Synthesis and Structural Characterization of Macrocyclic Plasmin Inhibitors

Simon J.A. Wiedemeyer, Guojie Wu, T. L. Phuong Pham, Heike Lang-Henkel, Benjamin Perez Urzua, James C. Whisstock, Ruby H.P. Law, Torsten Steinmetzer

Research output: Contribution to journalArticleResearchpeer-review


Two series of macrocyclic plasmin inhibitors with a C-terminal benzylamine group were synthesized. The substitution of the N-terminal phenylsulfonyl group of a previously described inhibitor provided two analogues with sub-nanomolar inhibition constants. Both compounds possess a high selectivity against all other tested trypsin-like serine proteases. Furthermore, a new approach was used to selectively introduce asymmetric linker segments. Two of these compounds inhibit plasmin with Ki values close to 2 nM. For the first time, four crystal structures of these macrocyclic inhibitors could be determined in complex with a Ser195Ala microplasmin mutant. The macrocyclic core segment of the inhibitors binds to the open active site of plasmin without any steric hindrance. This binding mode is incompatible with other trypsin-like serine proteases containing a sterically demanding 99-hairpin loop. The crystal structures obtained experimentally explain the excellent selectivity of this inhibitor type as previously hypothesized.

Original languageEnglish
Article numbere202200632
Number of pages16
Issue number6
Publication statusPublished - 14 Mar 2023


  • crystal structures
  • plasmin
  • protease inhibitors
  • slow-binding inhibition
  • trypsin

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