d-Peptides as inhibitors of PR3-membrane interactions

Abstract

Proteinase 3 (PR3) is a neutrophil serine protease present in cytoplasmic granules but also expressed at the neutrophil surface where it mediates proinflammatory effects. Studies of the underlying molecular mechanisms have been hampered by the lack of inhibitors of the PR3 membrane anchorage. Indeed while there exist inhibitors of the catalytic activity of PR3, its membrane interfacial binding site (IBS) is distinct from its catalytic site. The IBS has been characterized both by mutagenesis experiments and molecular modeling. Through docking and molecular dynamics simulations we have designed d-peptides targeting the PR3 IBS. We used surface plasmon resonance to evaluate their effect on the binding of PR3 to phospholipid bilayers. Next, we verified their ability of binding to PR3 via fluorescence spectroscopy and isothermal titration calorimetry. The designed peptides did not affect the catalytic activity of PR3. A few peptides bound to PR3 hydrophobic pockets and inhibited PR3 binding to lipids. While the (KFF)₃K d-peptide inconveniently showed a significant affinity for the lipids, another d-peptide (SAKEAFFKLLAS) did not and it inhibited the PR3-membrane binding site with IC₅₀ of about 40μM. Our work puts forward d-peptides as promising inhibitors of peripheral protein-membrane interactions, which remain high-hanging fruits in drug design.

Keywords: Computational docking and molecular dynamics simulations; Isothermal titration calorimetry; Peripheral membrane binding; Proteinase 3 membrane binding site; Surface plasmon resonance; d-Peptides as inhibitors.