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April 15, 2009 (Vol. 29, No. 8)

Detection Method for Conformational Change

Second-Harmonic Generation Provides a Molecular-Level, Functional Readout in Real Time

  • Applications

    Click Image To Enlarge +
    Figure 3. ATP induces a conformational change in adenylate kinase and a change in the baseline SHG signal.
    (Produced with permission from the PCCP Owner Societies)

    A range of applications are enabled by the platform. One can screen for allosteric compounds directly because SHG detects molecules that block or induce conformational change, not simply those that are competitive at the active site. SHG is also useful for detecting oligomerization and complex formation. 

    Biodesy is currently offering both services (assay development and screening), as well as a beta instrument for testing and screening. The instrument, Artemis™, is capable of reading 1,000–1,500 compounds per day in wells templated on microscope slides. The slide must be manually changed by a technician after each read cycle. Throughput is more than adequate for focused libraries and fragment screens, and we plan to scale up to HT.

    With controls, an SHG assay can provide rapid answers to questions such as: is a compound an agonist or an antagonist; does a compound act allosterically; does a compound stabilize a specific conformation; and does a compound prevent or induce oligomerization or complex formation?

  • SHG Detection

    Click Image To Enlarge +
    Figure 4. Inhibitor APSA triggers a conformational change when it binds to adenylate kinase and subsequently prevents ATP-induced conformational change.
    (Produced with permission from the PCCP Owner Societies)

    The general scheme for SHG detection in label format is shown in Figure 2. Data from a typical SHG assay is shown in Figures 3 and 4. Wild-type adenylate kinase, which catalyzes the transfer of a phosphate group from ATP to AMP, is labeled nonsite specifically with an SH-active dye and coupled to a surface.

    A baseline signal is generated by the label on the protein. ATP triggers a conformational change and changes the SHG signal. A known inhibitor, AP5A, prevents a subsequent ATP-induced change. The SH signal change can either increase or decrease in a given assay, depending on whether the average orientation of the labels moves toward or away from the surface.

    We have used SHG to detect ligand- and drug-induced conformational changes in a variety of protein targets such as amyloids, integrins, enzymes, and membrane receptors. We have also successfully completed several small screening projects for customers.

    SHG is a break from incremental advances in drug discovery since it provides a molecular-level, functional readout in real-time. This platform is expected to be a central engine for identifying and validating both conventional and allosteric compounds, at lower cost and in cases where few other methods exist.

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