanisms. Hence, this collection of putative neurotransmitter and neuromodulator signal mediators represents a substantial and untapped resource for novel drug discovery. Recently, various methodologies have accelerated the discovery of novel ligands for these orphan receptors, identifying the basic components required for further physiological ligand/receptor system characterisation. Equipped with proven ligand identification strategies, the characterisation of all orphan GPCRs and the exploitation of their exciting potential as targets for the discovery of novel drugs is anticipated.     

Structure, pharmacology and therapeutic prospects of family C G-protein coupled receptors

Family C of G-protein coupled receptors (GPCRs) from humans is constituted by eight metabotropic glutamate (mGlu(1-8)) receptors, two heterodimeric gamma-aminobutyric acid(B) (GABA(B)) receptors, a calcium-sensing receptor (CaR), three taste (T1R) receptors, a promiscuous L-alpha-amino acid receptor (GPRC6A), and five orphan receptors. Aside from the orphan receptors, the family C GPCRs are characterised by a large amino-terminal domain, which bind the endogenous orthosteric agonists. Recently, a number of allosteric modulators binding to the seven transmembrane domains of the receptors have also been reported. Family C GPCRs regulate a number of important physiological functions and are thus intensively pursued as drug targets. So far, two drugs acting at family C receptors (the GABA(B) agonist baclofen and the positive allosteric CaR modulator cinacalcet) have been marketed. Cinacalcet is the first allosteric GPCR modulator to enter the market, which demonstrates that the therapeutic principle of allosteric modulation can also be extended to this important drug target class. In this review we outline the structure and function of family C GPCRs with particular focus on the ligand binding sites, and we present the most important pharmacological agents and the therapeutic prospects of the receptors.     

G protein-coupled receptors in drug discovery

G protein-coupled receptors (GPCRs) represent one of the most important drug discovery targets such that compounds targeted against GPCRs represent the single largest drug class currently on the market. With the revolutionary advances in human genome sciences and the identification of numerous orphan GPCRs, it is even more important to identify ligands for these orphan GPCRs so that their physiological and pathological roles can be delineated. To this end, major pharmaceutical industries are investing enormous amounts of time and money to achieve this object. This review is a bird's eye view on the various aspects of GPCRs in drug discovery.     

An ultra-HTS process for the identification of small molecule modulators of orphan G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) are the most successful target proteins for drug discovery research to date. More than 150 orphan GPCRs of potential therapeutic interest have been identified for which no activating ligands or biological functions are known. One of the greatest challenges in the pharmaceutical industry is to link these orphan GPCRs with human diseases. Highly automated parallel approaches that integrate ultra-high throughput and focused screening can be used to identify small molecule modulators of orphan GPCRs. These small molecules can then be employed as pharmacological tools to explore the function of orphan receptors in models of human disease. In this review, we describe methods that utilize powerful ultra-high-throughput screening technologies to identify surrogate ligands of orphan GPCRs.     

Constitutively activated G protein-coupled receptors: a novel approach to CNS drug discovery

G protein-coupled receptors (GPCRs) represent a major class of signal transduction proteins that modulate various biological functions. GPCRs are one of the most common targets for drug development-currently, 39 of the top 100 marketed drugs in use act directly or indirectly through activation or blockade of GPCR-mediated receptors. Nearly 160 GPCRs have been identified based on their gene sequence and their ability to interact with known endogenous ligands. However, an estimated 500-800 additional GPCRs have been classified as "orphan" receptors (oGPCRs) because their endogenous ligands have not yet been identified. Given that known GPCRs have proven to be such clinically useful drug targets, these oGPCRs represent a rich group of receptor targets for the development of novel and improved medicines. To develop ligands for these potential drug targets requires the ability to identify groups or pools of GPCRs that are likely to be involved in a specific disease process (obesity, schizophrenia, depression, etc.) and to dissect out the pharmacological and signal transduction differences between these GPCR subtypes. It also requires the development of assays to detect ligands of GPCRs even when the endogenous ligands are unidentified. This paper will review novel strategies to identify clinically interesting oGPCRs and to screen for small molecules that act as ligands without prior knowledge of endogenous ligands. This involves the use of constitutively activated GPCRs, a technology that provides a unique opportunity to identify several classes of pharmacological agents, including agonists, inverse agonists and allosteric modulators.     

The future of G protein-coupled receptors as targets in drug discovery

G protein-coupled receptors (GPCRs) represent the most abundant drug targets today. A large number of GPCR-based drugs have already been developed for a variety of indications in human disease. However, orphan receptors with unidentified ligands serve as potential targets still to be explored. Moreover, research on the interaction of GPCRs with different molecules in the signal transduction pathways, and further studies on receptor dimerization may also lead to the discovery of new drugs. Structure-based drug design will eventually play a key role in generating better and more selective drugs more rapidly when high-resolution structures of GPCRs can be provided by expression, purification and crystallography technologies.     

