Inositol Triphosphate Assays

Whereas most HTS labs utilize gene reporter or calcium fluorophore technologies to detect Gq- and or Gi-coupled receptors, some technologies detect inositol-triphosphate (IP3) formation in cells. Historically, IP3 assays depended on 3H inositol loading into cells. Radioactivity was then detected in aqueous and organic fractions after column separation. This technology is not readily amenable for HTS. IP3 formation is determined by measuring the displacement of a labeled IP3 analog from an IP3...

The Cysteine Shuffle

To test the feasibility of tagging receptor cysteines with a thiol-labeling reagent, the redox sensitivity of a radioligand receptor assay (RRA) for the 25-kDa glycoprotein hormone TSH was established. Unlike the 12 cysteines in the amino terminal extension of the related LH receptor, TSH (and FSH) receptors have only 11 extracellular cysteines, beyond the 2 involved in the conserved cystine bridge, suggesting that a free thiol may be available and influence ligand binding. Indeed, low...

Challenges in Evaluating Orphan GPCRs in the CNS

Receptor localization can provide the first clues to orphan GPCR function in normal and disease states, but it can be misleading in the brain. High throughput expression profiling is widely used to map orphan GPCR mRNA transcripts in the CNS, but receptor expression measured at the protein level often reveals different distributions because many receptors such as CB1 are localized to neuronal axon terminals distant from their origins of synthesis in cell bodies.201 Receptor distribution can...

Gpcr Internalization And Trafficking

Prolonged exposure of an agonist to a receptor leads to decreased sensitivity of the receptor to a subsequent agonist challenge. Several distinct mechanisms are involved in this process, for example, uncoupling of receptors from G proteins and reduction in the number of cell surface receptors by internalization or desensitization.81 This phenomenon controls the initiation and termination of the signal and regulates the intensity of response. An early event in the desensitization process is the...

Satiety and Glucose Homeostasis

Satiety signals are produced in the gastrointestinal (GI) tract in response to the presence of nutrients. Several satiety signals (cholecystokinin, pancreatic polypeptide, peptide YY, and amylin) are peptides that interact with their cognate GPCRs to induce a sense of fullness resulting in decreased food intake and, in some cases, modulation of nutrient metabolism. The receptors for satiety-inducing peptides may reside locally in the GI tract, on peripheral nerves running from the GI tract to...

Rhodopsin Subfamilies Bind Different Classes of Ligand

Interestingly, the concordance of phylogenies (Figure 10.2) and the 7TMR classification into four classes according to ligand size (Figure 10.1) produces a biologically relevant interpretation for the designations of a, p, g and 5 a for aminergic ligands, p for brain-gut peptides, g ligands for their Gi preference, and 5 ligands because they are the most diverse. Thus, the a family of rhodopsin receptors interacts predominantly with ami-nergic-like ligands, typified by monoamine...

Drugs In Development And Novel Drug Targets

Cardiovascular diseases constitute a major focus area for drug discovery and development. They are very prevalent in all populations, and many therapeutic needs are still unmet despite the many successes of the past 50 years. It is evident from Table 4.1 that relatively few GPCRs are currently exploited as cardiovascular drug targets, even if p-adrenoceptor and AT1 receptor antagonists represent two of the most widely used drug classes in modern medicine. Table 4.2 summarizes information on...

Intracellular Calcium Assays

The cell-based calcium flux assay is considered one of the most important screening techniques used in pharmaceutical drug discovery. Gq-coupled receptor-mediated increases in intracellular Ca2+ release can be measured by calcium-sensitive fluorescent dyes, bioluminescent indicators, or reporter gene assays. Gq-coupled receptor activation can elevate a transient increase in intracellular calcium to 400 to 1000 nM from a basal cytosolic level of 10 to 100 nM. Most common calcium-sensitive dyes...

Abcdef Classification of GPCRs

The conventional classification of the GPCR superfamily into six families (A-F) is maintained at the primary GPCR resource (GPCRDB, www.gpcr.org), which has moved to the University of Nijmegen, The Netherlands, with its cofounder Gerrit Vriend. The American mirror GPCRDB4 has suffered the fate of so many comprehensive scientific databases and has been privatized. The 7TMRs and GPCRs are assigned to class 2 in the IUPHAR receptor database (www.iuphar.org), with individual receptors encoded in a...

Screening For Modulators Of Orphan Receptors

Seven hundred twenty genes belong to the GPCR super family in the human genome and approximately half of them encode sensory receptors. Of the remaining 360 receptors, ligands have been identified for 210 the other 150 are considered orphan receptors89-92 (described in detail in Chapter 16). Because several known GPCR modulators have proven clinically useful drugs, the orphan GPCRs are viewed by the pharmaceutical and biotechnology industries as rich sources of novel drug targets. Thus, many...

