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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

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    Vernalis has quantified the strengths and limitations of various experimental approaches.

    Surface plasmon resonance, or SPR, is used to measure changes in molecular weight. Thus, it has  long been used to study protein-protein interactions by registering the association and dissociation of a ligand (prey) binding to an immobilized protein (bait). 

    SPR has recently been revitalized by its aptitude for fragment-based lead discovery. Fragments are low-molecular-weight compounds (<350 kD) that bind to targets with low affinity (µM–mM range), yet they exhibit high ligand efficiency—each atom contributes by directly contacting the target’s binding site.

    Once a hit is identified, the fragment is grown or combined with other fragments, usually by structure-guided design and synthesis, to quickly generate a much more potent lead. Fragments are simple, but libraries composed of them can represent many diverse compounds and different chemistries. It is hoped that discovering drugs in this piecemeal manner will shortly yield therapeutic breakthroughs.

    Biacore (now part of GE Healthcare) claims to be  the first company to apply SPR to label-free protein-protein interaction analysis with its Biacore™ systems, according to Stefan Lofas, Ph.D., principle R&D scientist. The system’s various applications venture beyond protein-protein interactions to include protein-DNA interactions, peptide interactions, the binding properties of antibodies, drugs, and small molecules, and even interaction of viruses with whole cells, he adds. 

    Biacore makes different machines for different purposes, including screening and drug discovery. Moreover, GE Healthcare recently acquired Microcal, a market leader in calorimetry, whose instruments can detect the number of binding sites and measure the enthalpy (DH) and entropy (DS) of binding. This is a “perfect complementary technique to Biacore systems,” Dr. Lofas explains.

    Tony Giannetti, Ph.D., research scientist at Genentech, uses Biacore systems to identify promiscuous binders and remove them from high-throughput screens where they would create false positives. This saves valuable time, money, and effort that might otherwise be spent crystallizing them, he notes. 

    Biacore systems have the ability to recognize all of the hallmarks of promiscuous binders, he insists: greater than 1:1 stoichiometry, a high Hill slope, irreversible binding, aggregation, and sensitivity to detergent. Dr. Giannetti utilized the latest methods in biosensor operation to develop a high-throughput procedure for hit identification from fragment libraries all the way to lead-generation chemistry. Key features include the ability to screen and verify thousands of compounds in a short time, the large dynamic range of the assay (kd from 200 pM to 20 mM), and the small amount of protein necessary for fragment screening (<0.5 mg protein from assay development through hit validation).

    He warns of the “need to align crystallographic and SPR conditions,” lest the PEG often present in crystallography buffer destroy the binding observed in Biacore experiments; PEG should be included in the initial Biacore screen. Dr. Giannetti points out that Biacore systems can also be used to complement enzyme assays by confirming potencies. Using Biacore systems, he has identified binders to baits as varied as kinases, polymerases, cytokines, receptors, proteases, and oxidases.

    Vernalis’ approach to fragment-based drug discovery is called SeeDs, for structural exploitation of experimental drug startpoints. It includes the design of a fragment library, identification of fragments that bind competitively to a target, and evolution of these hits into leads. 

    Unique to the SeeDs strategy is displacement of bound fragments by a potent competitor, notes Roderick Hubbard, Ph.D., of the University of York and Vernalis. This feature ensures that the fragment is binding to the target’s active site and dramatically increases the quality of the initial hits, he says. 

    SPR has been successfully used in two phases of the SeeDs protocol. First, it has been used to identify compounds that bind, or if NMR was used for that, to validate binding. Since it is rapid and sensitive, SPR is an especially effective way to prescreen, or triage, a library before crystallization is attempted. It also allows for the screening of more hydrophobic fragments than NMR does. 

    Once binding has been established, the SeeDs protocol calls for SPR to characterize binding kinetics and thermodynamics.  Applying SPR to the SeeDs method has successfully ascertained why isoxazole is a much more potent growth inhibitor than pyrazole, although they both have the same IC50 against Hsp90 (pyrazole is twofold faster on, but isoxazole is 15-fold slower off).

    The FujiFilm Life Sciences Affinity Screening System, or AP-3000, was specifically designed and implemented for high throughput, sensitive analysis of small molecule binding, according to the company. The target protein is immobilized on disposable sensor sticks, with a short flow path to diminish reagent use but a wide bore to alleviate clogging.

    There are six parallel channels, so six compounds and their corresponding blanks are analyzed simultaneously. It offers high-throughput (3,840 compounds in 24 hours) and high sensitivity (it can detect interactions as weak as 10 mM), and it is fully automated, from protein immobilization all the way through to data analysis, according to Don Janezic, the SPR business development manager. In addition, it can find hits, run dose response curves, and obtain affinity constants in less than two weeks, he says.

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