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August 01, 2013 (Vol. 33, No. 14)

Removing the Receptor Deconvolution Bottleneck

Powerful Tool Aims to Uncover Novel Disease-Relevant Drug Targets

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    Figure 1. Schematic representation of Retrogenix’ cell microarray technology

    Progress in the study of normal biological processes, or the mechanism of disease, has traditionally been hampered by the immense challenge in identifying the specific binding partners for a natural ligand or pathogen. Similarly, in drug discovery, the success of phenotypic screening approaches ultimately relies on deconvoluting the targets of any phenotypic molecules before these leads can be progressed.

    In the case of antibodies or peptides (i.e., biologics), the binding targets will generally be receptors or other types of cell surface protein. Until recently, the hit rate for accurately identifying a cell surface protein interaction was approximately 10% using techniques such as standard protein chips or immunoprecipitation and mass spectrometry-based methodologies. However, a validated high-throughput approach—using a cell microarray technology developed by Retrogenix—has now increased the likelihood of success to more than 60% and has already contributed to breakthroughs in malaria research and the deconvolution of receptor(s) for many antibody ligands.

    The elegance of the Retrogenix microarray system lies in expressing full-length human membrane proteins in the context of human cells. This allows for normal trafficking of the proteins to the cell surface, correct folding in the plasma membrane, and natural post-translational modification. Once expressed in their native state on the cell surface, these proteins are then available to interact with the test ligand. Representation of a large proportion of the plasma membrane proteome in a physiologically relevant system underpins the success of this technology in detecting specific hits for a variety of applications.

    This tool utilizes a proprietary library of expression vectors containing open reading frames (ORFs) encoding more than 3,500 full-length human plasma membrane proteins, as well as a green fluorescent protein (GFP). Each vector is combined with a lipid and the complexes are spotted in distinct locations on glass slides. Human cells are then grown to confluence over the spotted slides.

    Reverse-transfection of the cells sitting over each expression vector results in overexpression of each respective membrane protein, along with GFP. The ligand (e.g., protein, virus, phenotypic molecule) is applied to the cell microarrays and putative receptor targets identified by analyzing “gain-of-binding” using an appropriate detection system (Figure 1).

    The GFP acts to determine spot coordinates, as well as provide a quality control to ensure that the minimum transfection threshold has been exceeded. Typically, further tests are then undertaken to determine that the receptor “hit” is reproducible and confirm specificity to the test ligand.

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    Figure 2. All subclasses of plasma membrane protein are represented in the Retrogenix library. Within some of the main subclasses, the percentage coverage of all known members is shown.

    The current set of 3,500 plasma membrane proteins spans a broad spectrum of membrane protein subclasses, and is biased toward membrane proteins with an extracellular domain (ECD). As shown in Figure 2, good coverage across all subclasses of plasma membrane protein has been achieved.

    Of these 3,500 full-length ORFs, about 2,600 represent different genes, while the others represent alternative variants of the same genes, particularly where variants in the ECD exist. This collection is the largest set of commercially accessible plasma membrane protein expression clones, and currently represents approximately 60% of the known human plasma membrane proteome.

  • Notable Applications

    This technology has proven successful in uncovering the cell surface receptors of orphan ligands, which play a key role in normal or disease processes. Such ligands include natural soluble proteins and peptides, viruses, and cell surface-derived ligands (i.e., those mediating cell:cell interactions).

    A study published in Nature in June 2013 describes how Retrogenix’ technology identified the key receptor responsible for severe childhood malaria.

    This receptor, known as endothelial protein C receptor (EPCR), is responsible for binding and sequestration of malaria parasite-infected erythrocytes to blood vessels in the brain, likely to be interfering with anti-inflammatory and antithrombotic actions of EPCR. Of the 2,500 human plasma membrane proteins screened, EPCR was identified as the single specific hit—an event described as a “true eureka moment” by the lead researcher at the University of Copenhagen.

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    Figure 3. A typical phenotypic drug discovery approach using antibodies, yielding novel disease-relevant targets and antibody leads.

    Despite this contribution to malaria research, to date, the Retrogenix technology has been more extensively used for deconvoluting the cell surface targets of phenotypic antibodies that have been discovered using a phenotypic screening approach (Figure 3). One advantage of phenotypic screening over the alternative, target-led approach to drug discovery, is that completely new disease-relevant targets and pathways are being discovered.

    Retrogenix’ technology has identified many new drug targets on behalf of its partners. These have included receptor-type kinases, GPCRs, immunoglobulin superfamily members, anchor proteins, and others. In some cases, these have even been multisubunit receptors such as integrins, the additional subunits presumably being provided by the cells endogenous machinery, highlighting a further advantage of physiological systems over traditional protein chips.

    In addition to isolating a primary target, Retrogenix’ technology provides a tool to uncover the secondary or off-target activities of antibody and protein drug leads. This assists in candidate selection as it reduces the risk of future, costly failures that are due to off-target driven toxicities.

  • A Versatile and Scalable Technology

    As well as its applicability to a wide range of ligand types (e.g., antibodies, proteins, viruses), Retrogenix has developed and validated a range of detection systems for protein ligands (anti-His, anti-Flag, anti-V5, anti-Fc, biotin-streptavidin, directly fluorescently tagged, and radiolabeled), and for different species and isotypes of phenotypic antibodies.

    Crucially, the technology is highly sensitive, capable of detecting micromolar affinity ligand-receptor interactions even in the absence of any amplification strategies. Speed and throughput are not sacrificed for sensitivity and specificity; Retrogenix can identify and confirm specificity of ligand receptors in a matter of weeks, screening many molecules in parallel. Throughput is further enhanced by screening “pools” of up to 20 molecules for binding against the 3,500 membrane proteins, then subsequently matching each molecule to each receptor target.

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