Proteinase 3 (PR3) is a neutrophil enzyme that has been identified as a drug target for chronic inflammatory diseases. PR3 is expressed in cytoplasmic granules but also at plasma membranes where it mediates the inflammatory effects. The membrane interface binding site (IBS) of PR3 is distinct to its catalytic site therefore, targeting IBS would provide a more specific way to inhibit only the membrane-expressed PR3. We have designed D-peptides to target IBS by applying docking and molecular dynamics simulations. Next, we verified if these D-peptides bind to PR3 using isothermal titration calorimetry and fluorescence spectroscopy. Further, we performed surface plasmon resonance experiments to determine if these D-peptides inhibit the binding of PR3 to lipids. We verified that the designed D-peptides did not affect the catalytic activity of PR3. A few peptides bound to PR3 hydrophobic pockets and inhibited PR3 binding to lipid vesicles. However, the (KFF)3K peptide (both in the L- and D-form) bound also to the membrane. One D-peptide with the SAKEAFFKLLAS sequence inhibited the PR3-membrane binding site with IC50 of about 40 µM and did not bind to the membrane itself. This work provides a direct connection between the computational studies and experimental verification of D-peptides. We also suggest possible inhibition pockets in PR3 other than the catalytic site.

D-peptides as inhibitors of Proteinase 3-membrane interactions

253rd National Meeting (2017)

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