title: Mastering the canonical loop of serine protease inhibitors: enhancing potency by optimising the internal hydrogen bond network.
abstract: BACKGROUND
  Canonical serine protease inhibitors commonly bind to their targets through a rigid loop stabilised by an internal hydrogen bond network and disulfide bond(s). The smallest of these is sunflower trypsin inhibitor (SFTI-1), a potent and broad-range protease inhibitor. Recently, we re-engineered the contact β-sheet of SFTI-1 to produce a selective inhibitor of kallikrein-related peptidase 4 (KLK4), a protease associated with prostate cancer progression. However, modifications in the binding loop to achieve specificity may compromise structural rigidity and prevent re-engineered inhibitors from reaching optimal binding affinity.
  
  
  RESULTS
  In this study, the effect of amino acid substitutions on the internal hydrogen bonding network of SFTI were investigated using an in silico screen of inhibitor variants in complex with KLK4 or trypsin. Substitutions favouring internal hydrogen bond formation directly correlated with increased potency of inhibition in vitro. This produced a second generation inhibitor (SFTI-FCQR Asn(14)) which displayed both a 125-fold increased capacity to inhibit KLK4 (K(i) = 0.0386±0.0060 nM) and enhanced selectivity over off-target serine proteases. Further, SFTI-FCQR Asn(14) was stable in cell culture and bioavailable in mice when administered by intraperitoneal perfusion.
  
  
  CONCLUSIONS
  These findings highlight the importance of conserving structural rigidity of the binding loop in addition to optimising protease/inhibitor contacts when re-engineering canonical serine protease inhibitors.
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia.
authors.forename: Joakim E
authors.initials: JE
authors.lastname: Swedberg
authors.forename: Simon J
authors.initials: SJ
authors.lastname: de Veer
authors.forename: Kei C
authors.initials: KC
authors.lastname: Sit
authors.forename: Cyril F
authors.initials: CF
authors.lastname: Reboul
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Buckle
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Harris
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D006860:Hydrogen Bonding; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D056004:Molecular Dynamics Simulation; D011471:Prostatic Neoplasms / Q000473:pathology*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D018411:Spodoptera
chemical_list: D015842:Serine Proteinase Inhibitors