GPCR Target Validation

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Approximately 30 of the 210 liganded GPCRs are currently targets for drugs now on the market. Additionally, approximately 160 non-liganded or orphan receptors exist, resulting in over 300 potential targets for which new chemical entities can be created for potential therapeutic interventions.8 The challenge is to match the receptor with its physiological function and role in pathophysiological disorders. A number of parallel approaches have been utilized to validate GPCR drug targets including bioinformatic methods as described above. Some predictions can be gained from comparing gene sequences of known GPCRs with orphan GPCRs, such as family types, potential endogenous ligands, and accessory protein interaction domains.

Until actual protein structure information is obtained for GPCRs that will allow the accurate identification of ligand-binding domains, predictions of potential ligands based on sequence homologies and modeling are employed. Several examples of ligand types have been predicted for closely related orphan GPCRs after a ligand for one of the related orphan GPCRs has been elucidated. Examples include the histamine H4 receptor25 following identification of the H3 receptor, the trace amine receptors,26 and lysophospholipid receptors.27 However, there are other examples, where sequence homology similarities have resulted in improper classification of orphan GPCRs as receptors for ligands different from their cognate ligands including the histamine H3 receptor as a muscarinic receptor28 and the BLTj leukotriene B4 receptor as a purinergic receptor,29 indicating the limitations to this type of approach.

GPCRs have also been validated as therapeutic targets for a number of diseases based on the finding that naturally occurring mutations of the receptor can result in either a gain or loss of function of the receptor leading to an association with a disease state.30-32 An excellent review by Seifert and Wenzel-Seifert32 provides an overview of receptors that exhibit increased constitutive activity (gain of function) including (with associated disease in parentheses): rhodopsin (retinitis pigmentosa), cholecystokinin-2 receptor (gastric carcinoid tumor), KSHV-GPCR (Kaposi's sarcoma), chemokine receptor US28 (atherosclerosis), thyroid-stimulating hormone receptor (hyperthyroidism), luteinizing hormone receptor (precocious male puberty), parathyroid hormone receptor (dwarfism, hypercalcemia, hypophosphatemia), and calcium-sensing receptor (hypocalcemia). Naturally occurring mutations of GPCRs can also lead to loss of function of the receptor with associated pathophysiology.31 Familial nephrogenic diabetes insipidus has been associated with an inactive truncated human vasopressin V2 receptor33 as well as a single amino acid mutation (R137H) that results in the rapid desensitization of the receptor.34

Other human GPCRs in which a naturally occurring single point mutation results in an inactive receptor and association with a disease31 are the R16G mutant of the p2-adrenergic receptor (nocturnal asthma),35 the W64R mutant of the p3-adrenergic receptor (obesity),36 and the R60L mutant of thromboxane A2 (bleeding disorders).37 A deletion mutation of the human P2Y12 purinergic receptor is associated with bleeding disorders,38 and an autosomal recessive mutation in the canine orexin-2 receptor was found to underlie narcolepsy in dogs,39 suggesting a role for orexin in human narcolepsy.

These data suggest that these diseases result from mutations of the GPCRs and also that the wild-type GPCR plays a pivotal role in the normal physiological regulation of the signaling pathway and function deranged by the mutated receptor. Linkage of other inherited diseases such as those discussed above with liganded and orphan GPCRs may reveal novel therapeutic opportunities for particular receptors, leading to the discovery of new drugs to treat the associated disease states.

The identification of naturally occurring GPCR mutants and their altered functions are important to our understanding of the roles of GPCRs in disease processes. However, these mutants are relatively few in number and are generally expressed in a limited number of tissues, with an obvious lack of mutant GPCRs expressed in the central nervous system (CNS) where they are most abundant.

The ability to create transgenic animals in which GPCR genes of interest can be activated or inactivated allows researchers to generate a wide variety of mouse models amenable to testing the role of a single knocked-in or knocked-out GPCR in an array of functional and behavioral models. A recent review by Karasinska et al.40 lists nearly 90 Family A GPCR gene knock-out mouse models and their associated phenotypes, indicating levels of interest in determining the in vivo functions of this important class of receptors. A comparison of the GPCR knock-out phenotype with the efficacy of drugs in development and used in the clinic targeting that receptor demonstrates a very good correlation.41,42 The therapeutic indications correlate very well with the phenotype of the receptor knock-out animal model for some of the best-selling drugs listed in Table 3.3. 41

