MOLECULAR-BASIS FOR THE INHIBITION OF HUMAN ALPHA-THROMBIN BY THE MACROCYCLIC PEPTIDE CYCLOTHEONAMIDE-A

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 alpha-thrombin, a protease central to the bioregulation of thrombosis and hemostasis, was studied by x-ray crystallography. This work (2.3-angstrom 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 S1 specificity pocket; formation of a hydrogen-bonded two-strand antiparallel beta-sheet with Ser214-Gly216) and the alpha-keto amide group of CtA are primarily responsible for binding to thrombin, with the alpha-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 Trp60D as part of an ''aromatic stacking chain.'' Biochemical inhibition data (K(i) values at 37-degrees-C) were obtained for CtA with thrombin and a diverse collection of serine proteases. Thus, CtA is just a moderate inhibitor of human alpha-thrombin (K(i) = 0.18 muM) but a potent inhibitor of trypsin (K(i) = 0.023 muM) and streptokinase (K(i) = 0.035 muM). 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.