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November 2004 From Concept to Development
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Volume 7, Issue 11
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It’s a GPCR world


Cell-based screening assays and structural studies are fueling G-protein coupled receptors as one of the most popular classes of investigational drug targets.

David Filmore Download PDF | Table of Contents  


Rhodopsin Image; Protein Data BankIf you had to make a wild guess about the target of a certain drug, your best odds are with “G-protein coupled receptor.” Drugs targeting members of this integral membrane protein superfamily, which transmit chemical signals into a wide array of different cell types, represent the core of modern medicine. They account for the majority of best-selling drugs and about 40% of all prescription pharmaceuticals on the market. Notable examples include Eli Lilly’s Zyprexa, Schering-Plough’s Clarinex, GlaxoSmithKline’s Zantac, and Novartis’s Zelnorm.

And there is broad consensus that GPCRs will remain at the hub of drug development activities for the foreseeable future. According to a recent report by market analysis firm Navigant Consulting, “GPCRs are among the most heavily investigated drug targets in the pharmaceutical industry.”

These proteins are active in just about every organ system and present a wide range of opportunities as therapeutic targets in areas including cancer, cardiac dysfunction, diabetes, central nervous system disorders, obesity, inflammation, and pain. Consequently, GPCRs are prominent components of pipelines in small and large drug companies alike, and many drug discovery firms focus exclusively on these receptors.

But the path to novel GPCR-targeted medicines is not routine. Most GPCR-modulating drugs on the market weren’t initially targeted to a specific protein but were developed on the basis of functional activity observed in an assay. That they activated or inhibited a GPCR specifically was only later discovered. Post-Human Genome Project, however, targets are the starting points for most drug discovery endeavors. And there is still much to be learned about how GPCRs work and how they can be selectively modulated. Fortuitously, technologies designed specifically to tackle the GPCR challenge are blossoming.

Cell-based screening assays

The bread-and-butter of GPCR high-throughput screening is cell-based assays. Tools such as fluorescent imaging plate readers (commonly referred to as FLIPRs) allow multiwell plate analysis of GPCR activation events, which give good hints of small-molecule drug leads.

GPCRs exist at the interface of a cell’s external and internal environments. When the matching natural ligand—which for the range of GPCRs could be an amine, ion, nucleoside, lipid, peptide, protein, or, for optical receptors, light—comes along, it binds to a receptor’s active site and causes a conformational change in the protein to form its active state. This signals the G protein coupled to the receptor inside the cell to release components that set some predefined cellular mechanism in motion.

The trick for high-throughput cellular screening is to find a robust marker to monitor in cells overexpressing the GPCR of interest.

Calcium ions are one popular choice. Ca2+ is naturally produced in cells upon activation of GPCRs coupled to Gq proteins—one of the three main families of G proteins.

The Brussels-based company Euroscreen, for instance, has developed the AequoScreen assay to fuel its own GPCR-based drug discovery programs—as well as those of companies that purchase its technology. AequoScreen is based on a jellyfish-derived photoprotein called aequorin, which displays photoactivity proportional to Ca2+ concentration. Screening a library against an array of GPCR-overexpressing cells mixed with aequorin provides a quantitative means of assessing a compound’s ability to activate a GPCR (or its ability to antagonize activation).

Even though intracellular Ca2+ levels rise directly only from Gq-protein receptor activation, genetic expression methods have been developed that allow Ca2+ production to proceed upon activation of GPCRs coupled to other G protein types (i.e., Gi/Go or Gs). Thus, fluorescent Ca2+ screening has become somewhat of a universal approach to screening small-molecule libraries against GPCRs.

Cyclic adenosine monophosphate (cAMP), which controls myriad cellular metabolic pathways, is one of the most important “second messenger” compounds of the GPCR activation process. It also makes a good high-throughput screening marker. Numerous commercially available and individually made cell-based GPCR assays use luminescent tags that bind to cAMP.

Arena Pharmaceuticals’ entire drug screening program, for example, is based around the cAMP approach, although the company’s system doesn’t require the use of tagging compounds. With its Melanophore technology, it expresses GPCR targets in frog skin cells containing a pi