BackgroundCanonical 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 FindingsIn 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 (K i = 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/SignificanceThese 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.

Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P = 3.9 × 10−22). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P = 1.9 × 10−34). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-011-0981-1) contains supplementary material, which is available to authorized users.

The insulin receptor (IR) plays critical roles in metabolism and growth, directed by the binding of insulin. Decades of research to understand the mechanism of insulin binding and activation of the IR have identified a region of the receptor, the C-terminal (CT) peptide, to be crucial for insulin binding. In particular, a truncated IR consisting of the first three domains fused to the CT peptide was found to bind insulin with nanomolar affinity, with undetectable binding in the absence of fused or soluble CT peptide. Problematically, all current crystal structures of the IR indicate the fusion point of the CT peptide to the three domains is located far from the position of the CT peptide as resolved in such structures. We have attempted to address this problem using molecular modelling and dynamics simulations. The results led to the identification of a potential inter-domain interaction between the L2 domain and the CT peptide that is not observed in any of the crystal structures of the IR. Investigations into this new interaction found a conformational change that could potentially be in response to insulin binding. Additionally, further simulation work with the new conformation demonstrated its compatibility with the position and orientation of insulin from the latest insulin-bound IR crystal structure.

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