Investigating Polypharmacology through Targeting Known Human Neutrophil Elastase Inhibitors to Proteinase 3.

Using a combination of multisite λ-dynamics (MSλD) together with in vitro IC50 assays, we evaluated the polypharmacological potential of a scaffold currently in clinical trials for inhibition of human neutrophil elastase (HNE), targeting cardiopulmonary disease, for efficacious inhibition of Proteinase 3 (PR3), a related neutrophil serine proteinase. The affinities we observe suggest that the dihydropyrimidinone scaffold can serve as a suitable starting point for the establishment of polypharmacologically targeting both enzymes and enhancing the potential for treatments addressing diseases like chronic obstructive pulmonary disease.

Phospholipid Membrane Interactions of Model Ac-WL-X-LL-OH Peptides Investigated by Solid-State Nuclear Magnetic Resonance.

The role of aromatic amino acids in peripheral protein membrane binding has been reported to involve cation-π interactions with choline lipids. In this study, we have investigated the interactions of the model pentapeptide Ac-WL-X-LL-OH (where X = L, Y, F, or W) with the phospholipid membrane using solid-state NMR. The effect of guest residue X on the peptide-lipid interactome was complementary to the seminal report on the interfacial hydrophobicity scale by Wimley and White. We found that the phospholipids retained a lamellar phase in the presence of each of the peptides with an aromatic X residue, whereas the Leu peptide perturbed the bilayer to an extent where an additional isotropic phase was observed. The solid-state NMR 13C and 31P data provide additional information on the influence of these short peptides on the membrane that has not been previously reported. The magnitude of membrane perturbation was in the order of guest residue X = L > Y~F > W, which is consistent with the relative amino acid interfacial affinity reported by Wimley and White. Further work is, however, required to uncover the behavior of the peptide and localization in the membrane domain due to ambiguity of the 13C NMR data. We have launched efforts in this regard for the objective of better understanding the role of aromatic amino acids in peripheral membrane protein binding.

Expression and purification of human neutrophil proteinase 3 from insect cells and characterization of ligand binding.

Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug discovery efforts targeting PR3 require active enzyme for in vitro characterization, such as inhibitor screening, enzymatic assays, and structural studies. Recombinant expression of active PR3 overcomes the need for enzyme supplies from human blood and in addition allows studies on the influence of mutations on enzyme activity and ligand binding. Here, we report the expression of recombinant PR3 (rPR3) using a baculovirus expression system. The purification and activation process described resulted in highly pure and active PR3. The activity of rPR3 in the presence of commercially available inhibitors was compared with human PR3 by using a fluorescence-based enzymatic assay. Purified rPR3 had comparable activity to the native human enzyme, thus being a suitable alternative for enzymatic studies in vitro. Further, we established a surface plasmon resonance-based assay to determine binding affinities and kinetics of PR3 ligands. These methods provide valuable tools for early drug discovery aiming towards treatment of lung inflammation.