S. Lorraine Martin

Irreversible inhibition of the bacterial cysteine protease-transpeptidase sortase (SrtA) by substrate-derived affinity labels

We report on the first synthesis, kinetic evaluation and application of novel substrate-derived inhibitors against the Staphylococcus aureus cysteine protease-transpeptidase, sortase (staphylococcal surface protein sorting A, SrtA). The peptidyl-diazomethane and peptidyl-chloromethane analogues, Cbz (benzyloxycarbonyl)-Leu-Pro-Ala-Thr-CHN(2) (I) and Cbz-Leu-Pro-Ala-Thr-CH(2)Cl (II) respectively were found to act as time-dependent irreversible inhibitors of recombinant sortase (SrtA(DeltaN)). The peptidyl-chloromethane analogue (II) was the most powerful with an inhibitor specificity constant (k(i)/K(i)) of 5.3x10(4) M(-1).min(-1), approx. 2-fold greater than that determined for the peptidyl-diazomethane (I). Additionally, using Western-blot analysis, we have been able to demonstrate that a biotinylated version of the peptidyl-diazomethane analogue, biotin-Ahx (aminohexanoyl)-Leu-Pro-Ala-Thr-CHN(2) (III), can be used as an affinity label to detect the presence of wild-type SrtA in crude cell lysates prepared from S. aureus.

Neutrophil Elastase Activity is Associated with Exacerbations and Lung Function Decline in Bronchiectasis

Rationale: Sputum neutrophil elastase and serum desmosine, which is a linked marker of endogenous elastin degradation, are possible biomarkers of disease severity and progression in bronchiectasis. This study aimed to determine the association of elastase activity and desmosine with exacerbations and lung function decline in bronchiectasis. Methods: This was a single‐center prospective cohort study using the TAYBRIDGE (Tayside Bronchiectasis Registry Integrating Datasets, Genomics, and Enrolment into Clinical Trials) registry in Dundee, UK. A total of 433 patients with high‐resolution computed tomography‐confirmed bronchiectasis provided blood samples for desmosine measurement, and 381 provided sputum for baseline elastase activity measurements using an activity‐based immunosassay and fluorometric substrate assay. Candidate biomarkers were tested for their relationship with cross‐sectional markers of disease severity, and with future exacerbations, mortality and lung function decline over 3 years. Measurement and Main Results: Elastase activity in sputum was associated with the bronchiectasis severity index (r = 0.49; P < 0.0001) and was also correlated with the Medical Research Council dyspnea score (r = 0.34; P < 0.0001), FEV1% predicted (r = −0.33; P < 0.0001), and the radiological extent of bronchiectasis (r = 0.29; P < 0.0001). During a 3‐year follow‐up, elevated sputum elastase activity was associated with a higher frequency of exacerbations (P < 0.0001) but was not independently associated with mortality. Sputum elastase activity was independently associated with FEV1 decline (&bgr; coefficient, −0.139; P = 0.001). Elastase showed good discrimination for severe exacerbations with an area under the curve of 0.75 (95% confidence interval [CI], 0.72‐0.79) and all‐cause mortality (area under the curve, 0.70; 95% CI, 0.67‐0.73). Sputum elastase activity increased at exacerbations (P = 0.001) and was responsive to treatment with antibiotics. Desmosine was correlated with sputum elastase (r = 0.42; P < 0.0001) and was associated with risk of severe exacerbations (hazard ratio 2.7; 95% CI, 1.42‐5.29; P = 0.003) but not lung function decline. Conclusions: Sputum neutrophil elastase activity is a biomarker of disease severity and future risk in adults with bronchiectasis.

From sentencing to execution--the processes of apoptosis.

Objectives Cell proliferation and apoptosis play a major role in maintaining homeostasis and as such any disruption within these processes can lead to disease states. Apoptosis occurs in three non‐distinct phases – induction, effector and degradation – and can be executed through both the extrinsic and intrinsic pathways in addition to recognised sub‐pathways such as the p53 and lysosomal pathways. This review article highlights these pathways, incorporating an overview of the molecular regulators of apoptosis.

