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Nov 15, 2009 (Vol. 29, No. 20)

Strategies for Companies Targeting GPCRs

Emerging Tools and Methodologies for Screening and Discovery Are Improving Odds for Success

  • Screening Platforms

    Click Image To Enlarge +
    Photo protein assay principle: To reduce the M4 receptor expression level, CHO-aequorin-Ga16-M4 cells were serum-starved for two days. PerkinElmer used a double-injection protocol.

    One of those screening platforms is the HitHunter cAMP, which, according to the company’s senior director of cell biology Tom Wehrman, Ph.D., “is a sensitive and reliable platform that detects intracellular cyclic adenosine monophosphate (cAMP) using chemiluminescence.”

    More recently, DiscoveRx used the EFC platform to develop an assay to detect ß-arrestin recruitment to activated GPCRs, a primary rather than secondary signaling event that enables rapid deorphanization of a GPCR target.

    “Over the past five years, the rate of de-orphanization has declined, suggesting that the standard second messenger deorphanization platforms have not resulted in a large number of receptor-ligand pairings,” said Dr. Wehrman. “With the introduction of the PathHunter Arrestin technology, we are now seeing a renewed interest in deorphanization programs given that arrestin is known to interact with the vast majority of GPCRs.”

    Dr. Wehrman’s presentation focused on the advantages of using multiple GPCR screening approaches such as G-protein-dependent cAMP/calcium screening along with G-protein-independent PathHunter ß-arrestin and generation of novel hits and pharmacology resulting from such comprehensive studies. His presentation also included a discussion of the next generation of tools developed based on PathHunter technology.

    These tools provide either a quantitative measurement of GPCR trafficking (total endocytosis GPCR assay system) or GPCR heterodimerization (PathHunter GPCR dimerization assay). All of the PathHunter technologies are whole cell-based and use a single-step, no-wash, high-throughput screening-friendly, chemiluminescence-based assay format.

    PathHunter GPCR dimerization assays are designed to study the interactions between different pairs of GPCR molecules or between GPCR heterodimers. “These assays are unique in that they measure the functional coupling of two GPCRs through the arrestin pathway and enable screens for modulators of GPCR activity,” commented Dr. Wehrman. The Endocytosis GPCR assay system measures GPCR internalization by quantifying the amount of receptor in the endosomal compartment, thus providing a nonimaging method to screen compounds for their ability to cause endocytosis of the receptor.

    “A more complete view of compound activity is obtained using arrestin binding in conjunction with second messenger assays. The arrestin and second messenger pathways can lead to disparate biological effects, making it increasingly important to monitor arrestin binding, as well as G-protein activation.”

    Charles Lunn, Ph.D., research fellow, department of new lead discovery, Schering-Plough, concured that “there is an interest in looking at GPCRs in ß-arrestin-dependent systems, partly to interrogate G-protein-independent as well as G-protein-dependent signal transduction mechanisms. But, we appreciate that every GPCR assay format can bias the types of modulators you identify. Label-free profiling of cell-ligand interactions may moderate this bias.” 

    “People are also going into more label-free technologies, especially for GPCRs,” added Suresh Poda, Ph.D., senior scientist, Lundbeck Research. “The good thing about these label-free technologies is that you don’t need to overexpress your target. You don’t need to co-express any promiscuous G-proteins in your cell lines.”

    PerkinElmer is developing tools and technologies for the screening and discovery of compounds targeting GPCRs. “Our flagship GPCR-related technology is the luminescent photo-protein, Aequorin,” said Martina Bielefeld Sévigny, Ph.D., vp and GM of drug discovery and research reagent solutions.

    Aequorin-producing cell lines generate luminescence in response to an increase in intracellular calcium levels, thus enabling researchers to measure GPCR-induced signal-transduction pathways. Very high signal-to-noise ratios are typically obtained, enabling the discovery and development of allosteric modulators. PerkinElmer also offers over 300 GPCR cell lines, which are validated for different signaling pathways.

  • Drug Delivery

    Stephen W. Hunt III, Ph.D., svp, discovery research at Ascent Therapeutics, presented data on the firm’s Pepducin® technology.

    “Pepducin is a biotherapeutics platform that may overcome many of the obstacles to current drug discovery approaches. It provides an opportunity to pharmacologically address the entire family of GPCR targets including those that have been intractable to date,” Dr. Hunt said.

    Its lead program targets CXCR4, a GPCR and chemokine receptor that has become an attractive drug target due to its involvement in cancer and HIV. Among the observations that make CXCR4 an important cancer target include the fact that it is the receptor for stromal cell-derived factor 1 (SDF1α), which is found in high concentrations in the bone marrow, it is expressed in hematopoietic stem cells, along with many mature leukocytes, and SDF1α-CXCR4 interaction leads to the retention of these cells in the marrow. “Interrupting this interaction can facilitate release of the cells into the systemic circulation,” explained Dr. Hunt.

    “Pepducins work by a completely unique mechanism of cell entry and allosteric modulation of GPCR activity. Pepducins enter the cell in seconds via a mechanism that we call insertion and inversion. Pepducin’s lipid tail serves to insert and anchor the Pepducin in the external leaflet of the cell membrane. Charges in the peptidic portion of the molecule are neutralized, thus allowing the pepducin to traverse the membrane and anchor in the intracellular leaflet of the cell membrane. Once inside the cell, the Pepducin is in a position to encounter its target GPCR.”

    GPCRs have become extremely popular drug targets for the pharmaceutical industry. Expansion beyond classical agonist and antagonist drug classes into the realm of GPCR modulators has created new opportunities for GPCR drug discovery. Structural studies utilizing state-of-the-art biophysical methods to analyze GPCRs have enabled enhanced drug design.

    Additionally, current screening platforms allow for the measurement of GPCR-induced second messenger systems with higher throughput, higher sensitivity, and in a label-free format. Finally, new delivery technologies are enabling more accurate targeting of internal GPCR moieties.


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