Fragment-based screening using surface plasmon resonance technology.
J Biomol Screen, 2009/4;14(4):337-49.
Perspicace S[1], Banner D, Benz J, Müller F, Schlatter D, Huber W
Affiliations
PMID: 19403917DOI: 10.1177/1087057109332595
Abstract
Surface plasmon resonance (SPR) technology has emerged as a new and powerful technique to investigate the interaction between low-molecular-weight molecules and target proteins. In the present work, the authors assemble from a large compound collection a library of 2226 molecules (fragments having low molecular weights between 100 and 300 Da) to screen them for binding to chymase, a serine protease. Both the active chymase and a zymogen-like form of the protein were used in parallel to distinguish between specific and unspecific binding. The relative ligand-binding activity of the immobilized protein was periodically measured with a reference compound. The screening experiments were performed at 25 degrees C at a fragment concentration of 200 microM in the presence of 2% DMSO. Applying the filter cascade, affinity-selectivity-competition (competition with reference compounds and cross-competition with fragments), 80 compounds show up as positive screening hits. Competition experiments between fragments show that they bind to different parts of the active site. Of 36 fragments co-crystallized for X-ray studies, 12 could be located in the active site of the protein. These results validate the authors' library and demonstrate that the application of SPR technology as a filter in fragment screening can be achieved successfully.
MeSH terms
Adsorption; Chymases; Crystallography, X-Ray; Fluorescence; Humans; Ligands; Molecular Weight; Protein Binding; Reference Standards; Reproducibility of Results; Small Molecule Libraries; Surface Plasmon Resonance; Temperature; Titrimetry
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