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.

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Methicillin-resistant S. aureus (MRSA) frequently causes disease outbreaks and has become endemic in many regions, adding to the morbidity, mortality, and cost of care associated with hospital-acquired infections. Enhanced surveillance and infection control measures have been adopted by healthcare institutions (HCIs) to address this unresolved problem (5). In particular, reporting of bloodstream infections (BSI) by MRSA is often mandatory and reduction of BSI rates is a performance target (5,12,21).In the Centre region of France, an extensive, prospective, longitudinal, region-wide survey of BSI has been under way since 2000. Data are collected for 3 months of each year in a large number of HCIs to establish a comprehensive picture of the epidemiology of severe hospital-acquired infections. MRSA BSI and methicillin-sensitive S. aureus (MSSA) BSI are extensively studied within this framework. All of the S. aureus strains isolated during successive study periods are sent to our central laboratory for susceptibility testing, molecular typing, and analysis of virulence genes with the aim of determining the spread and diversity of S. aureus strains in the region. The results obtained during the first 4 years of surveillance (2000 to 2003) of MRSA BSI have been reported previously (27).Here we report the data from 2004 to 2006. We looked for any major changes in the epidemiology of antibiotic resistance and of virulence genes in strains of S. aureus responsible for BSI. We identify a need to focus efforts on preventing both MRSA and MSSA BSI infections and raise the issue of whether the use of fluoroquinolones (FQs) has contributed to the acquisition of resistance and virulence genes by S. aureus strains. MATERIALS AND METHODSBSI epidemiological survey method. A BSI surveillance program in the Centre region of France (2.5 million inhabitants) and a microbiological study of S. aureus strains isolated from BSI cases have been conducted since 2000. Thirtytwo HCIs, comprising 6,027 short-stay beds, participated in this annual 3-month survey of all cases of BSI. Here, we report results for the years 2004 to 2006. The survey covered 2,007,681 patient days (PD). The methods, study design, and data for the years 2000 to 2003 have been reported elsewhere (27). Briefly, the variables studied included patient age and sex, portal of entry, community-or hospital-acquired BSI, occurrence of death within 7 days of BSI diagnosis, and duration of hospital stay. Data were analyzed with Epi Info v.6 software. Data were analyzed with a 2 test with five degrees of freedom. The incidences of community-acquired and nosocomial BSI were determined with respect to the number of PD.Microbiological methods. (i) Bacteriology. Three hundred fifty-eight BSIassociated S. aureus strains were collected during the three survey periods (2004, 2005, and 2006). The strains were sent to the reference laboratory of the Relais d'Hygiène du Centre. The isolates were identified as S. aureus according to previously described procedures (27).(ii) Antimicrobial ...

Greglin is an 83-residue serine protease inhibitor purified from the ovaries of the locust Schistocerca gregaria. Greglin is a strong inhibitor of subtilisin and human neutrophil elastase, acting at sub-nanomolar and nanomolar concentrations, respectively; it also inhibits neutrophil cathepsin G, a-chymotrypsin and porcine pancreatic elastase, but to a lesser extent. In the present study, we show that greglin resists denaturation at high temperature (95°C) and after exposure to acetonitrile and acidic or basic pH. Greglin is composed of two domains consisting of residues 1-20 and 21-83. Mass spectrometry indicates that the N-terminal domain (1-20) is post-translationally modified by phosphorylations at three sites and probably contains a glycosylation site. The crystal structure of the region of greglin comprising residues 21-78 in complex with subtilisin was determined at 1.75 Å resolution. Greglin represents a novel member of the non-classical Kazal inhibitors, as it has a unique additional C-terminal region (70-83) connected to the core of the molecule via a supplementary disulfide bond. The stability of greglin was compared with that of an ovomucoid inhibitor. The thermostability and inhibitory specificity of greglin are discussed in light of its structure. In particular, we propose that the C-terminal region is responsible for non-favourable interactions with the autolysis loop (140-loop) of serine proteases of the chymotrypsin family, and thus governs specificity. DatabaseThe atomic coordinates and structure factors for the greglin-subtilisin complex have been deposited with the RCSB Protein Data Bank under accession number 4GI3. Structured digital abstractGreglin and Subtilisin Carlsberg bind by X-ray crystallography (View interaction)

Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases, large quantities of which are stored in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to degrade engulfed microorganisms inside phagolysosomes. Active forms of these proteases are also externalized during neutrophil activation at inflammatory sites, thus helping to regulate inflammatory and immune responses. A fraction of secreted neutrophil serine proteases (NSPs) remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities using sensitive ortho-aminobenzoyl-peptidyl-N-(2,4-dinitrophenyl) ethylenediamine fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. These are used to measure subnanomolar concentrations of free or membrane-bound NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids. We describe the synthesis of FRET substrate, neutrophil purification, and kinetic experiments on activated neutrophils. The protocol for measuring NSP activity on the surface of activated neutrophils can be adapted to measure NSP activities in whole biological fluids. Such data clarify the contributions of individual NSPs to the development of inflammatory diseases. Ultimately, these proteases may be shown to be targets for therapeutic inhibitors.

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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.