Characterization of Matrix Metalloproteinase Inhibitors: Enzymatic Assays

Patrick A. Marcotte1, Steven K. Davidsen1

1 Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.7
DOI:  10.1002/0471141755.ph0307s13
Online Posting Date:  August, 2001
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Abstract

The matrix metalloproteinases (MMPs) are a family of tightly regulated proteases that are involved in the catabolic aspect of remodeling and maintenance of normal tissue, and more than 20 human MMPs have been identified thus far. The MMPs collectively degrade a broad range of protein components of the extracellular matrix. While some substrate overlap exists, individual MMPs have been shown to process certain substrates more efficiently than others. These differences raise the critical issue of whether broad‐spectrum inhibitors, active against all MMPs, or selective inhibitors, targeted to a subset of enzymes, represent the optimal therapeutic strategy for a given disease. This suggests the need to assess the inhibition potency of test compounds across a range of MMP family members. Described in this unit is a method for the in vitro characterization of MMP inhibitors. The is used to determine the potency of test compounds as inhibitors of 8 representative MMPs through the measurement of their inhibition of cleavage of a fluorogenic substrate. Since this substrate is efficiently hydrolyzed by all MMPs in the screening assays presented here, the method is convenient for assessing the selectivity of inhibitors against multiple enzymes. A describes the activation of MMP zymogens.

     
 
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Table of Contents

  • Basic Protocol 1: In Vitro Analysis of MMP Inhibition by Test Compounds
  • Support Protocol 1: Activation of MMP Zymogens
  • Reagents and Solutions
  • Commentry
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Vitro Analysis of MMP Inhibition by Test Compounds

  Materials
  • MMP buffer 1, pH 7.4 (see recipe)
  • MMP buffer 2, pH 6.5 (see recipe)
  • Test inhibitors in DMSO
  • 1 to 10 mM marimastat (BB‐2516) (Calbiochem) in DMSO, store indefinitely at –20°C
  • 1 to 10 mM ABT‐770 (Abbott Laboratories) in DMSO, store indefinitely at –20°C
  • Dilution buffer: 10% (v/v) HPLC‐grade DMSO in appropriate MMP buffer, room temperature
  • Enzymes (see recipe)
  • 48 µM MMP substrate working solution (see recipe)
  • 400‐µl polypropylene RIA vials and a 96‐tube aluminum block holder (Sarstedt)
  • Automated pipet for serial dilution of inhibitor solutions in RIA vials and transfer of inhibitor solutions from RIA vials to microtiter plates (e.g., Cetus ProPette, Perkin‐Elmer)
  • Single and 8‐channel electronic repeater pipets (e.g., Biohit, Eppendorf) for addition and transfer of solutions to multiple wells
  • Flat‐bottomed white microtiter plates (e.g., Microfluor, DYNEX)
  • Orbital shaker
  • Clear plate sealer
  • Fluorescent plate reader (e.g., Fluoroskan, Titertek Instruments; f max, Molecular Devices; or equivalent) with software that performs capture of the fluorescent readings and calculation of the rate of each reaction
  • Filters for excitation (335 nm with a 35‐nm band pass) and emission (485 nm with a 55‐nm band pass). These filters can be specially ordered from Titertek Instruments. Standard filters near these wavelengths could also be used with some decrease in sensitivity.

Support Protocol 1: Activation of MMP Zymogens

  Materials
  • Zymogen form of MMP (see recipe for Enzymes)
  • Trypsin
  • Soybean trypsin inhibitor or aprotinin (bovine pancreatic trypsin inhibitor) (optional for trypsin activation)
  • Soybean trypsin inhibitor/agarose column (optional for trypsin activation)
  • Para‐aminophenylmercuric acetate (APMA or pAPMA, Sigma)
  • 50 mM NaOH
  • 1 M acetic acid (optional for pAPMA activation)
  • Gel‐filtration column (e.g., Pharmacia PD‐10; optional)
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Figures

