Novel Barbiturate-Nitrate Compounds Inhibit the Upregulation of Matrix Metalloproteinase-9 Gene Expression in Intestinal Inflammation through a cGMP-Mediated Pathway.

Shane O’Sullivan,Jun Wang,Marek W Radomski,Carlos Medina,John F Gilmer

doi:10.3390/biom10050808

Shane O’Sullivan, Jun Wang + Show 3 more

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https://doi.org/10.3390/biom10050808

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Journal: Biomolecules	Publication Date: May 25, 2020
Citations: 7	License type: CC BY 4.0

Affiliation: Trinity College Dublin

Abstract

Matrix metalloproteinase-9 is upregulated in inflammatory bowel disease. Barbiturate nitrate hybrid compounds have been designed to inhibit MMP secretion and enzyme activity. In this study, we investigated the mechanism of action of barbiturate-nitrate hybrid compounds and their component parts using models of intestinal inflammation in vitro. Cytokine-stimulated Caco-2 cells were used in all in vitro experiments. The NO donors SNAP and DETA-NONOate were used to study the effect of NO on MMP-9 mRNA. Mechanistic elucidation was carried out using the soluble guanylate cyclase (sGC) inhibitor, ODQ, and the cGMP analogue, 8-Bromo-cGMP. Further experiments were carried out to elucidate the role of NF-κB. NO donors exerted an inhibitory effect on MMP-9 mRNA in cytokine-stimulated cells. While the non-nitrate barbiturates had a limited effect on MMP-9 expression, the hybrid compounds inhibited MMP-9 expression through its NO-mimetic properties. No effect could be observed on mRNA for MMP-1 or MMP-2. The sGC inhibitior, ODQ, abolished the nitrate-barbiturate inhibition of MMP-9 gene expression, an effect which was reversed by 8-Br-cGMP. This study shows that the barbiturate scaffold is suitable for hybrid design as an MMP-9 inhibitor in cytokine-stimulated Caco-2 cells. The inhibition of MMP-9 levels was largely mediated through a reduction in its mRNA by a sGC/cGMP pathway mediated mechanism.

Highlights

The matrix metalloproteinases are a group of endopeptidases capable of digesting the extracellular matrix (ECM), basement membrane as well as having an immunomodulatory role related to activation of other proteases and inflammatory mediators [1,2,3]
We found that the inhibition of MMP-9 mRNA was largely mediated through a reduction in its mRNA by a soluble guanylate cyclase (sGC)/cGMP-mediated pathway
The nitrate-barbiturates (10 μM) caused a statistically signifificant reduction in MMP-9 mRNA in cytokine-stimulated Caco-2 cells after 24 h compared to the untreated, stimmuullaatteedd cceellllss ((FFiigguurree 22))

Summary

Introduction

The matrix metalloproteinases are a group of endopeptidases capable of digesting the extracellular matrix (ECM), basement membrane as well as having an immunomodulatory role related to activation of other proteases and inflammatory mediators [1,2,3]. MMP-9 is known to be up-regulated in IBD [4,5,6,7] and is associated with disruption of the epithelial barrier and activation of pro-inflammatory mediators [8,9,10,11] Inhibition of this enzyme may aid in reducing the severity of the disease. The development of clinically useful synthetic inhibitors has, to date, been disappointing, mainly due to dose-limiting side-effects but it is true that the efficacy evident in animal models has not translated [12,13] This has led to a revaluation of the precise role of specific MMPs in a given pathological setting and to investigate new strategies for modulating dysregulated MMP activity in disease tissue. We found that the inhibition of MMP-9 mRNA was largely mediated through a reduction in its mRNA by a sGC/cGMP-mediated pathway

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