Shiny and Active: Advanced Bioconcept's Fluo-peptides for Labeling Receptors

By Deborah Wilkinson

Contents
Introduction
All the Right Elements
Spotting the G
In the Image
On the Flow
Born To Bind
Deals and Squeals
References

Introduction (Back to Top)
Last autumn, NEN Life Science Products Inc. (Boston, MA) purchased Advanced Bioconcepts Ltd., a formerly privately held company in Montreal. The Canadian subsidiary offers a unique line of fluorescent peptides (Fluo-peptides) of interest to the life sciences community. Fluo-peptides belong to a growing class of products termed "biologically active peptides." Virtually countless numbers of peptides responsible for carrying out a plethora of biological activities have been identified during the past couple of decades. What's more, methodologies for generating large quantities of these critical peptide reagents are available.

The needs of researchers for growing numbers of different peptide reagents coupled with technologies for mass-production is the stuff of capitalists' dreams. Indeed, increasing numbers of biologically active peptides are offered by biotechnology firms of all sizes. There seems to be no end to the possibilities for different reagents of this class, a fact that likely appeals to scientist-entrepreneurs. Opportunities for niche markets abound. Advanced Bioconcept's Fluo-peptide line of reagents caters primarily to research groups studying peptide hormones and their receptors.

All the Right Elements (Back to Top)
"Many biologically active peptides occur naturally; however, many others have been designed to have specific activities—often modeled on the functional regions of characterized proteins," explains science writer Paul Diehl (1). Additionally, biologically active peptides can be altered for use in different types of experiments. For example, peptides can be tagged with moieties that transform them into molecular probes.

Advanced Bioconcept was formed in the early 1990s to develop and commercialize novel fluorescent probes, in keeping with the growing trend to shun radioactive-based methods in favor of those amenable to high-throughput applications. One of the company's major goals was to advance the use of fluorescent-tagged peptide hormones for labeling the respective receptors (2–5). These efforts paid off for the Canadian enterprise, which now markets more than 40 fluorescent peptides. Most of these reagents, dubbed Fluo-peptides, are peptide hormone derivatives, all of which retain the ability to bind their cognate receptors with high affinity. Fluo-peptides can be used to study peptide-receptor interactions via different techniques including cellular imaging, flow cytometry, and receptor-ligand binding assays. These nontoxic reagents have significant potential for high throughput applications in diagnostics and drug discovery.

Spotting the G (Back to Top)
Many Fluo-peptides bind or are thought to bind to members of the G protein-coupled receptor (GPCR) family. These transmembrane receptors are key controllers of processes spanning the spectrum of a cell's activities (reviewed in 6–8). Examples of GPCR involvement are found in neurotransmission, cellular growth, differentiation, metabolism, secretion, inflammation, and immunity. Scientists aren't generally known for irrational exuberance or unbridled enthusiasm, even when merited. However, the import of GPCRs cannot be overemphasized. Thus, it's not entirely surprising and perhaps quite appropriate that these receptors were hailed as "the single most important drug targets for medical therapy" in a 1997 report from researchers at Duke University Medical Center (9).

Advanced Bioconcept's product listing reads like a "Who's Who" among GPCR-binding hormones. Offerings in this category include fluorescent derivatives of angiotensin II/IV, somatostatin, corticotropin-releasing factor, growth hormone releasing hormone, neurotensin, glucagon-like peptide 1, and gastrin-releasing peptide. "Fluo-peptides are one of the best available tools for studying GPCRs because these monolabeled peptides retain their biological activity. Fluo-peptides bind to their receptors with high affinities and can be effectively applied to a broad range of experimental applications," asserts Jonathan Suk, a technical sales specialist at Advanced Bioconcept.

In the Image (Back to Top)
Receptor imaging using Fluo-peptides enables the identification, localization, and characterization of specific receptors on cells and tissues. One advantage of Fluo-peptides has to do with size. These reagents are significantly smaller than are the antibodies requisite for conventional immunolabeling studies. Also, researchers can directly visualize the location of receptors bound to Fluo-peptides. This direct approach enables higher resolution imaging of receptors than is possible via traditional in vitro autoradiography techniques. Any suitable fluorescence microscope can be used to visualize receptors tagged with Fluo-peptides. Confocal microscopes afford resolutions between that of conventional microscopy and electron microscopy, and are ideally suited for Fluo-peptide applications. Not surprisingly, the most impressive results are obtained when individual cells are enriched for the labeled receptor. Thus, Fluo-peptides are particularly suited for labeling tissue- or cell- specific receptors, as well as for probing receptors expressed in transfected cell lines.

On the Flow (Back to Top)
Flow cytometry is a standard research and diagnostic tool used to analyze fluorescently labeled cell populations. It is most commonly performed using cells labeled with fluorescent antibodies directed against cell surface antigens. Antibodies specifically directed against a given peptide hormone receptor types can be difficult to generate. As a result, antibodies against many peptide receptors are simply not commercially available.

Fluo-peptides provide previously unavailable markers for peptide receptors. Because Fluo-peptides bind directly to their receptors, the intermediate steps involved in using antibody "sandwich" detection methods are not needed. Unlike labeled antibodies, each Fluo-peptide binds specifically to one hormone-binding site on an activated receptor. The 1:1 molar ratio between the fluorescent label and the hormone-binding site enables receptor quantification. Cells tagged with Fluo-peptides can also be isolated from mixtures of cells via fluorescence-activated cell sorting (FACS). "Because Fluo-peptides are nontoxic, the potential for sorting live cells for therapeutic applications becomes apparent," points out Advanced Bioconcept.

