Abstract
The membrane-bound matrix metalloproteinase 14 (MMP14, also known as MT1-MMP) plays important roles in the remodeling of the extracellular matrix during various cellular processes such as cancer metastasis, angiogenesis, and wound healing through its proteolytic activity. There are no known MMP14-specific inhibitors to date, and hence identification of MMP14-specific inhibitors will be beneficial for finding potential therapeutics for various diseases, including cancer and inflammation. High-throughput screening (HTS) assays have become a common way to search for new small compounds, peptides, and natural products. Enzymatic assays are highly amenable to HTS because most enzyme activities are quantifiable with the effect of many small molecules of interest on a specific target enzyme. Here, we describe a fluorescence-based enzymatic assay that can be applied as a large-scale HTS and a follow-up enzyme kinetics assay to find MMP14-specific inhibitors.
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References
Hwang IK, Park SM, Kim SY et al (2004) A proteomic approach to identify substrates of matrix metalloproteinase-14 in human plasma. Biochim Biophys Acta 1702:79–87
Ries C, Egea V, Karow M et al (2007) MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines. Blood 109:4055–4063
Gonzalez-Molina J, Gramolelli S, Liao Z et al (2019) MMP14 in sarcoma: a regulator of tumor microenvironment communication in connective tissues. Cell 8(9):991
Holmbeck K, Bianco P, Caterina J et al (1999) MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell 99:81–92
Devel L, Rogakos V, David A et al (2006) Development of selective inhibitors and substrate of matrix metalloproteinase-12. J Biol Chem 281:11152–11160
Gupta SP, Patil VM (2012) Specificity of binding with matrix metalloproteinases. Exp Suppl 103:35–56
Schechter I, Berger A (1967) On the size of the active site in proteases. I Papain. Biochem Biophys Res Commun 27:157–162
Gimeno A, Beltran-Debon R, Mulero M et al (2020) Understanding the variability of the S1' pocket to improve matrix metalloproteinase inhibitor selectivity profiles. Drug Discov Today 25:38–57
Laronha H, Caldeira J (2020) Structure and function of human matrix metalloproteinases. Cell 9(5):1076
Cooney MJ (2017) Kinetic measurements for enzyme immobilization. Methods Mol Biol 504:215–232
Han KY, Dugas-Ford J, Lee H et al (2015) MMP14 cleavage of VEGFR1 in the cornea leads to a VEGF-trap antiangiogenic effect. Invest Ophthalmol Vis Sci 56(9):5450–5456
Han KY, Chang JH, Lee H et al (2016) Proangiogenic interactions of vascular endothelial MMP14 with VEGF receptor 1 in VEGFA-mediated corneal angiogenesis. Invest Ophthalmol Vis Sci 57:3313–3322
Hiratsuka S, Minowa O, Kuno J et al (1998) Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. PNAS 95:9349–9354
Gabhann FM, Popel AS (2008) Systems biology of vascular endothelial growth factors. Microcirculation 15:715–738
Santamaria S, Nagase H (2018) Measurement of protease activities using fluorogenic substrates. Methods Mol Biol 1731:107–122
Copeland RA (2005) Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem Anal 46:1–265
Macarron R, Hertzberg RP (2011) Design and implementation of high throughput screening assays. Mol Biotechnol 47(3):270–285
Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73
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Lee, H., Ibrahimi, L., Han, KY. (2024). Fluorescence-Based Peptidolytic Assay for High-Throughput Screening of MMP14 Inhibitors. In: Santamaria, S. (eds) Proteases and Cancer. Methods in Molecular Biology, vol 2747. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3589-6_18
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DOI: https://doi.org/10.1007/978-1-0716-3589-6_18
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