表面プラズモン共鳴装置を用いたフラグメントスクリーニング

Abstract

A surface plasmon resonance (SPR) optical biosensor is a label-free biophysical device which can detect molecular interaction in real time. SPR is an emerging technology for fragment screening, the first step in fragment-based drug discovery. Low levels of protein consumption and the ability to detect interactions with K(d) as low as mM make this technology particularly attractive. Inherently small SPR responses due to fragment binding had been an issue but, owing to well-established experimental protocols, such responses have become readily detectable. Medium-throughput instruments are now on the market from several manufacturers that enable complete screening of a library with several thousand compounds within a few days. In fragment screening, test compounds are injected at a high concentration because of the low affinity expected for small molecules, making it likely that many false positives arise from non-specific binding to an unrelated part of the target protein. Such false positives have to be eliminated by a well-designed assay cascade so as to select true hits which can then be subjected to X-ray crystallization to obtain detailed structural information. SPR-based direct binding assays have to be developed with a sufficient binding capacity and good reproducibility with a Z'-factor larger than 0.6. In selecting hit candidates from fragment primary screens, the shape of sensorgrams, binding stoichiometry and response level to reference proteins when available must be carefully evaluated. The selected compounds from primary screening need to be further examined in terms of dose-dependence and binding competition against tight binding reference compounds to ensure that they bind to the designated site of the target protein.