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Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro
Published on: February 16, 2020
Bivalent Inhibitor with Selectivity for Trimeric MMP-9 Amplifies Neutrophil Chemotaxis and Enables Functional Studies
Elisa Nuti1, Armando Rossello1, Doretta Cuffaro1
1Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy.
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.
Insights
A novel inhibitor targeting trimeric matrix metalloproteinase-9 (MMP-9) unexpectedly amplified inflammation. This suggests MMP-9 trimers may temper excessive neutrophil migration, revealing a new therapeutic target.
Area of Science:
- Immunology
- Biochemistry
- Pharmacology
Background:
- Leukocyte migration is crucial for immune responses to infection and injury.
- Matrix metalloproteinases (MMPs), particularly MMP-9, are key regulators of inflammatory cell migration.
- Targeting MMPs offers a potential strategy for controlling inflammation.
Purpose of the Study:
- To evaluate a bivalent carboxylate inhibitor's selective inhibition of trimeric MMP-9.
- To compare its efficacy against a monovalent inhibitor.
- To investigate the in vivo effects of selective trimeric MMP-9 inhibition on inflammation.
Main Methods:
- In vitro enzyme inhibition assays to determine IC50 values and selectivity.
- Mouse models of chemotaxis and endotoxin shock to assess in vivo inflammatory responses.
- Microscopy and flow cytometry to quantify neutrophil infiltration.
Main Results:
- The bivalent inhibitor potently inhibited trimeric MMP-9 (IC50 = 0.1 nM) with >500-fold selectivity over monomers.
- Unexpectedly, the inhibitor amplified leukocyte and neutrophil influx in a chemotaxis model.
- Mice treated with the inhibitor showed increased plasma and lung MMP-9 levels, indicating heightened inflammation.
Conclusions:
- Trimeric MMP-9 may play a novel role in suppressing excessive neutrophil migration.
- A highly selective inhibitor for trimeric MMP-9 was identified, enabling further research into MMP-9 proteoform functions.
- This discovery opens new avenues for understanding and potentially modulating inflammatory processes.