Insect Cells

Insect cells obviously resemble mammalian cells in many aspects and are therefore attractive for recombinant protein production. Development of baculovirus vectors for heterologous gene expression has been very successful.36 Generally, the strong polyhedrin promoter from Autographa californica has been applied for expression in several insect cell lines from organisms such as Spodoptera frugiperda (Sf9 and Sf21 cells) and Trichoplusia ni (Tni and High FiveTM cells).37 Insect cells possess many...

References

The druggable genome. Nat. Rev. Drug Disc. 2002, 1 727-730. 2. Bockaert, J. and Pin, J.P Molecular tinkering of G protein-coupled receptors an evolutionary success. EMBO J. 1999, 18 1723-1729. 3. Foord, S.M. Receptor classification post genome. Curr. Opin. Pharmacol. 2002, 2 561-566. 4. Chalmers, D.T. and Behan, D.P. The use of constitutively active GPCRs in drug discovery and functional genomics. Nat. Rev. Drug Disc. 2002, 1 599- 608. 5. Vassilatis, D.K.,...

Homogeneous Radioactive Binding Assays

Examples of multiple platforms for homogeneous radioactive binding assays include SPA beads Amersham Biosciences , LEADseeker beads Amersham Biosciences , FlashBlue beads PerkinElmer , and FlashPlate PerkinElmer . All these formats are scintillation proximity assays SPAs . In this type of assay, an isotope-labeled ligand is brought into close proximity to a scintillant embedded in beads or plate plastics by binding to a receptor immobilized onto the beads or plates.17 SPA is a simple assay format and is well suited to automation because it requires no separation step. The scintillant in SPA beads is incorporated into fluomicro-spheres. SPA beads are made of either polyvinyl toluene PVT or yttrium silicate Ysi . Beads are available with several surface coatings to immobilize membranes including PVT-WGA wheat germ agglutinin , Ysi-WGA, PVT-PEI polyethylen-imine -WGA, and Ysi-poly-D-lysine. Beads coated with WGA bind cell membranes through surface carbohydrates. Beads coated with...

Gpcr Drug Discovery

Selection of a potential GPCR as a target for a preclinical drug discovery effort involves a number of factors viewed from both business and scientific perspectives. This section deals with the scientific criteria used for the selection of a target. It should be stated that a target should obviously fit a company's business plan including alignment with the therapeutic expertise of the company and the capabilities of other preclinical facilities chemical libraries, high throughput screening,...

Gpcrtractability Current Therapeutics

These cell surface receptors have historically been attractive drug targets for pharmaceutical companies. More than 30 of all prescription drugs are directed at fewer than 50 known receptors.5,7,8 One feature of this receptor family that makes it a tractable drug target is the host of physiological functions including homeostatic mechanisms cardiovascular, gastrointestinal, and metabolic processes , central nervous system function, and growth that are regulated through the activation of GPCRs...

GRAFS Classification of 7TMRs

Grafs Classification

The five main families identified from multiple sequence alignment, with high bootstrap support, are designated glutamate, rhodopsin, adhesion, frizzled taste2, and secretin. The first letters of the five family names constitute the GRAFS acronym designation. The rhodopsin and glutamate designations conform to the original A and C families, whereas family B has been split into the secretin GPCR family and the related but distinct non-GPCR adhesion family, with 7TMR sequences characterized by...

Novel Gpcr Features And Impact On Drug Discovery Approaches

Recent advances in GPCR research have provided additional opportunities for drug discovery beyond the single conventional binding site approach used to date for all currently marketed drugs that target liganded GPCRs. The discoveries of many yet-to-be defined orphan GPCRs, the finding that GPCRs can form homo- and or hetero-oligomeric complexes with other GPCRs and the ever-growing number of accessory proteins found to interact with and influence the GPCR signaling complex mean that the number...

Gpcr Family Overview

A wide variety of extracellular stimuli including photons, ions, neurotransmitters, peptides, and lipids can elicit intracellular signaling events through activation of cell surface GPCRs. These receptors all share a common heptahelical transmembrane-spanning structure that allows the interaction of external stimuli at selective binding sites in extracellular domains or within the transmembrane-spanning domains of GPCRs. This binding in turn conveys conformational changes in the GPCRs,...

GPCR Target Validation

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...

Coupling To G Proteins

The classical mechanism by which GPCRs are functionally active is defined by their coupling to guanine nucleotide-binding proteins or G proteins.7 Typically, a GPCR is in its resting or low affinity state in the absence of agonists. The binding of an agonist to a receptor triggers a modification in the receptor conformation and the formation of a complex with the intracellular G proteins Figure 2.1 . The hetero-trimeric G protein consists of a membrane-associated Ga subunit and the more...

GPCR Druggability

The potential to create a potent and selective human therapeutic agent for a GPCR can also influence the selection of that receptor as a target. Biological factors to consider prior to advancing any screening effort for a GPCR target include a number of those already discussed above such as the localization and distribution of the GPCR, the nature of the receptor family type, structure of the receptor, endogenous ligands i.e., small molecule versus large peptide , and type of the...