Molecular Basis for the Inhibition of Human α-Thrombin by the Macrocyclic Peptide Cyclotheonamide A

• Published in: Proceedings of the National Academy of Sciences - PNAS 1993-09, Vol.90 (17), p.8048-8052
• Main Authors: Maryanoff, Bruce E., Qiu, Xiayang, Padmanabhan, K. P., Tulinsky, Alexander, Almond, Harold R., Andrade-Gordon, Patricia, Greco, Michael N., Kauffman, Jack A., Nicolaou, K. C., Liu, Aijn, Brungs, Peter H., Fusetani, Nobuhiro
• Format: Article
• Language: English
• Subjects: Amides; Amino Acid Sequence; Biochemistry; Biological and medical sciences; Blood. Blood coagulation. Reticuloendothelial system; Carbon; Crystallography; Enzymes; Humans; Hydrogen Bonding; Hydrogen bonds; Kinetics; Ligands; Marine; Medical sciences; Models, Molecular; Molecular Sequence Data; Molecular Structure; Peptides, Cyclic - chemistry; Peptides, Cyclic - pharmacology; Pharmacology. Drug treatments; Plasma spectra; Polysorbates; Protein Structure, Secondary; Serine Proteinase Inhibitors - chemistry; Substrate Specificity; Theonella; Thrombin - antagonists & inhibitors; Thrombin - chemistry; X-Ray Diffraction
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.17.8048

The macrocyclic peptide cyclotheonamide A (CtA), isolated from the marine sponge Theonella sp., represents an unusual class of serine protease inhibitor. A complex of this inhibitor with human α-thrombin, a protease central to the bioregulation of thrombosis and hemostasis, was studied by x-ray crystallography. This work (2.3-Å resolution) confirms the structure of CtA and reveals intimate details about its molecular recognition within the enzyme active site. Interactions due to the "Pro-Arg motif" (Arg occupancy of the S1specificity pocket; formation of a hydrogen-bonded two-strand antiparallel β-sheet with Ser214-Gly216) and the α-keto amide group of CtA are primarily responsible for binding to thrombin, with the α-keto amide serving as a transition-state analogue. A special interaction with the "insertion loop" of thrombin (Tyr60A-Thr60I) is manifested through engagement of the hydroxyphenyl group of CtA with Trp60Das part of an "aromatic stacking chain." Biochemical inhibition data (Kivalues at 37⚬C) were obtained for CtA with thrombin and a diverse collection of serine proteases. Thus, CtA is just a moderate inhibitor of human α-thrombin (Ki= 0.18 μM) but a potent inhibitor of trypsin (Ki= 0.023 μ M) and streptokinase (Ki= 0.035 μM). The relative lack of potency of CtA as a thrombin inhibitor is discussed with respect to certain structural features of the enzyme complex. We also report the total synthesis of CtA, by a convergent [2 + 3] fragment-condensation approach, to serve the preparation of cyclotheonamide analogues for structure-function studies.