Molecular manipulation of G-protein-coupled receptors: a new avenue into drug discovery

During the past 10 years or so, associated with the introduction of molecular biology techniques to G protein-coupled receptor (GPCR) research, outstanding progress has been made in understanding the mechanisms of action of these key proteins and their physiological functions. in-vivo manipulation of levels of GPCRs using transgenic and gene knock-out approaches have been particularly successful in assessing the roles of specific GPCRs in animal physiology. Drug discovery is aiming to produce highly specific compounds based on subtle definition of receptor subtypes which can best be studied using heterologous expression of wild type or mutated forms of cDNA or genes encoding these proteins. Furthermore, new therapeutic opportunities may be provided by investigation of orphan receptors, the natural ligands for which remain unidentified. Some human diseases have been shown to be associated with rare mutations of GPCRs and the possibility that widely distributed polymorphisms in GPCR genes may allow selective therapeutic strategies for population subgroups is driving the development of the science of pharmacogenetics.     

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.     

G蛋白偶联受体固有活性研究进展与新药开发

G蛋白偶联受体(G-prote in-coup led receptor,GPCR)是与G蛋白有信号连接的一大类受体家族,是人体内最大的膜受体蛋白家族,是一类具有7个跨膜螺旋的跨膜蛋白受体。GPCR的结构特征和在信号传导中的重要作用决定了其可以作为很好的药物靶标。目前世界药物市场上有三分之一的小分子药物是GPCR的激活剂(agon ist)或拮抗剂(antagon ist)。以其为靶点的药物在医药产业中占据显著地位。在当今前50种最畅销的上市药物中,20%属于G蛋白受体相关药物。近来的研究发现,大多数G蛋白偶联受体具有一个很重要的特性,就是具有固有活性(Constitutive ac-tivity),即无激动剂条件受体自发的维持激活并维持下游信号传导通路的活性。固有活性涉及受体、G蛋白及下游信号通路之间的关系。该文就G蛋白偶联受体固有活性概念、研究进展、反相激动剂与固有活性研究、固有活性与新药开发4个方面,进行以下论述。     

Ligand function at constitutively active receptor mutants is affected by two distinct yet interacting mechanisms

It has been proposed that mutations that induce constitutive activity in G-protein-coupled receptors (GPCRs) concomitantly enhance the ability of partial agonists to trigger second-messenger signaling. Using the cholecystokinin type 2 receptor (CCK-2R) as a model system, we have explored whether this association applies to a diverse set of activating mutations. Consistent with established principles, constitutively active CCK-2Rs resulting from amino acid substitutions within the third intracellular loop each systematically increased partial agonist activities versus corresponding wild-type values. In contrast, activating mutations within transmembrane domain segments near the extracellular loops led to an increase in efficacy of only a subset of compounds but decreased or did not change the function of others. When transmembrane domain amino acid substitutions were introduced in combination with intracellular amplifying mutations, observed changes in ligand activity were defined by the product of two discernible factors 1) systematic amplification caused by an equilibrium shift from the inactive to the active receptor conformation and 2) ligand-specific alterations in signaling, which probably result from mutation-induced changes in the putative binding pocket. These findings illustrate functional heterogeneity among GPCR mutants with ligand-independent signaling. A subgroup of activating mutations facilitates receptor isomerization to the active state and in parallel perturbs ligand receptor interactions. These mutants do not adhere to the previously proposed "hallmark criteria" of constitutive activity.     

Recognition of privileged structures by G-protein coupled receptors

Privileged structures are ligand substructures that are widely used to generate high-affinity ligands for more than one type of receptor. To explain this, we surmised that there must be some common feature in the target proteins. For a set of class A GPCRs, we found a good correlation between conservation patterns of residues in the ligand binding pocket and the privileged structure fragments in class A GPCR ligands. A major part of interior surface of the common ligand binding pocket of class A receptors, identified in many GPCRs, is lined with variable residues that are responsible for selectivity in ligand recognition, while other regions, typically located deeper into the binding pocket, are more conserved and retain a predominantly hydrophobic and aromatic character. The latter is reflected in the chemical nature of most GPCR privileged structures and is proposed to be the common feature that is recognized by the privileged structures. Further, we find that this subpocket is conserved even in distant orthologs within the class A family. Three pairs of ligands recognizing widely different receptor types were docked into receptor models of their target receptors utilizing available structure- activity relationships and mutagenesis data. For each pair of ligands, the ligand-receptor complexes reveal that the nature of the privileged structure binding pocket is conserved between the two complexes, in support of our hypothesis. Only part of the privileged structures can be accommodated within the conserved subpocket. Some contacts are established between the privileged structure and the nonconserved parts of the binding pocket. This implies that any one particular privileged structure can target only a subset of receptors, those complementary to the full privileged structure. Our hypothesis leads to a valuable novelty in that ligand libraries can be designed without any foreknowledge of the structure of the endogenous ligand, which in turn means that even orphan receptors can in principle now be addressed as potential drug targets.     

Orphan G-protein-coupled receptors: the next generation of drug targets?

The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery. Large scale DNA sequencing has allowed the identification of a plethora of DNA sequences distantly related to known G protein-coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets. In most cases the extent of sequence homology is insufficient to assign these `orphan'' receptors to a particular receptor subfamily. Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors. Briefly, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections. The functional genomics approach involves the use of `humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery. Both systems provide an excellent platform for identifying novel receptor ligands. Once activating ligands are identified they can be used as pharmacological tools to explore receptor function and relationship to disease.     

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