Inhibition of escherichia coli glucosamine synthetase by novel electrophilic analogues of glutamine—comparison with 6-diazo-5-oxo-norleucine

A series of electrophilic glutamine analogues based on 6-diazo-5-oxo-norleucine has been prepared, using novel synthetic routes, and evaluated as inhibitors of Escherichia coli glucosamine synthetase. The gamma-dimethylsulphonium salt analogue of glutamine was found to be one of the most potent inactivators of this enzyme yet reported, with an apparent second order rate constant (k2/Ki) of 3.5 x 10(5) M(-1) min(-1).

Anti-inflammatory effects of DX-890, a human neutrophil elastase inhibitor.

BACKGROUND Neutrophil elastase (NE)-mediated inflammation contributes to lung damage in cystic fibrosis (CF). We investigated if DX-890, a small-protein NE inhibitor, could reduce neutrophil trans-epithelial migration and reduce activity released from neutrophils and NE-induced cytokine expression in airway epithelial cells. METHODS Activated blood neutrophils (CF and healthy) treated ±DX-890 were assayed for NE activity. Transmigration of calcein-labeled neutrophils was studied using a 16HBE14o(-) epithelial monolayer. IL-8 release from primary nasal epithelial monolayers (CF and healthy) was measured after treatment ±DX-890 and NE or CF sputum. RESULTS DX-890 reduced NE activity from neutrophils (CF and healthy) and reduced neutrophil transmigration. DX-890 pre-treatment reduced IL-8 release from epithelial cells of healthy or CF subjects after stimulation with NE and CF sputum sol. All improvements with DX-890 were statistically significant (p<0.05). CONCLUSIONS DX-890 reduces NE-mediated transmigration and inflammation. NE inhibition could be useful in managing neutrophilic airway inflammation in CF.

Proteases implicated in apoptosis: old and new.

Objectives The role of proteases in the regulation of apoptosis is becoming increasingly apparent. Whilst many of these proteases are already characterised, some have yet to be identified. Traditionally caspases held the traditional role as the prime mediators of apoptosis; however, attention is now turning towards the contribution made by serine proteases.

Inhibition of Protease–Epithelial Sodium Channel Signaling Improves Mucociliary Function in Cystic Fibrosis Airways

RATIONALE In cystic fibrosis (CF) a reduction in airway surface liquid (ASL) height compromises mucociliary clearance, favoring mucus plugging and chronic bacterial infection. Inhibitors of the epithelial sodium channel (ENaC) have therapeutic potential in CF airways to reduce hyperstimulated sodium and fluid absorption to levels that can restore airway hydration. OBJECTIVES To determine whether a novel compound (QUB-TL1) designed to inhibit protease/ENaC signaling in CF airways restores ASL volume and mucociliary function. METHODS Protease activity was measured using fluorogenic activity assays. Differentiated primary airway epithelial cell cultures (F508del homozygotes) were used to determined ENaC activity (Ussing chamber recordings), ASL height (confocal microscopy), and mucociliary function (by tracking the surface flow of apically applied microbeads). Cell toxicity was measured using a lactate dehydrogenase assay. MEASUREMENTS AND MAIN RESULTS QUB-TL1 inhibits extracellularly located channel activating proteases (CAPs), including prostasin, matriptase, and furin, the activities of which are observed at excessive levels at the apical surface of CF airway epithelial cells. QUB-TL1-mediated CAP inhibition results in diminished ENaC-mediated Na(+) absorption in CF airway epithelial cells caused by internalization of a prominent pool of cleaved (active) ENaCγ from the cell surface. Importantly, diminished ENaC activity correlates with improved airway hydration status and mucociliary clearance. We further demonstrate QUB-TL1-mediated furin inhibition, which is in contrast to other serine protease inhibitors (camostat mesylate and aprotinin), affords protection against neutrophil elastase-mediated ENaC activation and Pseudomonas aeruginosa exotoxin A-induced cell death. CONCLUSIONS QUB-TL1 corrects aberrant CAP activities, providing a mechanism to delay or prevent the development of CF lung disease in a manner independent of CF transmembrane conductance regulator mutation.

