Osteoarthritis (OA) is the most prevalent joint disease affecting most of the elderly, imposing a major socio- economic burden on society. The hallmark of the disease is the breakdown of articular cartilage by elevated proteinase activities that degrade major extracellular matrix molecules, aggrecans and collagens, but currently there are no effective treatments for OA, except joint replacement surgery. Our long-term objective is to find ways to specifically inhibit these proteinases and apply them for clinical intervention in OA. The key enzymes involved are matrix metalloproteinases (MMPs) and adamalysins with thrombospondin motif (ADAMTSs). Their activities are regulated by the endogenous tissue inhibitors of metalloproteinases (TIMPs). Our recent studies have shown that TIMP-3 variants that selectively inhibit aggrecanases prevent the progression of OA in an animal model, whereas wild-type TIMP-3 that broadly inhibits MMPs and ADAMTSs was not effective. This suggests that highly selective inhibitors are essential for therapeutic development. To achieve this goal two major aims are proposed. The first is to understand the molecular basis for selectivity in our TIMP-3 variants using biochemical, biophysical and structural methods, and to use this to further develop highly discriminating inhibitors of metalloproteinases. Specifically, we will rationally design TIMP-3 variants that differentially inhibit the main two aggrecanases, ADAMTS-4 and -5, and screen human antibody phage display libraries for """"""""exosite inhibitors"""""""" of aggrecanases and collagenases. The prototype exosite inhibitors will be further refined by mutation. The mechanism by which calcium pentosan polysulfate (CaPPS), an exosite inhibitor of aggrecanases, enhances the interactions between TIMP-3 and aggrecanases will be also elucidated. The second major aim is to evaluate the efficacy of available and newly developed metalloproteinase inhibitors using in vitro and in vivo models of OA and to validate target enzymes in human OA using these inhibitors. To this end, we will continue characterizing TIMP-3 and [-1A]TIMP-3 transgenic mice to delineate the molecular basis of their differential effects on blocking OA progression by analyzing extracellular matrix components protected by these inhibitors using a combination of immunological and proteomic approaches. Newly developed exosite inhibitors and TIMP variants will be tested for their efficacy to protect cartilage from degradation using an in vitro cartilage explant culture system and the in vivo mouse model of OA induced by medial meniscotibial ligament transaction. Effective inhibitors will be also tested in human cartilage in culture to further validate their efficacy. Using available inhibitors we will identify and characterize a novel TIMP-1- sensitive aggrecanase as it is considered as a potential therapeutic target in humans. Accomplishment of these goals will provide new principles for developing therapeutic interventions for OA.

Public Health Relevance

Osteoarthritis (OA) is a disease that afflicts approximately 21 million people in the US over the age of 25, generating an enormous economic cost that is growing rapidly as the population ages. This project is aimed at developing and evaluating a novel approach for OA treatment employing engineered proteins and other molecules that specifically block the enzymes responsible for degrading cartilage in OA. We will investigate the mechanisms through which they act and assess their effectiveness using animal models of the disease and human tissues derived from joint replacement surgery.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR040994-16
Application #
7797562
Study Section
Skeletal Biology Structure and Regeneration Study Section (SBSR)
Program Officer
Tyree, Bernadette
Project Start
1991-09-01
Project End
2014-02-28
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
16
Fiscal Year
2010
Total Cost
$511,500
Indirect Cost
Name
Florida Atlantic University
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
004147534
City
Boca Raton
State
FL
Country
United States
Zip Code
33431
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Yamamoto, Kazuhiro; Santamaria, Salvatore; Botkjaer, Kenneth A et al. (2017) Inhibition of Shedding of Low-Density Lipoprotein Receptor-Related Protein 1 Reverses Cartilage Matrix Degradation in Osteoarthritis. Arthritis Rheumatol 69:1246-1256
Watanabe, Kenta; Hirata, Michiko; Tominari, Tsukasa et al. (2016) BA321, a novel carborane analog that binds to androgen and estrogen receptors, acts as a new selective androgen receptor modulator of bone in male mice. Biochem Biophys Res Commun 478:279-285
Zou, Haiyin; Wu, Ying; Brew, Keith (2016) Thermodynamic Basis of Selectivity in the Interactions of Tissue Inhibitors of Metalloproteinases N-domains with Matrix Metalloproteinases-1, -3, and -14. J Biol Chem 291:11348-58
Yamamoto, Kazuhiro; Okano, Hiroshi; Miyagawa, Wakako et al. (2016) MMP-13 is constitutively produced in human chondrocytes and co-endocytosed with ADAMTS-5 and TIMP-3 by the endocytic receptor LRP1. Matrix Biol 56:57-73
Doherty, Christine M; Visse, Robert; Dinakarpandian, Deendayal et al. (2016) Engineered Tissue Inhibitor of Metalloproteinases-3 Variants Resistant to Endocytosis Have Prolonged Chondroprotective Activity. J Biol Chem 291:22160-22172
Watanabe, Kenta; Hirata, Michiko; Tominari, Tsukasa et al. (2016) The MET/Vascular Endothelial Growth Factor Receptor (VEGFR)-targeted Tyrosine Kinase Inhibitor Also Attenuates FMS-dependent Osteoclast Differentiation and Bone Destruction Induced by Prostate Cancer. J Biol Chem 291:20891-20899
Lund, Jacob; Troeberg, Linda; Kjeldal, Henrik et al. (2015) Evidence for restricted reactivity of ADAMDEC1 with protein substrates and endogenous inhibitors. J Biol Chem 290:6620-9
Seubert, Bastian; Gr├╝nwald, Barbara; Kobuch, Julia et al. (2015) Tissue inhibitor of metalloproteinases (TIMP)-1 creates a premetastatic niche in the liver through SDF-1/CXCR4-dependent neutrophil recruitment in mice. Hepatology 61:238-48
Gardiner, M D; Vincent, T L; Driscoll, C et al. (2015) Transcriptional analysis of micro-dissected articular cartilage in post-traumatic murine osteoarthritis. Osteoarthritis Cartilage 23:616-28

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