Matrix metalloproteinases (MMPs), and TIMPs that inhibit them, control tissue remodeling in cancer, arthritis, arterial disease, pulmonary disease, reproductive events, and wound healing. Mechanisms of action of two key natural MMP inhibitors are not adequately understood, i.e., the human protein TIMP-1 and the cancer preventative EGCG from green tea. The mechanisms of action of these two with therapeutic potential will be investigated further. The previous project found the high affinity of a typical TIMP and MMP to be driven by entropy gain, largely configurational. The backbone of the beta-barrel of N-TIMP-1 is mobilized upon binding of MMP-3. For a more complete spatial and temporal view of flexibility changes linked to their binding, NMR studies of mobility will be expanded to free and N-TIMP-1-bound states of MMP-3, to side chain methyl groups, and to longer time scales where thermodynamic inferences become more robust. Engineering of N-TIMP-1 to enhance therapeutic potential has neglected its undesirable growth factor activity. The previous project found a conserved surface on TIMPs hypothesized to mediate this activity. This patch will be mutagenized in an effort to remove N-TIMP-1's activity of stimulating cell proliferation. Signaling pathways and receptor mediating TIMP-1's growth factor activity will be identified. Inhibition of a few MMPs was found among the chemopreventative bioactivities of the main polyphenol in green tea, EGCG. Which MMPs are inhibited by EGCG will be surveyed. The novel mechanism of MMP inhibition by EGCG and its structural mode of binding an MMP will be investigated. A contemporary drug target for cardiovascular and pulmonary conditions is MMP-12. MMP-12 has distinctively high activity upon the elastin component of blood vessels and lungs and upon the elastin-preserving inhibitor of elastase known as a1-anti-trypsin. Sequence determinants of MMP-12's activity upon elastin and a1-anti-trypsin are not known and will be probed. Sites in MMP-12 that interact with a1-anti-trypsin will be mapped by NMR. Elastase specificity will be engineered out of MMP-12, and then transferred into MMP-3. Discovery of features that confer the unique specificity to MMP-12 should aid design of more selective inhibitors and may suggest determinants of specificities of other MMPs. ? ?
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