title: Structure-based understanding of ligand affinity using human thrombin as a model system.
abstract: Kinetic study of a series of compounds containing the thrombin-directed peptide D-Phe-ProboroArg-OH had indicated that the structure of the N-terminal blocking group may be correlated with binding [Kettner, C., Mersinger, L., & Knabb, R. (1990) J. Biol. Chem. 265, 18289-18297]. In order to further study this phenomenon, a second series of compounds that contains a C-terminal methyl ester in place of the boronic acid was synthesized, binding measured, and the three-dimensional structure in complex with human thrombin determined by X-ray crystallography. Incubation of Ac-D-Phe-Pro-Arg-OMe, Boc-D-Phe-Pro-Arg-OMe, and H-D-Phe-Pro-Arg-OMe resulted in the formation of thrombin-product complexes within the crystal. Ki values for the corresponding products (free carboxylic acids) were 60 +/- 12 microM, 7.8 +/- 0.1 microM, 0.58 +/- 0.02 microM, respectively, indicating that the nature of the N-terminal blocking group has a significant effect on affinity. Examination of the crystal structures indicated that the higher affinity of the H-D-Phe peptide is due to rearrangement of one residue comprising the S3 site (Glu192) in order to maximize electrostatic interactions with the "NH3(+)-" of H-D-Phe. The relative affinity of Boc-D-Phe-Pro-Arg-OH is due to favorable hydrophobic interactions between thrombin and the bulky butyl group. However, this results in less favorable binding of Arg-P1 in the oxyanion hole as shown by long hydrogen-bonding distances. This work gave rise to some general observations applicable to structure-based drug design: (1) altering the structure of an inhibitor at one site can affect binding at an unchanged distal site; (2) minor alteration of inhibitor structure can lead to small, but significant reorganization of neighboring protein structure; (3) these unexpected reorganizations can define alternate binding motifs.
authors.affiliation: Department of Chemical and Physical Sciences, DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880, USA. nienabev@crow.pprd.abbott.com
authors.forename: V L
authors.initials: VL
authors.lastname: Nienaber
authors.forename: L J
authors.initials: LJ
authors.lastname: Mersinger
authors.forename: C A
authors.initials: CA
authors.lastname: Kettner
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004563:Electrochemistry; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008024:Ligands; D009842:Oligopeptides; D013917:Thrombin