Mapping biological to clinical phenotypes during the development (21 days) and resolution (21 days) of experimental gingivitis

AIM To characterize and map temporal changes in the biological and clinical phenotype during a 21-day experimental gingivitis study. MATERIALS AND METHODS Experimental gingivitis was induced over 21 days in healthy human volunteers (n = 56), after which normal brushing was resumed (resolution phase). Gingival and plaque indices were assessed. Gingival crevicular fluid was collected from four paired test and contra-lateral control sites in each volunteer during induction (Days 0, 7, 14 and 21) and resolution (Days 28 and 42) of experimental gingivitis. Fluid volumes were measured and a single analyte was quantified from each site-specific, 30s sample. Data were evaluated by analysis of repeated measurements and paired sample tests. RESULTS Clinical indices and gingival crevicular fluid volumes at test sites increased from Day 0, peaking at Day 21 (test/control differences all p < 0.0001) and decreased back to control levels by Day 28. Levels of four inflammatory markers showed similar patterns, with significant differences between test and control apparent at Day 7 (substance P, cathepsin G, interleukin-1β, elastase: all p < 0.03) and peaking at Day 21 (all p < 0.002). Levels of α-1-antitrypsin showed no pattern. CONCLUSIONS Levels of substance P, cathepsin G, interleukin-1β and neutrophil elastase act as objective biomarkers of gingival inflammation induction and resolution that typically precede phenotypical changes.

Synthesis and kinetic evaluation of peptide α-keto-β-aldehyde-based inhibitors of trypsin-like serine proteases

New, synthetic peptide analogues bearing a C‐terminal basic α‐keto‐β‐aldehyde moiety were prepared as novel inhibitors of the trypsin‐like serine proteases. The compounds, Ac‐Leu‐Leu‐Arg‐COCHO, Ac‐Arg‐Gln‐Arg‐COCHO and Boc‐Val‐Leu‐Lys‐COCHO were evaluated kinetically against trypsin and three other trypsin‐like serine proteases, tryptase, plasmin and thrombin, all of which are implicated as mediators of important disease processes. Results illustrate that α‐keto‐β‐aldehydes are potent inhibitors, with similar potency to comparable peptide aldehydes, and intriguingly, appear to act, in some instances, by a novel mechanism of action. Ac‐Leu‐Leu‐Arg‐COCHO, an analogue of the natural product leupeptin, is a potent, tight‐binding inhibitor of trypsin (Ki(final) = 1.9 μm), plasmin (Ki(final) = 4.9 μm) and tryptase (Ki(final) = 1.2 μm) and an irreversible inactivator of thrombin (k2nd 4500 m−1. min−1). Boc‐Val‐Leu‐Lys‐COCHO was found to be a tight‐binding inhibitor of its target protease plasmin (Ki(final) = 3.1 μm) and was inactive against thrombin. Ac‐Arg‐Gln‐Arg‐COCHO was a slow‐binding inhibitor of tryptase (Ki(final) = 1.6 μm) and also irreversibly inactivated trypsin (k2nd = 8920 m−1 min−1). Peptides or peptidomimetics with a C‐terminal basic α‐keto‐β‐aldehyde function thus provide a useful new molecular template for the development of new therapeutic agents against a wide range of disorders, such as coagulopathies and asthma, which may be mediated by the aberrant activity of trypsin‐like serine proteases.

Ion channels as targets to treat cystic fibrosis lung disease

Lung health relies on effective mucociliary clearance and innate immune defence mechanisms. In cystic fibrosis (CF), an imbalance in ion transport due to an absence of chloride ion secretion, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) and a concomitant sodium hyperabsorption, caused by dyregulation of the epithelial sodium channel (ENaC), results in mucus stasis which predisposes the lungs to cycles of chronic infection and inflammation leading to lung function decline. An increased understanding of CFTR structure and function has provided opportunity for the development of a number of novel modulators targeting mutant CFTR however, it is important to also consider other ion channels and transporters present in the airways as putative targets for drug development. In this review, we discuss recent advances in CFTR biology which will contribute to further drug discovery in the field. We also examine developments to inhibit the epithelial sodium channel (ENaC) and potentially activate alternative chloride channels and transporters as a multi-tracked strategy to hydrate CF airways and restore normal mucociliary clearance mechanisms in a manner independent of CFTR mutation.