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Apr 15, 2012 (Vol. 32, No. 8)

Bringing GPCR Research to the Fore

  • Ruben Abagyan, Ph.D., professor at the Skaggs School of Pharmacy & Pharmacology at the University of California, San Diego, described efforts to expand the x-ray crystallographic structures of the dozen GPCRs determined so far to predictive models of hundreds of GPCRs in different conformational states.

    Dr. Abagyan noted that with recent work the community now has the crystal structures for nine GPCRs and will soon have the crystal structures for two or three more. This represents an “incredible advance,” he said, over a decade ago when we had the crystal structure of rhodopsin, but of no other GPCR. Nevertheless, there are estimated to be approximately 1,000 different GPCRs in the human genome, and so the current structural knowledge of a handful of GPCRs represents only 1% of the total.

    Dr. Abagyan emphasized that improved modeling techniques promise to expand the scope of GPCR proteins whose structures are understood, as well as increase the understanding of how a given GPCR binds to chemically different agonists, antagonists, inverse or partial agonists, and allosteric modulators.

    In particular, he described three different levels of structure-ligand binding computational predictions that can be carried out today. The easiest approach is if the crystal structure of the GPCR is known. In such cases, Dr. Abagyan showed that a virtual ligand screening approach using the latest docking tools and ICM (Internal Coordinate Mechanics) software demonstrated an over 90% success rate in predicting the correct ligand binding pose in a docking exercise to a single cognate pocket determined by crystallography, and 90% reliability in a more realistic cross-docking exercise if multiple experimental pocket conformations were considered.

    This represents “an outstanding result,” Dr. Abagyan said, when compared with the typical cross-docking success rates of 30% to 70% obtained with previous approaches. Moreover, in addition to the docking pose of active molecules, he showed for two better-characterized GPCRs that the docking scores are now capable of separating thousands of actives from inactive molecules.

    The second, more challenging approach applies when the GPCR crystal structure is not known, but that of a homologue is. In such cases, knowledge of previously discovered receptor ligands provides key information that can be used for helping the modeling process and improving docking and screening performance of the receptor.

    Dr. Abagyan demonstrated how the so-called “ligand-guided optimization of homology models” improves the initially inaccurate models of the ligand-binding pockets into an ensemble of highly predictive models. The new implementation, abbreviated as Alibero or LiBERO, improves the outcome of the procedure, Dr. Abagyan said.

    The third and most difficult approach is that for the overall model of a GPCR or an allosteric pocket that forms upon binding. For these cases, Dr. Abagyan’s group and his colleagues at Molsoft have developed an improved homology modeling approach that includes several essential elements: (i) a new force field that was tested for a better ability to predict loop conformations, (ii) a new membrane implicit solvation model, and, (iii) an adequate atom-level transfer of the distance restraints from a template to the model.

    The approach can be further assisted with what Dr. Abagyan termed as “fumigation” because it offers the potential of opening up the binding site for interaction with a potential small molecule modulator.

  • Fluorescent Ligands

    Click Image To Enlarge +
    Co-localization of ligand and GPCR: Human embryonic kidney cells (HEK-293T) that express a SNAP-tagged human adenosine-A3 receptor (green, upper right quadrant). The cells have been incubated with red fluorescent adenosine-A3 receptor antagonists (red, upper left quadrant). The bottom left quadrant is the merged image (yellow indicates co-localization of ligand and receptor). The bottom right quadrant image is the co-localization analysis, indicating black pixels where ligand and receptor are co-localized. [Stephen Hill, University of Nottingham Medical School]

    Stephen Hill, Ph.D., head of the School of Biomedical Sciences and professor of molecular pharmacology at the University of Nottingham Medical School, described the effective use of fluorescent ligands to investigate the function of GPCRs at the single living cell and single-molecule levels.

    Dr. Hill said that this important advance has been made possible by progress in confocal microscopy, quantitation of fluorescence intensity at the cell membrane, and the development of fluorescent ligands that retain their pharmacological activity and whose fluorescence is much brighter when the ligand binds to a cell surface receptor than when free in solution.

    Dr. Hill noted that the ability to work at the single-molecule and single-living-cell levels “will be critical when working with limited numbers of cells, such as is often the case in clinical investigations.”

    In addition, the approach allows detailed kinetic studies to be undertaken with the receptor in its native environment within the cell membrane. Also, the increased resolution and temporal capability of these techniques can be applied to native cells endogenously expressing the receptor of interest.

    Using live CHO cells bearing recombinant human adenosine A1 or adenosine A3 GPCRs, Dr. Hill demonstrated that a receptor-specific agonist or antagonist could inhibit the binding of a fluorescent ligand to cell surface receptors at the single-cell level. He further demonstrated that this approach could be used in a 96-well plate for high-throughput screening with a confocal imaging plate reader to investigate how well a nonfluorescent drug might inhibit GPCR-ligand-specific fluorescence in live cells.

    The approach could also be used to quantitate the effect of small molecular fragments on fluorescent ligand binding in fragment-based drug discovery, he noted. This successful use of fluorescence with live cells represents a major advance over the current approach of radiologic labeling of GPCRs in membrane homogenates, he added.

    Dr. Hill went on to show that the same fluorescent approach could be used to label adenosine A3 receptors in live human neutrophils where these GPCRs are endogenously expressed. In neutrophils, the adenosine A3 receptors are known to coordinate chemotaxis and, not surprisingly, Dr. Hill said, observation of the fluorescent signals indicated that the adenosine A3 receptors were located at the leading edge of the neutrophil.


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