The authors present fragment screening data obtained using a label-free parallel analysis approach where the binding of fragment library compounds to 4 different target proteins can be screened simultaneously using surface plasmon resonance detection. They suggest this method as a first step in fragment screening to identify and select binders, reducing the demanding requirements on subsequent X-ray or nuclear magnetic resonance studies, and as a valuable "clean-up" tool to eliminate unwanted promiscuous binders from libraries. A small directed fragment library of known thrombin binders and a general 500-compound fragment library were used in this study. Thrombin, blocked thrombin, carbonic anhydrase, and glutathione-Stransferase were immobilized on the sensor chip surface, and the direct binding of the fragments was studied in real time. Only 12 μg of each protein is needed for screening of a 3000-compound fragment library. For screening, a binding site-blocked target as reference facilitates the identification of binding site-selective hits and the signals from other reference proteins for the elimination of false positives. The scope and limitations of this screening approach are discussed for both target-directed and general fragment libraries. (Journal of Biomolecular
A set of compounds designed to bind to the S2−S3 pockets of thrombin was prepared. These compounds included examples with no interactions in the S1 pocket. Proline, a common P2 in many thrombin inhibitors, was combined with known P3 residues and P1 substituents of varying size and lipophilicity. Binding constants were determined using surface plasmon resonance (SPR) biosensor technology and were found to be in good agreement with results from an enzyme assay. A dramatic increase in affinity (100−1000 times) was seen for compounds incorporating an amino group capable of forming a hydrogen bond with gly216 in the protein backbone. The ligand efficiency was increased by including substituents that form stronger hydrophobic interactions with the P1 pocket. The binding mode was confirmed by X-ray analysis, which revealed the anticipated binding motif that included hydrogen bonds as well as a tightly bound water molecule. A QSAR model indicated that hydrogen bonding and lipophilicity were important for the prediction of binding constants. The results described here may have implications for how directed compound libraries for shallow protein pockets, like S2 and S3 in serine proteases, can be designed.
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