Abstract
Matrix metalloproteinases (MMPs) are a family of metzincin enzymes that act as the principal regulators and remodelers of the extracellular matrix (ECM). While MMPs are involved in many normal biological processes, unregulated MMP activity has been linked to many detrimental diseases, including cancer, neurodegenerative diseases, stroke, and cardiovascular disease. Developed as tools to investigate MMP function and as potential new therapeutics, matrix metalloproteinase inhibitors (MMPIs) have been designed, synthesized, and tested to regulate MMP activity. This chapter focuses on the use of enzyme kinetics to characterize inhibitors of MMPs. MMP activity is measured via fluorescence spectroscopy using a fluorogenic substrate that contains a 7-methoxycoumarin-4-acetic acid N-succinimidyl ester (Mca) fluorophore and a 2,4-dinitrophenyl (Dpa) quencher separated by a scissile bond. MMP inhibitor (MMPI) potency can be determined from the reduction in fluorescent intensity when compared to the absence of the inhibitor. This chapter describes a technique to characterize a variety of MMPs through enzyme inhibition assays.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jin Y, Roycok MD, Bosco DB et al (2013) Matrix metalloproteinase inhibitors based on the 3-mercaptopyrrolidine core. J Med Chem 56:4357–4373. doi:10.1021/jm400529f
Hooper NM (1994) Families of zinc metalloproteases. FEBS Lett 354:1–6
Lee S, Park HI, Sang Q-X A (2007) Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion. Biochem J 403:31–42. doi:10.1042/BJ20061390
Woessner J, Nagase H (2000) Matrix metalloproteinases and TIMPs. Oxford University Press, New York
Nagase H, Woessner FJ Jr (1999) Matrix metalloproteinases. J Biol Chem 274:21491–21494
Hurst DR, Schwartz MA, Jin Y et al (2005) Inhibition of enzyme activity of and cell-mediated substrate cleavage by membrane type 1 matrix metalloproteinase by newly developed mercaptosulphide inhibitors. Biochem J 392:527–536. doi:10.1042/BJ20050545
Park HI, Turk BE, Gerkema FE et al (2002) Peptide substrate specificities and protein cleavage sites of human endometase/matrilysin-2/matrix metalloproteinase-26. J Biol Chem 38:35168–35175. doi:10.1074/jbc.M205071200
Savinov AY, Remacle AG, Golubkov VS et al (2006) Matrix metalloproteinase 26 proteolysis of the NH2-terminal domain of the estrogen receptor β correlates with the survival of breast cancer patients. Cancer Res 66:2716–2724
Zhao YG, Xiao AZ, Park HI et al (2004) Endometase/matrilysin-2 in human breast ductal carcinoma in situ and its inhibition of breast cancer invasion. Cancer Res 64:590–598
Lee S, Desai KK, Ickowski KA et al (2006) Coordinated peak expression of MMP-26 and TIMP-4 in preinvasive human prostate tumor. Cancer Res 16:750–758
Hurst DR, Schwartz MA, Ghaffari MA et al (2004) Catalytic- and ecto-domains of membrane type 1-matrix metalloproteinase have similar inhibition profiles but distinct endopeptidase activities. Biochem J 877:775–779
Sang Q-X A, Jin Y, Newcomer RG et al (2006) Matrix metalloproteinase inhibitors as prospective agents for the prevention and treatment of cardiovascular and neoplastic diseases. Curr Top Med Chem 6:289–316
Candelario-Jalil E, Yang Y, Rosenburg GA (2009) Diverse roles of matrix metalloproteinase and tissue inhibitors of metalloproteinases in neuroinflammation and cerebral ischemia. Neuroscience 158:983–994
Lehrke M, Greif M, Broedl UC et al (2009) MMP-1 serum levels predict coronary atherosclerosis in humans. Cardiovasc Diabetol 8:50
Tuomainen AM, Nyyssonen K, Laukkanen JA et al (2005) Serum matrix metalloproteinase-8 concentrations are associated with cardiovascular outcome in men. Arterioscler Thromb Vasc Biol 27:2722–2728
Baker AH, Edwards DR, Murphy G (2002) Metalloproteinase inhibitors: biological actions and therapeutic opportunities. J Cell Sci 115:3719–3727
Fields GB (2010) Using fluorogenic peptide substrates to assay matrix metalloproteinases. In: Clark IM, Young DA, Rowan AD (eds) Matrix metalloproteinase protocols, vol 2, Methods in molecular biology. Springer, New York, pp 393–433
Gershkovich AA, Kholodovych VV (1996) Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS). J Biochem Biophys Methods 33:135–162
Roycik MD, Myers JS, Newcomer RG et al (2013) Matrix metalloproteinase inhibition in atherosclerosis and stroke. Curr Mol Med 13:1299–1313
Park HI, Jin Y, Hurst DR et al (2003) The intermediate S1’ pocket of the endometase/matrilysin-2 active site revealed by enzyme inhibition kinetic studies, protein sequence analyses, and homology modeling. J Biol Chem 51:51646–51653. doi:10.1074/jbc.M310109200
Hu J, Van den Steen PE, Sang Q-X A et al (2007) Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases. Nat Rev Drug Discov 6:480–498
Muroski ME, Roycik MD, Newcomer RG et al (2008) Matrix metalloproteinase-9/gelatinase B is a putative therapeutic target of chronic obstructive pulmonary disease and multiple sclerosis. Curr Pharm Biotechnol 9:34–46
Park HI, Lee S, Ullah A et al (2010) Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker. Anal Biochem 396:262–268
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Lively, T.J., Bosco, D.B., Khamis, Z.I., Sang, QX.A. (2016). Assessment of Synthetic Matrix Metalloproteinase Inhibitors by Fluorogenic Substrate Assay. In: Cao, J. (eds) Breast Cancer. Methods in Molecular Biology, vol 1406. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3444-7_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3444-7_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3442-3
Online ISBN: 978-1-4939-3444-7
eBook Packages: Springer Protocols