Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Identification of bikunin as a scaffold for a humanised aprotinin analogue using molecular dynamics simulations (#159)

Alex Jackson 1 , Cameron Grant 1 , Alex Duff 1 , Darren Leahy 1 , Jonathan Harris 1
  1. Faculty of Health, Queensland University of Technology, Kelvin Grove, Queensland, Australia

Massive bleeding during invasive surgeries is a key issue within clinical settings. Hyperfibrinolysis, or excessive proteolytic breakdown of fibrin clots, is the major driver of massive bleeding and requires clinical intervention during invasive cardiac surgeries. In 2008 the potent antifibrinolytic/antiprotease drug Trasylol® based on the bovine Kunitz inhibitor aprotinin was removed from clinical use following reports of severe anaphylactic reactions and hypersensitivity1-5, as a result of its bovine origin6, 7. Accordingly, this project focused on the redesign of a human protein as a replacement for Trasylol®. Bikunin, a dual-domain Kunitz protease inhibitor was identified for this purpose. Bikunin’s dual-domain structure allows for targeting of Trasylol®’s cognate protease plasmin, while providing a secondary anti-inflammatory activity potentially due to inhibition of plasma kallikrein in conjunction with further pro-inflammatory enzymes for which it has an innate specificity8, 9. To adapt bikunin as a plasmin inhibitor, in silico molecular dynamics simulations were performed comparing aprotinin-protease and bikunin-protease complexes. Structures of domain 1 from wild-type bikunin, a bikunin domain 1 variant containing the aprotinin interacting loop and aprotinin were independently superimposed into the active sites of plasmin and plasma kallikrein. Significant increases in the interaction energies and H-bonds formed from the wild-type bikunin to the humanised aprotinin bikunin (haBik) variant for both plasmin and plasma kallikrein simulations (p = <0.0001) were observed, with no significant difference between haBik and aprotinin (p = >0.05) when interacting with plasmin. The simulations with plasma kallikrein showed that there was still a significant difference between haBik and aprotinin’s interaction efficiency (p = <0.0001). However, we hypothesised that haBik’s anti-inflammatory secondary domain will in part account for this difference. Finally, we have begun production of bikunin variants with substantial yields in a Pichia pastoris expression system, purifying the inhibitors to homogeneity and carrying out preliminary kinetic characterisation.

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