Videos

Literature Cited

Literature Cited
   Barlow, R. and Blake, J.F. 1989. Hill coefficients and the logistic equation. Trends Pharm. Sci. 10;440‐441.
   Beekman, B., Drijfhout, J.W., Bloemhoff, W., Ronday, H.K., Tak, P.P., and te Koppele, J.M. 1996. Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media. FEBS Lett. 390;221‐225.
   Bottomley, K.M., Johnson, W.H., and Walter, D.S. 1998. Matrix metalloproteinase inhibitors in arthritis. J. Enzyme Inhib. 13:79‐101.
   Cha, J. and Auld, D.S. 1997. Site‐directed mutagenesis of the active site glutamate in human matrilysin. Investigation of its role in catalysis. Biochemistry 36:16019‐16024.
   Clendeninn, N.J. and Appelt, K. 2000. Matrix Metalloproteinase Inhibitors in Cancer Therapy. Humana Press, Totowa, N.J.
   Curtin, M.L., Garland, R.B., Davidsen, S.K., Marcotte, P.A., and Albert, D.H., Magoc, T.J., and Hutchins, C. 1998. Broad spectrum matrix metalloproteinase inhibitors. An examination of succinamide hydroxamate inhibitors with P1 C‐alpha gem‐disubstitution. Bioorg. 8:1443‐1448.
   Curtin, M.L., Florjancic, A.S., Heyman, H.R.,, Michaelides, M.R., Garland, R.B., Holms, J.H., Steinman, D.H., Dellaria, J.F., Gong, J., Wada, C.K., Guo, Y., Elmore, I.B., Tapang, P., Albert, D.H., Magoc, T.J., Marcotte, P.A., Bouska, J.J., Goodfellow, C.L., Bauch, J.L., Marsh, K.C., Morgan, D.W., and Davidsen, S.K. 2001. Discovery and characterization of a potent, selective and orally bioavailable MMP inhibitor ABT‐770 Bioorg. Med. Chem. Lett. 11:1557‐1560.
   Gershkovich, A.A. and Kholodovych, V.V. 1996. Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS). J. Biochem. Biophys. Methods 33:135‐162.
   Johnson, L.L., Pavlovsky, A.G., Johnson, A.R., Janowicz, J.A., Man, C.F., Ortwine, D.F., Purchase, C.F., White, A.D., and Hupe, D.J. 2000. A rationalization of the acidic pH dependence for stromelysin‐1 (matrix metalloproteinase‐3) catalysis and inhibition J. Biol. Chem. 275:11026‐11033.
   Knight, C.G. 1995. Fluorimetric assays of proteolytic enzymes. In Proteolytic Enzymes: Aspartic and Metallo Peptidases, Methods in Enzymology, Vol. 248 (A.J. Barrett, ed.), pp. 18‐34. Academic Press, San Diego.
   Lynch, K. 1999. Theoretical and practical difficulties of developing a tumouristatic drug in the treatment of cancer. Int. J. Pharm. Med. 13:127‐136.
   Marcotte, P.A., Elmore, I.N., Guan, Z., Magoc, T.J., Albert, D.H., Morgan, D.W., Curtin, M.L., Garland, R.B., Guo, Y., Heyman, H.R., Holms, J.H., Sheppard, G.S., Steinman, D.H., Wada, C.K., and Davidsen, S.K. 1999. Evaluation of the inhibition of other metalloproteinases by matrix metalloproteinase inhibitors. J. Enzyme Inhib. 14:425‐435.
   Matayoshi, E.D., Wang, G.T., Krafft, G.A., Erickson, J. 1990. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 247:954‐958.
   Mookhtiar, K.A. and Van Wart, H.E. 1990. Purification to homogeneity of latent and active 58‐kilo‐Dalton forms of human neutrophil collagenase. Biochemistry 29:10620‐10627.
   Nagase, H. and Woessner, J.F. 1999. Matrix metalloproteinases. J. Biol. Chem. 274:21491‐21494.
   Netzel‐Arnett, S., Mallya, S.K., Nagase, H., Birkedal‐Hansen, H., VanWart, H.E. 1991. Continuously recording fluorescent assays optimized for five human matrix metalloproteinases. Anal. Biochem. 195:86‐92.
