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Structure-based understanding of ligand affinity using human thrombin as a model system

V L Nienaber1, L J Mersinger, C A Kettner

  • 1Department of Chemical and Physical Sciences, DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880, USA. nienabev@crow.pprd.abbott.com

Biochemistry
|July 30, 1996
PubMed
Summary

Modifying the N-terminal blocking group of thrombin inhibitors significantly impacts binding affinity. Structural analysis reveals how these changes induce protein rearrangements, offering insights for drug design.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Drug Design

Background:

  • Previous studies indicated N-terminal blocking groups influence thrombin inhibitor binding.
  • Understanding these interactions is crucial for developing effective anticoagulants.

Purpose of the Study:

  • To synthesize and evaluate a new series of thrombin inhibitors with C-terminal methyl esters.
  • To elucidate the structural basis for varying inhibitor affinities using X-ray crystallography.

Main Methods:

  • Synthesis of Ac-D-Phe-Pro-Arg-OMe, Boc-D-Phe-Pro-Arg-OMe, and H-D-Phe-Pro-Arg-OMe.
  • Measurement of inhibition constants (Ki) for corresponding free carboxylic acids.
  • X-ray crystallography to determine the three-dimensional structures of thrombin-inhibitor complexes.

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Main Results:

  • Ki values varied significantly based on the N-terminal group: 60 µM (Ac), 7.8 µM (Boc), and 0.58 µM (H).
  • The H-D-Phe peptide exhibited higher affinity due to electrostatic interactions with Glu192.
  • The Boc group enhanced affinity via hydrophobic interactions but hindered optimal P1 binding.

Conclusions:

  • The N-terminal blocking group critically modulates thrombin inhibitor affinity.
  • Minor structural modifications can induce significant protein conformational changes.
  • These findings provide valuable insights for structure-based drug design, highlighting allosteric effects and alternative binding modes.