Born To Bind (Back to Top)
Receptor-ligand binding assays are typically performed for the study of receptor pharmacology and physiology. Other applications include drug screening as well as expression cloning. These assays require the use of labeled ligands. The binding affinity of a test compound is determined by measuring its ability to displace the labeled ligand from its receptor. Traditionally, such ligands have been labeled with radioactive isotopes. Fluo-peptide technology provides a safer, non-isotopic alternative for the study of receptors that bind peptide hormones. Several different methods can be used to quantify receptor binding with Fluo-peptides.

In filtration assays, fluorescence intensity can be measured directly via fluorometery. In the filtration assay format, Fluo-peptides function in exactly the same fashion as do radiolabeled peptides. Unlike filtration formats, homogeneous assays such as those based on fluorescence polarization do not require separation of bound versus free ligand. The elimination of washing and filtering steps enhances detection of low affinity interactions, and renders homogeneous assays amenable to automation. The potential of Fluo-peptides to aid in the discovery of novel receptor-ligand interactions and therapeutic compounds is especially compelling, given recent advances in high-throughput screening and fluorescence detection technologies

Deals and Squeals (Back to Top)
NEN Life Science Products' acquisition of Advanced Bioconcept is very much in keeping with the Boston behemoth's aim to "aggressively… solidify its leadership in research and drug discovery markets." Fluo-peptides extend the NEN line of fluorescent products, which also includes nucleotides, immunohistochemical reagents, and the patented tyramide signal amplification system (TSA). TSA is widely used in immunohistochemistry and in situ hybridization techniques to enhance fluorescence or chromogenic signals up to 100-fold without loss of resolution. Advanced Bioconcept reports that a significant increase in the sensitivity of receptor localization studies can be obtained by using its parent company's TSA in conjunction with standard Fluo-peptide imaging protocols. Two different basic classes of TSA systems (direct versus indirect) are available. TSA Direct is generally preferable when less signal amplification is necessary. Applications might include the visualization of medium to high receptor expressing cells, especially when viewing under a confocal microscope.

TSA Indirect provides greater signal amplification than TSA Direct, and is useful for visualizing receptors expressed at low levels, or when using a conventional fluorescence microscope.

Advanced Bioconcept is benefiting substantially from NEN's well-established marketing and selling organization. NEN can also help augment Advanced Bioconcept's research endeavors by providing strong financial support and the benefit of its extensive experience in product development. But will the once privately held company be able to maintain the character that made it successful? Advanced Bioconcept thinks so. "Our focus will be to broaden our offering by increasing the number of Fluo-peptides available, and by developing novel assays for high-throughput screening, imaging, and other applications," maintains Martin LeBlanc, general manager. "Advanced Bioconcept also offers custom synthesis of Fluo-peptides as well as assay development services for high-throughput screening applications."

References (Back to Top)

1. Diehl, P., "Biologically active peptides: Who makes them and who sells them?" The Scientist, 12(18): 16, Sept. 14, 1998.
2. Faure, M.P. et al., "Synthesis of a biologically active fluorescent probe for labeling neurotensin receptors." The Journal of Histochemistry and Cytochemistry, 42: 755, June 1994.
3. Faure, M. P. et al., "Somatodendritic internalization and perinuclear targeting of neurotensin in the mammalian brain." J. Neurosci., 15: 4140, June 1995.
4. Nouel, D. et al., "Fluorescent ligands for studying neuropeptide receptors by confocal microscopy." Endocrinology, 138: 296, Jan. 1997.
5. Beaudet, A. et al., "Fluorescent ligands for studying neuropeptide receptors by confocal microscopy." Braz. J. Med. Biol. Res., 31: 1479, Nov. 1998.
6. Hebert, T.E. and Bouvier, M., "Structural and functional aspects of G protein-coupled receptor oligomerization." Biochem. Cell Biol., 76: 1, 1998.
7. Luttrell, L.M. et al., "Regulation of tyrosine kinase cascades by G-protein-coupled receptors." Current Opinion in Cell Biology, 11: 177, April 1999.
8. Bunemann, M. and Hosey, M. M., "G-protein coupled receptor kinases as modulators of G-protein signalling," J. Physiol. (London), 517: 5, May 15, 1999.
9. Barak, L. S. et al., "A beta-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation." J. Biol. Chem ., 272: 27497, Oct. 1997.

Deborah Wilkinson earned a Ph.D. in biochemistry at the University of Tennessee, then completed her first postdoctoral fellowship in U.T.'s Division of Hematology. After conducting research at St. Jude's Children's Research Hospital (Memphis, TN) for three years on the molecular basis of pediatric leukemias, she became a freelance writer and educator. She currently resides in Birmingham, AL, and can be reached at deborahwilkinson@sprynet.com.

For more information: Martin LeBlance, General Manager, Advanced Bioconcept, 1440 St. Catherine St. W., Ste. 424, Montreal, PQ H3G 1R8 Canada. Tel: 514-874-5434 or 888-FLUOTAG. Fax: 514-874-9077, Toll Free. Or contact: Elaine Carraher, Marketing Communications Manager, NEN Life Science Products, 549 Albany Street, Boston, MA 02118-2512. Tel: 800-551-2121.