Phage ELISA test of nineteen selected clones after four panning rounds, peptide sequences and estimated apparent afffinity.

<p>A) Optical density responses detected after the binding of peptide-phage to immobilized Kly18 are shown. Background binding to BSA has been subtracted for each clone. Clones with signals above 0.5 were deemed positive and further sequenced. B) Peptide-phage clones were tested in inhibition ELISA for estimation of affinity. All clones (except clone 13) shared the WXP motif.</p

The tetrapeptide displays a Ki value similar to that of the peptide 15 lead structure.

<p>Shown are chemdraw structure models of the peptide versions 15-0, 15-5, 15-6 and 15-7. All truncated versions of peptide15 and intact peptide15 had low micromolar Ki values.</p

Peptide15 inhibits the proteolytic activity of Kly18.

<p>Peptide15 was pre-incubated with Kly18 in varying concentrations followed by addition of the substrate FITC-casein. The peptide15 inhibitory effect was seen as a decrease in Relative Fluorescence Units (RFU) in a dose-response manner. Error bars represent standard deviations between three experiments.</p

Peptide15 displays the characteristics of a competitive inhibitor.

<p>Shown are 1/V-1/S plot of peptide15 inhibition of Kly18. Kly18 was pre-incubated with fixed concentrations of peptide15 (10 µM and 30 µM) for 30 minutes, followed by incubation with varying concentrations of FITC-casein (5–50 µg/ml). As seen from the Lineweaver-Burke plot the Y-intercept is approximately the same for un-inhibited Kly18 as Kly18 inhibited with 10 µM and 30 µM, respectively. This suggested that peptide15 was a competitive inhibitor of Kly18. Linear regression lines were made using SigmaPlot 11.0. Data represent mean values from three experiments per substrate concentration.</p

Screening of truncated peptides demonstrates that a tetra-peptide version of peptide15 can inhibit Kly18.

<p>Different length versions of peptide15 were tested for Kly18 inhibitory activity. All peptides were screened in a relatively high single concentration (87 µM) to reveal if omission of single amino acid residues displayed dramatic effects on Kly18 inhibition. The data demonstrated that peptide15 can be limited to (NH₂)-SWFP-(CONH₂) (structure 15-7) and still inhibit Kly18.</p

Peptide15-MBP is specific for Kly18. A)

<p>The peptide15-MBP fusion protein interacted exclusively with Kly18, thereby demonstrating that peptide15 was specific for Kly18. <b>B)</b> To demonstrate that binding to Kly18 was mediated by peptide15 and not by MBP itself, control experiments were performed using MBP instead of MBP-peptide15. Error bars represent standard deviations between three experiments.</p

Structural alignment of the catalytic domains from Karilysin (Kly18) and human Matrix Metalloprotease-3 (MMP-3).

<p>MMP-3 is seen in green and Kly18 is seen in grey. The figure was prepared using Pymol (DeLano Scientific LLC) and the coordinate files 1CQR (MMP-3) and 2XS3 (Kly18). The structure alignment was performed using the ‘align’ function in PyMol. The Kly18 was structurally similar to MMP-3 as well as other mammalian MMP’s (MMP-1 to 3, MMP-7 to MMP-14, MMP-16 and MMP-20) (Cerda-Costa <i>et al.</i> 2011). The figure displays the zinc (yellow sphere) that is coordinated by the three active site histidines (H155, H159 and H165). The specificity loop is indicated with a red circle.</p