Poster Presentation The 42nd Lorne Conference on Protein Structure and Function 2017

Understanding the role of rigidity in the potency of a Laskowski-mechanism protease inhibitor (#227)

Blake T Riley 1 , Olga Ilyichova 1 , Simon J de Veer 2 , Joakim E Swedberg 3 , Jonathan M Harris 2 , David E Hoke 1 , Ashley M Buckle 1
  1. Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia
  2. Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia
  3. Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia

Sunflower Trypsin Inhibitor (SFTI-1) is the smallest member of the Bowman-Birk protease inhibitor (BBI) family — a 14-residue cyclised peptide, comprising of two anti-parallel beta strands bridged by a disulfide linkage. As a Laskowski-mechanism inhibitor, it binds to the active site of its target protease where the catalytic triad cleaves its scissile bond (P1-P1'), and then reforms the bond in situ, with an equilibrium of 1:9 in favour of the intact bond[1].

As SFTI-1 has been reported to bind various S1 and S3 family serine proteases, it is a useful scaffold for designing specific inhibitors against serine protease targets of therapeutic interest. We have used the SFTI scaffold to engineer selective inhibitors against both kallikrein-4[2,3] and kallikrein-7, which have been implicated in tumorigenesis and metastasis of some cancers.

However, our understanding of how the structure of the SFTI scaffold contributes to its inhibitory potency is incomplete. It has been suggested that the ridigity delivered by the bicyclic structure and the backbone hydrogen-bond network is the main determinant of the potency of the scaffold. Strikingly, recent reports suggest that backbone cyclisation is not essential, and may actually reduce inhibitory potency[4].

Using crystal structures and extensive molecular dynamics simulations, we seek to understand the determining factors of rigidity and hydrolysis resistance in the SFTI scaffold and Laskowski-mechanism inhibitors. This provides useful knowledge for the engineering of specific and potent inhibitors using this versatile scaffold.

  1. Marx, U. C. et al. Enzymatic cyclization of a potent bowman-birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide. J. Biol. Chem. 278, 21782–21789 (2003).
  2. Swedberg, J. E. et al. Substrate-guided design of a potent and selective kallikrein-related peptidase inhibitor for kallikrein 4. Chem. Biol. 16, 633–643 (2009).
  3. Riley, B. T. et al. Direct and indirect mechanisms of KLK4 inhibition revealed by structure and dynamics. Sci. Rep. 6, 35385 (2016).
  4. Avrutina, O. et al. Between two worlds: a comparative study on in vitro and in silico inhibition of trypsin and matriptase by redox-stable SFTI-1 variants at near physiological pH. Org. Biomol. Chem. 10, 7753 (2012).