Biophysical methods are increasingly being adopted by the pharmaceutical industry as a way to improve the identification of high-quality hits and lead compounds.1 Fragment-based drug discovery uses structural methods and libraries of low-molecular-weight compounds (<300 Da) to identify low-affinity candidates for development into leads.2
Surface plasmon resonance (SPR) biosensors provide label-free, real-time analysis of drug-target interactions. Targets are immobilized onto a dextran-coated gold sensor chip and drug binding is detected via changes in mass concentration at the sensor surface—providing data on binding selectivity, stoichiometry, affinity, and kinetics. These instruments are widely used in the pharmaceutical industry, mainly downstream of secondary screening, in areas such as hit validation and lead optimization.3 Fragment libraries typically contain far fewer compounds than those used for high-throughput screening, making primary screening feasible for SPR systems with sufficient throughput capabilities.
Fragment screening presents several technical challenges, including high demands on structural methods (X-ray crystallography, NMR) that consume large amounts of target protein, and the low (typically millimolar) affinities exhibited by fragments, requiring high sample concentrations and associated solubility/non-specific binding issues. SPR-based strategies can be designed to overcome many of these issues. The entire library (typically 1,000-10,000 fragments) can be rapidly screened against the target proteins and a blank reference to remove problem compounds—such as those with solubility problems or those that exhibit sticky behavior. After this clean-up step, fragments can be examined at a single concentration against multiple immobilized proteins (targets, blank reference, and additional protein references) to identify true binders in one assay (Figure 1). Modification of the target protein to establish binding site selectivity can be either by mutation or chemical blocking of the active site.
These assays are automated and rapid (>1000 fragments in a 24-hour unattended run), with low target-protein consumption (low µg quantities typically sufficient for 1000 fragments). Libraries can also be analyzed using concentration series of fragments to validate that true saturable binding has occurred and for ranking based on affinity. Finally, using known target-binding control compounds in competition assays with the fragments gives important information on binding site mapping and further validates the hits. These assays can be run either prior to or in parallel with structural methods for efficient fragment screening.
SPR-based screening of fragment libraries provides an efficient, informative complement to structural methods such as X-ray crystallography or NMR. Direct binding measurements rapidly eliminate problem fragments and general protein binders, providing identification of verified hits, with data on selectivity, estimated affinity and binding site information. This is achieved with a throughput appropriate for fragment libraries, and with very low target consumption. Moreover, the same instrument can provide high quality characterization data (binding selectivity, affinity and kinetics) for lead selection and optimization during subsequent stages of the drug discovery process.4
At a glance
Company: GE Healthcare
Product: Biacore A100
More information: www.gelifesciences.com/biacore
1. Huber W. A new strategy for improved secondary screening and lead optimization using high-resolution SPR characterization of compound-target interactions. J Mol Recognit. 2005;18:273-281.
2. Hajduk P and Greer J. A decade of fragment-based drug design: strategic advances and lessons learned. Nature Rev Drug Discov. 2007;6:211-219.
3. Danielson H. Integrating surface plasmon resonance biosensor-based interaction kinetic analyses into the lead discovery and optimization process. Future Med Chem. 2009;1 (In Press).
4. Andersson K, et al. Label-free kinetic binding data as a decisive element in drug discovery. Expert Opin Drug Discov. 2006;1:439-446.
Filed Under: Drug Discovery