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Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including α2‐macroglobulin (α2‐M), serpins [α1‐proteinase inhibitor (α1‐PI)], monocyte neutrophil elastase inhibitor (MNEI), α1‐antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by α1‐PI and MNEI but not by SLPI. α2‐M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that α2‐M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2‐M and hPR3 is governed by a kass in the ≤ 105 m−1·s−1 range. Since α2‐M‐trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2‐M bait region (residues 690–728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39‐residue bait region of α2‐M (39pep‐α2‐M). Since the 39pep‐α2‐M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that α2‐M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well‐recognized function of major target autoantigen in granulomatosis with polyangiitis. [ABSTRACT FROM AUTHOR]

Neutrophils are one of the most important military services of the armed forces of the immune system, a crucial line of defense against bacterial or fungal onslaughts. One of their mechanisms of action relies on the production of serine proteases. One of these enzymes is proteinase 3 (PR3), which is engaged in the processing of pro-inflammatory cytokines, receptors, heat shock proteins and in the generation of antibacterial peptides. Despite its protective function, uncontrolled activity of PR3 has been associated with the progression of inflammation and tissue injury. Although PR3 was characterized at the beginning of 90's of the last century for the first time, the research on the development of its inhibitors is barely noticeable. Here we present the recent findings on the design, synthesis and the activity of phosphonic PR3 inhibitors together with the historical perspective. Image 1 • Proteinase 3 is considered an interesting drug target since its activity has been associated with different pathophysiological disorders. • Several classes of PR3 inhibitors such as isocoumarins, N -hydroxysuccinimides, thiatriazolidine and phosphonates have been developed. • Diaryl esters of phosphonic analogues of amino acids represent an interesting class of irreversible inhibitors of proteinase 3. • Phosphonic inhibitors can be easily turned into the activity-based probes useful for protease visualization either in vitro or in cell assays. [ABSTRACT FROM AUTHOR]

Neutrophils are among the first cells implicated in acute inflammation. Leaving the blood circulation, they quickly migrate through the interstitial space of tissues and liberate oxidants and other antimicrobial proteins together with serine proteinases. Neutrophil elastase, cathepsin G, proteinase 3 (PR3), and neutrophil serine protease 4 are four hematopoietic serine proteases activated by dipeptidyl peptidase I during neutrophil maturation and are mainly stored in cytoplasmic azurophilic granules. They regulate inflammatory and immune responses after their release from activated neutrophils at inflammatory sites. Membrane-bound PR3 (mbPR3) at the neutrophil surface is the prime antigenic target of antineutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a vasculitis of small blood vessels and granulomatous inflammation of the upper and/or lower respiratory tracts. The interaction of ANCA with mbPR3 results in excessive activation of neutrophils to produce reactive oxygen species and liberation of granular proteinases to the pericellular environment. In this review, we focus on PR3 and dipeptidyl peptidase I as attractive pharmacological targets whose inhibition is expected to attenuate autoimmune activation of neutrophils in GPA. [ABSTRACT FROM AUTHOR]

Abstract: In this study, chemical synthesis of the selective chromogenic/fluorogenic substrates for proteinase 3 is described. The substrates’ sequence was obtained using combinatorial chemistry methods. Deconvolution of the tripeptide library against proteinase 3 with general formula ABZ-X3-X2-X1-ANB-NH2 yielded the active sequence. Selected peptide was further modified on its C terminus to investigate the impact of chromophore moiety modification on enzyme–substrate interaction. To determine specificity, activity of selected substrates was characterized against proteinase 3 and neutrophil elastase. Finally, the peptide ABZ-Tyr-Tyr-Abu-ANB-NH2 displayed the highest value of specificity constant (k cat/K M =189×103 M−1 s−1) for proteinase 3. To the best of our knowledge, this is the first short peptide that undergoes selective proteolysis by proteinase 3 and displays no significant hydrolysis in the presence of human neutrophil elastase and cathepsin G. [Copyright &y& Elsevier]