These findings support the idea that GPCR knock-out animals serve an important role in developing new therapeutics. Not only does the inactivation of a GPCR gene reveal insight into the function of that particular receptor, it also allows for the determination of how other GPCRs may interact (heterologous desensitization, shared signaling pathways, up- or down-regulation, protein-protein and pharmacological interactions) with the GPCR in question by comparing the effects of pharmacological agents in wild-type and knock-out animals. GPCR knock-out animals also serve as valuable tools in determining the functions of closely related GPCR subtypes for which there are no selective pharmacological tools available and of orphan GPCRs whose endogenous ligands have yet to be identified.41


Correlation of Best-Selling Drugs and Phenotypes


Trade Name

H2 receptor antagonist Ranitidine m-opioid receptor agonist Angiotensin AT1

receptor antagonist Purinergic P2Y12

receptor antagonist Muscarinic receptor antagonist

Fentanyl Losartan Clopidrogel Tolterodine

Brand Name


Leukotriene antagonist

H1 receptor antagonist Fexofenadine Allegra

Cozaar Plavix Detrol


Gastroesophageal reflux disease

Montelukast Singulair Asthma Allergies Duragesic Pain

Hypertension Thrombosis Overactive bladder

Knock-Out Phenotype

Abolition of histamine-sensitive gastric acid release Decreased extravasation

Decreased T and B cell responsiveness Increased pain sensitivity Low blood pressure

Decreased platelet aggregation Increased urine retention

Animals with inactivated GPCRs can also be used to test for the selectivities of actions of drugs targeting that specific receptor in order to determine whether the in vivo effect of the drug is related to the target GPCR. However, one must be aware of certain cautions when interpreting the phenotypes of GPCR knock-out animals, especially in behavioral tests, including the contribution of the background strain of mouse to the responses and possible disruption of function of other receptors that contribute to the response phenotype that are affected by the inactivated GPCR during pre- and postnatal development and in mature animals.41 Other limitations of transgenic animals include cost, time, species that can be utilized (primarily mice), and the irreversibility of the inactivation (excepting conditional knock-outs).43

Another approach that can be used in the GPCR target validation process is the use of sequence-specific antisense oligonucleotides that target the mRNA of the specific GPCR being interrogated. To date, this technique has been used to effectively inhibit the expression of over 100 GPCR proteins both in vitro and in vivo.43 Several advantages of the antisense oligonucleotide approach include (1) the ability to selectively reduce or eliminate expression of GPCRs for which there are no selective ligands to determine the function of the receptor, (2) no receptor up-regulation caused by prolonged reduction in GPCR expression by the antisense oligonucleotides, (3) reversibility, and (4) applicability to any animal species. However, it should be noted that complete suppression of GPCR protein expression is never achieved. About 70% of the studies conducted on GPCRs produced 50% or less suppression of the receptor.43

In addition, a number of other disadvantages associated with this approach include sequence-independent effects, limited internalization of the oligonucleotides, narrow dose-range windows to elicit effect, and the need for continuous administration.43

It should also be noted that other strategies for inhibiting gene expression related to the antisense oligonucleotide approach include antisense RNA, triplex antigenes, ribozymes, and small interfering RNA (siRNA). The use of siRNA is now the preferred method for target validation in vitro. Continued improvements in delivery systems in vivo now allow the development of animal models suitable for target validation.44-46 Fewer reports published to date concern the knock-down of GPCR gene expression by siRNAs, but the expression of chemokine CCR5 and CXCR4 receptors was decreased by siRNAs targeting them, resulting in reduced HIV infection.47 Additionally, siRNA targeting the lysophospholipid G2A receptor decreased the expression of this receptor in T lymphoid cells, resulting in reduced chemotaxis,48 demonstrating the utility of this approach for GPCR research.

One final means of GPCR target validation is using pharmacological agents to test for desired therapeutic responses in animal models of disease. These tools can include known standard compounds that demonstrate potent and selective binding to the target in question if one is testing for a novel indication for an already well-characterized GPCR. Conversely, when little is known about the physiological role of the GPCR for which no pharmacological tools are available, compounds that exhibit activity in high throughput screening or from lead optimization testing can be evaluated in animal models to determine the physiological role of the GPCR. A variety of caveats associated with using novel test agents to validate GPCR targets must be considered when interpreting the results including receptor selectivity, pharmacokinetic properties, distribution, and metabolism, among others.

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Coping with Asthma

Coping with Asthma

If you suffer with asthma, you will no doubt be familiar with the uncomfortable sensations as your bronchial tubes begin to narrow and your muscles around them start to tighten. A sticky mucus known as phlegm begins to produce and increase within your bronchial tubes and you begin to wheeze, cough and struggle to breathe.

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