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Mar 1, 2009 (Vol. 29, No. 5)

Revitalizing Surface Plasmon Resonance

Resurgence Is Driven by SPR’s Aptitude for Fragment-Based Lead Discovery

  • Conventional SPR Shake-Up

    Click Image To Enlarge +
    Graffinity reports that its screening method turns conventional SPR methodologies upside down.

    Graffinity’s screening method literally turns conventional SPR methodologies upside down. Rather than immobilizing the target protein on the chip and running each compound in the library over it, it immobilizes each compound in its own sensor field and runs the target protein over them, explains Mathias Woker, CBO, who adds that this method has a number of advantages. 

    First, he says, it can vary the point at which the compound binds to the chip, presenting different surfaces to the target protein.

    Renate Sekul, Ph.D., head of research and development at Graffinity, thinks that “it is highly important to screen an active protein,” which is feasible in this system since the protein is in solution. This also makes standardizing the binding conditions across the library much easier.

    Second, it is fast, Woker notes, making it well suited to high-throughput screens. This is partially because chip regeneration is not as much of an issue, and partially because they measure light spectrum shifts rather than light angle shifts to minimize errors, Dr. Sekul said.

    Woker claims that “a Graffinity screen of 110,000 compounds takes 10 days where usually an experiment would take more than three months with strong limitations concerning the solubility of the compounds screened and the conformity of the results concerning consistent protein quality over the experiment time frame.”

    Since 2002, Graffinity has screened over 80 targets, including kinases, phospodiesterases, proteases, nuclear hormone receptors, and even RNAs, and found hits for all of them, Woker concludes.

  • Protein Interaction Studies

    Bernhard Geierstanger, Ph.D., group leader for NMR at the Genomics Institute of the Novartis Research Foundation, incorporates unnatural amino acids into proteins as site-specific NMR-active labels. This is “a unique way of putting an NMR active label at a chosen site in a protein,” he says, which is essential because in a “reasonable size protein—30 kD, for example—the number of signals is tremendous and impossible to resolve.” 

    Dr. Geierstanger, in collaboration with Peter Schultz, Ph.D., head of the institute, has created over 50 different unnatural amino acids with different purposes, along with their cognate orthogonal tRNA/aminoacyl-tRNA synthetase pairs. Some are fluorescent, some are photoactive, and some are labeled with fluorine or 15N or 13C. They can be used in lieu of any natural amino acid and placed exactly where the investigator chooses, which is a particular boon in large systems.

    A potential limitation is that only one label can be incorporated into each sample. Monitoring the chemical shift change in a series of such single resonances, however, allows site-directed screening for binders. This can reduce the number of false positives currently seen in drug development by ensuring that the drug is binding to the correct site.

    In an exciting twist, Dr. Geierstanger has made photocaged serine, cysteine, and tyrosine. Once incorporated at the desired site, the NMR label can be cut off of these residues. Thus, for the first time, individual amino acids can be labeled without altering the protein’s primary sequence, which alleviates any fears about changing the protein’s conformation or interactions. This allows him to use NMR for label-free binding, much like the other investigators use SPR.


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