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Background: Proteinase 3 activity is poorly controlled by physiological inhibitors, and its biological function is not well understood. Results:We have designed irreversible phosphonate inhibitors based on structural differences between proteinase 3 and elastase. Conclusion: They selectively inhibit proteinase 3 in biological fluids and can act as activity-based probes. Significance: These inhibitors will help clarify proteinase 3 function.

Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the alpha-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with alpha-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future

The neutrophilic serine protease proteinase 3 (PR3) is involved in inflammation and immune response and thus appears as a therapeutic target for a variety of infectious and inflammatory diseases. Here we combined kinetic and molecular docking studies to increase the potency of peptidyl-diphenyl phosphonate PR3 inhibitors. Occupancy of the S1 subsite of PR3 by a nVal residue and of the S4-S5 subsites by a biotinylated Val residue as obtained in biotin-VYDnV(O-CH-4-Cl) enhanced the second-order inhibition constant k/[I] toward PR3 by more than 10 times ( k/[I] = 73000 ± 5000 M s) as compared to the best phosphonate PR3 inhibitor previously reported. This inhibitor shows no significant inhibitory activity toward human neutrophil elastase and resists proteolytic degradation in sputa from cystic fibrosis patients. It also inhibits macaque PR3 but not the PR3 from rodents and can thus be used for in vivo assays in a primate model of inflammation.

Background: The pathophysiology of subclinical versus clinical rejection remains incompletely understood given their equivalent histological severity but discordant graft function. The goal was to evaluate serine hydrolase enzyme activities to explore if there were any underlying differences in activities during subclinical versus clinical rejection. Methods: Serine hydrolase activity-based protein profiling (ABPP) was performed on the urines of a case control cohort of patients with biopsy confirmed subclinical or clinical transplant rejection. In-gel analysis and affinity purification with mass spectrometry were used to demonstrate and identify active serine hydrolase activity. An assay for proteinase 3 (PR3/PRTN3) was adapted for the quantitation of activity in urine. Results: In-gel ABPP profiles suggested increased intensity and diversity of serine hydrolase activities in urine from patients undergoing subclinical versus clinical rejection. Serine hydrolases (n = 30) were identified by mass spectrometry in subclinical and clinical rejection patients with 4 non-overlapping candidates between the two groups (i.e. ABHD14B, LTF, PR3/PRTN3 and PRSS12). Western blot and the use of a specific inhibitor confirmed the presence of active PR3/PRTN3 in samples from patients undergoing subclinical rejection. Analysis of samples from normal donors or from several serial post-transplant urines indicated that although PR3/PRTN3 activity may be highly associated with low-grade subclinical inflammation, the enzyme activity was not restricted to this patient group. Conclusions: There appear to be limited qualitative and quantitative differences in serine hydrolase activity in patients with subclinical versus clinical renal transplant rejection. The majority of enzymes identified were present in samples from both groups implying that in-gel quantitative differences may largely relate to the activity status of shared enzymes. However qualitative compositional differences were also observed indicating differential activities. The PR3/PRTN3 analyses indicate that the activity status of urine in transplant patients is dynamic possibly reflecting changes in the underlying processes in the transplant. These data suggest that differential serine hydrolase pathways may be active in subclinical versus clinical rejection which requires further exploration in larger patient cohorts. Although this study focused on PR3/PRTN3, this does not preclude the possibility that other enzymes may play critical roles in the rejection process.

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent PR3. We developed selective phosphonate inhibitors with the structure: Ac‐peptidyl‐P(O‐C6H4‐4‐Cl)2. The peptidyl moiety was deduced from molecular modeling and kinetic studies comparing the substrate specificities of PR3 with a single residue PR3 mutant and HNE. The P4 and P2 positions were essential to discriminate between PR3 and HNE. We then synthesized N‐terminally biotinylated peptidyl phosphonates to identify PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils, despite their low molecular mass, suggesting that the conformation and reactivity of membrane‐bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease).

Neutrophil elastase (NE) is involved in the degradation of extracellular matrix molecules making it an attractive target for the development of anti‐inflammatory compounds in neutrophilic pulmonary diseases. It is mainly secreted extracellularly by activated neutrophils at inflammatory sites but a minor fraction is translocated to the membrane where it remains proteolytically active. We investigated the properties of the novel Protein Epitope Mimetic (PEM) POL6014, a medium sized fully synthetic macrocycle, towards soluble and membrane‐bound NE. Circulating human neutrophils from healthy volunteers were purified and used within 3 hours after activation from Calcium ionophore (A23187) as source of membrane bound NE. The enzymatic reaction using the Fluorescence Resonance Energy Transfer (FRET) Technology was started by addition of a specific human NE substrate (ABZ‐APEEIMRRQ‐EDDnp, 13 μM final) 5 minutes after incubation of POL6014 with human NE. We found that POL6014 inhibits membrane‐bound human NE and soluble human NE with similar Ki values in the sub‐nanomolar range (0.69 ± 0.31 nM (n=7) and 0.53 ± 0.37 nM (n=3), respectively). This means that POL6014 could inhibit human NE stoichiometrically in pathophysiological conditions so that no molar excess of inhibitor would be required to control NE activity. In addition we evaluated POL6014 activity on rat and non‐human primate (NHP) NE in order to qualify these species for in vivo testing. Lysates of circulating neutrophils from both species were used as source of NE. Species selective FRET substrates (ABZ‐QPMAVVQSVPQ‐Yno2 for rodent, ABZ‐APQQIMDDQ‐EDDnp for primate) were used to determine the inhibitory potency of POL6014. We found that POL6014 is also a potent low nanomolar inhibitor with IC50 = 1.0 ± 0.5 nM (n=4) against rat NE and IC50 = 2.2 ± 1.4 nM (n=3) against Cynomolgus (Macaca fascicularis) NHP NE, which makes these species appropriate to test the in vivo efficacy and toxicology of POL6014. In conclusion, POL6014 showed potent inhibition of soluble and membrane‐bound human NE and similar potency across human, rat and NHP.Support or Funding InformationThis work was supported by Polyphor Ltd.

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