Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Only a small number of proteases have been identified capable of efficient processing of triple-helical regions of collagens. Several members of the zinc metalloenzyme family, specifically matrix metalloproteinases (MMPs), possess collagenolytic activity. A mechanistic understanding of the cleavage of intact collagens has been pursued for many years;the results of such studies could lead to the development of truly selective MMP inhibitors. Our laboratory developed triple-helical peptides (THPs) as MMP substrates, with the goal of using these models to dissect collagenolytic behavior. Our work with THP substrates, along with prior studies from other research groups, have led to a """"""""conformational entropy shift"""""""" hypothesis explaining how MMPs process collagen without input from an external energy source. The research plan described herein focuses on testing our collagenolysis hypothesis by utilizing several biophysical approaches [NMR spectroscopy, hydrogen/deuterium exchange mass spectrometry (HDX MS), X-ray crystallography, and X-ray absorption spectroscopy (XAS)] in combination with site-specific mutagenesis and kinetic analyses to precisely determinate the roles of MMP regions and residues in the binding, unwinding, and hydrolysis of triple-helical structures. Variants of THPs will be created, by site-directed and combinatorial approaches, to obtain substrates that are selective within the collagenolytic MMPs. Based on the mechanistic results, we will compare the inhibitory capabilities of phosphinate-containing THPs using site-directed and combinatorial libraries to develop novel, selective MMP inhibitors. Co-crystallization and HDX MS of MMPs and THP inhibitors will be utilized to evaluate the sites of interaction between the two biomolecules, allowing for further optimization of lead compounds. Select inhibitors will be tested using cells overexpressing the targeted MMPs, in a model of angiogenesis, and in mouse models of breast carcinoma and melanoma. Ultimately, we would like to obtain inhibitors that target those proteases implicated in cancer progression (MMP-2, MMP-9, and MT1-MMP) while sparing proteases with host-beneficial functions (MMP-3 and MMP- 8). PUBLIC HEALTH REVELANCE: The present study is designed to create a novel class of therapeutic agents to selectively stop the action of tumor-associated enzymes that degrade proteins (proteases). These proteases have been shown to be important for cancer progression, and thus blocking their function will impair the spread of cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA098799-10
Application #
8038451
Study Section
Drug Discovery and Molecular Pharmacology Study Section (DMP)
Program Officer
Knowlton, John R
Project Start
2003-03-05
Project End
2013-02-28
Budget Start
2011-03-01
Budget End
2012-02-29
Support Year
10
Fiscal Year
2011
Total Cost
$352,963
Indirect Cost
Name
Torrey Pines Institute for Molecular Studies
Department
Type
DUNS #
605758754
City
Port Saint Lucie
State
FL
Country
United States
Zip Code
34987
Marcink, Tara C; Simoncic, Jayce A; An, Bo et al. (2018) MT1-MMP Binds Membranes by Opposite Tips of Its ? Propeller to Position It for Pericellular Proteolysis. Structure :
Stawikowski, Maciej J; Fields, Gregg B (2018) Tricine as a convenient scaffold for the synthesis of C-terminally branched collagen-model peptides. Tetrahedron Lett 59:130-134
Tokmina-Roszyk, Michal; Fields, Gregg B (2018) Dissecting MMP P10' and P11' subsite sequence preferences, utilizing a positional scanning, combinatorial triple-helical peptide library. J Biol Chem 293:16661-16676
Pahwa, Sonia; Bhowmick, Manishabrata; Amar, Sabrina et al. (2018) Characterization and regulation of MT1-MMP cell surface-associated activity. Chem Biol Drug Des :
Amar, Sabrina; Smith, Lyndsay; Fields, Gregg B (2017) Matrix metalloproteinase collagenolysis in health and disease. Biochim Biophys Acta Mol Cell Res 1864:1940-1951
Van Doren, Steven R; Marcink, Tara C; Koppisetti, Rama K et al. (2017) Peripheral membrane associations of matrix metalloproteinases. Biochim Biophys Acta Mol Cell Res 1864:1964-1973
Prior, Stephen H; Byrne, Todd S; Tokmina-Roszyk, Dorota et al. (2016) Path to Collagenolysis: COLLAGEN V TRIPLE-HELIX MODEL BOUND PRODUCTIVELY AND IN ENCOUNTERS BY MATRIX METALLOPROTEINASE-12. J Biol Chem 291:7888-901
Singh, Warispreet; Fields, Gregg B; Christov, Christo Z et al. (2016) Importance of the Linker Region in Matrix Metalloproteinase-1 Domain Interactions. RSC Adv 6:23223-23232
Iyer, Rugmani Padmanabhan; de Castro BrĂ¡s, Lisandra E; Patterson, Nicolle L et al. (2016) Early matrix metalloproteinase-9 inhibition post-myocardial infarction worsens cardiac dysfunction by delaying inflammation resolution. J Mol Cell Cardiol 100:109-117
Fields, Gregg B; Stawikowski, Maciej J (2016) Imaging Matrix Metalloproteinase Activity Implicated in Breast Cancer Progression. Methods Mol Biol 1406:303-29

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