Mastering the canonical loop of serine protease inhibitors : enhancing potency by optimising the internal hydrogen bond network

Swedberg, Joakim, De Veer, Simon, Sit, Kei Chun, Reboul, Cyril, Buckle, Ashley, & Harris, Jonathan (2011) Mastering the canonical loop of serine protease inhibitors : enhancing potency by optimising the internal hydrogen bond network. PLoS ONE, 6(4), pp. 1-11.

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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.

Methodology/Principal Findings 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 Asn14) which displayed both a 125-fold increased capacity to inhibit KLK4 (Ki = 0.0386±0.0060 nM) and enhanced selectivity over off-target serine proteases. Further, SFTI-FCQR Asn14 was stable in cell culture and bioavailable in mice when administered by intraperitoneal perfusion.

Conclusion/Significance 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.

Impact and interest:

30 citations in Scopus
32 citations in Web of Science®
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ID Code: 52334
Item Type: Journal Article
Refereed: Yes
ISSN: 1932-6203
Subjects: Australian and New Zealand Standard Research Classification > AGRICULTURAL AND VETERINARY SCIENCES (070000)
Australian and New Zealand Standard Research Classification > MEDICAL AND HEALTH SCIENCES (110000)
Divisions: Current > QUT Faculties and Divisions > Faculty of Health
Current > Institutes > Institute of Health and Biomedical Innovation
Copyright Owner: Copyright 2011 the authors
Copyright Statement: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Deposited On: 19 Jul 2012 06:29
Last Modified: 09 Apr 2014 12:21

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