Abstract: The tetrapeptide Phe-Asn-Pro-Arg (SEQ ID NO: 3) is a structurally-optimized sequence for binding to the active site of thrombin. By conjugating this tetrapeptide or variants thereof to a C-terminal fragment of hirudin, we were able to generate a series of new multivalent inhibitors of thrombin containing only genetically encodable natural amino acids. We found that synergistic binding to both the active site and an exosite of thrombin can be enhanced through substitutions of amino acid residues at the P4, P3 and P3? sites of the active-site directed sequence, Xaa (P4)-Yaa (P3)-Pro (P2)-Arg (P1)-Pro(P1?)-Gln(P2?)-Zaa(P3?). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P3? sites for multivalent and optimized bridge-binding.

Abstract: The tetrapeptide Phe-Asn-Pro-Arg (SEQ ID NO: 3) is a structurally-optimized sequence for binding to the active site of thrombin. By conjugating this tetrapeptide or variants thereof to a C-terminal fragment of hirudin, we were able to generate a series of new multivalent inhibitors of thrombin containing only genetically encodable natural amino acids. We found that synergistic binding to both the active site and an exosite of thrombin can be enhanced through substitutions of amino acid residues at the P4, P3 and P3? sites of the active-site directed sequence, Xaa (P4)-Yaa (P3)-Pro (P2)-Arg (P1)-Pro(P1?)-Gln(P2?)-Zaa(P3?). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P3? sites for multivalent and optimized bridge-binding.

Abstract: The tetrapeptide Phe-Asn-Pro-Arg is a structurally-optimized sequence for binding to the active site of thrombin. By conjugating this tetrapeptide or variants thereof to a C-terminal fragment of hirudin, we were able to generate a series of new multivalent inhibitors of thrombin containing only genetically encodable natural amino acids. We found that synergistic binding to both the active site and an exosite of thrombin can be enhanced through substitutions of amino acid residues at the P4, P3 and P3? sites of the active-site directed sequence, Xaa(P4)-Yaa(P3)-Pro(P2)-Arg(P1)-Pro(P1?)-Gln(P2?)-Zaa(P3?). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P3? sites for multivalent and optimized bridge-binding. Panning of the phage library has led to thrombin-inhibitory peptides possessing strong anti-clotting activities in the low nanomolar range and yet interfering only partially with the catalytic active site of thrombin.

Abstract: There is provided a method of quantitatively ranking transient ligand binding to target biomolecules by means of NMR relaxation dispersion profiles. The present invention also relates to a method to identify ligand site obeying two-state and more complex binding behavior in a transient complex of a ligand with a target molecule, still with the use of NMR. There is also provided an efficient method to quantitate fast dissociation rates of ligands containing at least one magnetic nuclei by performing NMR relaxation dispersion experiments at different protein concentrations, enabling the evaluation of populations and exchange rates, and extending the practical applicability of the NMR relaxation dispersion experiments.