Bivalent Inhibitor with Selectivity for Trimeric MMP-9 Amplifies Neutrophil Chemotaxis and Enables Functional Studies on MMP-9 Proteoforms.

Elisa Nuti,Armando Rossello,Rafaela Vaz Sousa Pereira,Mieke Gouwy,Doretta Cuffaro,Caterina Camodeca,Jens Van Bael,Pierre Fiten,Jennifer Vandooren,Ghislain Opdenakker,Dries Van Der Maat,Estefania Ugarte-Berzal,Erik Martens

doi:10.3390/cells9071634

Elisa Nuti, Armando Rossello + Show 11 more

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https://doi.org/10.3390/cells9071634

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Abstract

A fundamental part of the immune response to infection or injury is leukocyte migration. Matrix metalloproteinases (MMPs) are a class of secreted or cell-bound endopeptidases, implicated in every step of the process of inflammatory cell migration. Hence, specific inhibition of MMPs is an interesting approach to control inflammation. We evaluated the potential of a bivalent carboxylate inhibitor to selectively inhibit the trimeric proteoform of MMP-9 and compared this with a corresponding monovalent inhibitor. The bivalent inhibitor efficiently inhibited trimeric MMP-9 (IC50 = 0.1 nM), with at least 500-fold selectivity for MMP-9 trimers over monomers. Surprisingly, in a mouse model for chemotaxis, the bivalent inhibitor amplified leukocyte influxes towards lipopolysaccharide-induced inflammation. We verified by microscopic and flow cytometry analysis increased amounts of neutrophils. In a mouse model for endotoxin shock, mice treated with the bivalent inhibitor had significantly increased levels of MMP-9 in plasma and lungs, indicative for increased inflammation. In conclusion, we propose a new role for MMP-9 trimers in tempering excessive neutrophil migration. In addition, we have identified a small molecule inhibitor with a high selectivity for the trimeric proteoform of MMP-9, which will allow further research on the functions of MMP-9 proteoforms.

Highlights

Neutrophils are the most abundant circulating leukocytes and the first cells to arrive at an inflammatory site
We evaluated the ability of a bivalent carboxylate inhibitor to block the catalytic activity of stable Matrix metalloproteinases (MMPs)-9 trimers compared to monomers, and other proteases with significance in inflammation
To evaluate the specificity of the inhibitors in the context of inflammation, we investigated the effect of the monovalent and bivalent carboxylate inhibitors on other proteases secreted by infiltrating leukocytes or epithelial cells

Summary

Introduction

Neutrophils are the most abundant circulating leukocytes and the first cells to arrive at an inflammatory site. Neutrophil proteases mediate numerous steps of this inflammatory process They contribute to leukocyte recruitment and chemotaxis by shedding of selectins and integrins, degradation of cell junctional and extracellular matrix proteins and modulation of chemokine activity by limited proteolysis [4,5]. A major family of proteases implicated in inflammation are the matrix metalloproteinases (MMPs) [6] These zinc-dependent endopeptidases cleave and modulate the activities of a range of chemokines, resulting in activation or inactivation of chemokines [5]. Whereas their roles in inflammation are being recognized, MMPs were originally (± 20 years ago) studied as potent targets in cancer treatment, due to their ability to cleave different components of the extracellular matrix. Cancer treatment often involves long-term drug use and a better drug safety profile in contrast with shorter treatments such as those required for acute inflammations (e.g., sepsis syndromes) [7,10]

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