AN INVESTIGATION INTO NOVEL INHIBITORS OF CHANNEL ACTIVATING PROTEASES: IMPLICATIONS FOR CYSTIC FIBROSIS LUNG DISEASE.

Research output: Contribution to conferencePoster

Abstract

Introduction: Cystic fibrosis (CF) is an autosomal recessive disease, characterised by chronic lung infections, inflammation and irreversible lung damage. ENaC, a sodium channel found within the lung epithelia can be activated through proteolytic cleavage by channel activating proteases (CAPs) (1), leading to upregulated sodium absorption from the airways, contributing to the dehydration and thick mucus observed in CF (2,3). The inhibition of these CAPs presents a promising target for therapy with a hope of rehydrating the airways through reduced ENaC activation, thereby restoring airway surface liquid and effective mucociliary clearance.

Aims and Objectives: Previous work by Reihill et al has shown that QUB-TL1, an active site-directed protease inhibitor, effectively inhibits trypsin and trypsin-like enzymes, which reduces ENaC-mediated sodium absorption (4). The objective of this study therefore was to build upon this knowledge base by kinetically evaluating the mode of action of other novel protease inhibitors, NAP858, NAP1099, NAP1127 and NAP743 and investigating their downstream effects on a number of inflammatory markers.

Methods: The novel inhibitors’ activity against a range of proteases were profiled using fluorogenic substrates, followed by the calculation of second order rate constants in the case of NAP858 and NAP1099. Further to this, in order to visualize the binding of each novel inhibitor to recombinant trypsin, western blotting techniques were utilized. The cytotoxicity of each of the inhibitors at a range of concentrations through treatment of a CuFi cell line for 24 hours was observed by monitoring the release of the cytosolic enzyme lactate dehydrogenase (LDH). Downstream effects on expression levels of proteinase activated receptor 2 (PAR-2) and cytokines, IL-8 and IL-6, in CuFi cells treated with our inhibitors were investigated using real-time quantitative PCR (qPCR).

Results and Discussion: We report NAP858, NAP1099, NAP1127 and NAP743 to be effective irreversible inhibitors of trypsin, matriptase, prostasin and human airway trypsin-like protease (HAT). Additional kinetic studies, calculating second order rate constants for NAP858 and NAP1099 allowed direct comparison to QUBTL1 and shows these two compounds to be equal in inhibition of trypsin to QUBTL1 and superior against matriptase and HAT. Cytotoxicity studies performed on CuFi cells indicated that treatment of these cells with our compounds for 24 hours did not increase LDH release from cells when compared with vehicle alone. Initial qPCR studies have indicated that CuFi cell treatment with NAP858 and NAP1099 causes a reduction in IL-8 expression, a potentially beneficial outcome as this pro-inflammatory cytokine is found in elevated concentrations in the CF lung (5).

Conclusion: These novel inhibitor compounds are promising follow-up compounds to QUBTL1, especially regarding efficacy against a number of key trypsin-like proteases in CF. They, therefore, present a promising future therapeutic option to inhibit the activation of ENaC by CAPs.

References

1. Gaillard et al (2010) Eur J Physiol, 460, 1-17
2. Randell et al (2006) Am J Resp Cell Mol, 35, 20-28
3. Thibodeau et al (2013) Cell Tissue Res, 351, 309-32
4. Reihill JA et al (2016) Am J Respir Crit Care Med, 194, 701-710
5. Courtney et al (2004) J Cyst Fibros, 3, 223-231
Original languageEnglish
Publication statusPublished - 29 Mar 2017
Event14th European Cystic Fibrosis Society Basic Science Conference - Albufeira, Portugal
Duration: 29 Mar 201701 Apr 2017
https://www.ecfs.eu/conference/portugal2017

Conference

Conference14th European Cystic Fibrosis Society Basic Science Conference
CountryPortugal
CityAlbufeira
Period29/03/201701/04/2017
Internet address

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    Douglas, L., Ferguson, T., Reihill, J., & Martin, L. (2017). AN INVESTIGATION INTO NOVEL INHIBITORS OF CHANNEL ACTIVATING PROTEASES: IMPLICATIONS FOR CYSTIC FIBROSIS LUNG DISEASE.. Poster session presented at 14th European Cystic Fibrosis Society Basic Science Conference, Albufeira, Portugal.