   Parks, W.C. and Mecham, R.P. (eds.) 1998. Matrix Metalloproteinases. Academic Press, San Diego.
   Plantner, J.J., Smine, A., and Quinn, T.A. 1998. Matrix metalloproteinases and metalloproteinases in human interphotoreceptor matrix and vitreous. Curr. Eye Res. 17:132‐140.
   Powers, J.C. and Kam, C.‐M. 1995. Peptide thioester substrates for serine peptidases and metalloendopeptidases. In Proteolytic Enzymes: Aspartic and Metallo Peptidases, Methods in Enzymology, Vol. 248 (A.J. Barrett, ed.), pp. 3‐18. Academic Press, San Diego.
   Rasmussen, H.S. and McCann, P.P. 1997. Matrix metalloproteinase inhibition as a novel anticancer strategy. A review with special focus on batimastat and marimastat. Pharmacol. Ther. 75:69‐75.
   Rodbard, D., Rayford, P.L., Cooper, J.A., and Ross, G.T. 1968. Statistical quality control of radioimmunoassays. J. Clin. Endocrinol. Metab 28:1412‐1418.
   Stricklin, G.P., Bauer, E.A., Jeffrey, J.J., and Eisen, A.Z. 1977. Human skin collagenase. Isolation of precursor and active forms from both fibroblast and organ cultures. Biochemistry 16:1607‐1615.
   Tanzawa, K., Ishii, M., Ogita, T., and Shimada, K. 1992. Matlystatins, new inhibitors of type‐IV collagenases from Actinomadura atramentaria. II. Biological activities. J. Antibiot. 5:1733‐1737.
   Vu, T.H. and Werb, Z. 2000. Matrix metalloproteinases: Effectors of development and normal physiology. Genes Dev. 14:2123‐2133.
   Woessner, J.F., Jr. 1991. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 5:2145‐2154.
   Woessner, J.F., Jr 1999. Matrix metalloproteinase inhibition. From the Jurassic to the third millenium In Inhibition of Matrix Metalloproteinases: Therapeutic Applications. Vol. 878. (R.A. Greenwald S.. Zucker, and L.M.. Golub, eds.). pp. 388‐403. New York Academy of Sciences, N.Y.
   Ye, Q.‐Z., Johnson, L.L., Hupe, D.J., and Baragi, V.V. 1992. Purification and characterization of the human stromelysin catalytic domain expressed in Escherichia coli. Biochemistry 31:11231‐11235.
Key References
   Clendeninn, N.J. and Appelt, K. (eds.) 2000. See above.
  These books are excellent general references for MMPs, providing information on enzyme properties, catalytic specificity, natural substrates and inhibitors.
   Parks, W.C. and Mecham, R.P.. (eds.) 1998. See above.
  This symposium volume is a compilation of papers presented at a conference sponsored by the N.Y.A.S. in October, 1998. Many of the same authors who contributed to this symposium volume also contributed to the books edited by Parks & Mecham and Clendeninn & Appelt.
   Greenwald, R.A., Zucker, S., and Golub, L.M.. (eds.) 1999. Inhibition of Matrix Metalloproteinases: Therapeutic Applications. Annals of the New York Academy of Sciences, Vol. 878, New York Academy of Sciences, New York.
Internet Resources
  htt://www.calbiochem.com
  Calbiochem on‐line catalog.
  http://www.chemicon.com
  Chemicon on‐line catalog.
  http://www.oncresprod.com
  Oncogene Research Products on‐line catalog.
  http://www.chem.qmw.ac.uk/iupac/jcbn
  IUPAC‐IUBMB Joint Commission on Biochemical Nomenclature (JCBN).
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