Matrix Metalloproteinase Inhibition and Specificity

描述（由申请人提供）：基质金属蛋白酶（MMP）和抑制MMP的TIMPs控制癌症、关节炎、动脉疾病、肺病、生殖事件和伤口愈合中的组织重塑。两种关键的天然MMP抑制剂的作用机制尚未充分了解，即，人蛋白TIMP-1和来自绿色茶的防癌EGCG。这两种具有治疗潜力的作用机制将进一步研究。先前的项目发现典型TIMP和MMP的高亲和力由熵增益驱动，主要是构型。N-TIMP-1的β-桶骨架在MMP-3结合后被动员。为了更完整的空间和时间的灵活性变化与他们的结合，NMR研究的流动性将扩大到自由和N-TIMP-1结合状态的MMP-3，侧链甲基，并在更长的时间尺度，热力学推断变得更加强大。工程化N-TIMP-1以增强治疗潜力，忽略了其不期望的生长因子活性。先前的项目发现了一个保守的表面上的TIMPs假设介导这种活动。将对该贴片进行诱变，以去除N-TIMP-1刺激细胞增殖的活性。将鉴定信号通路和介导TIMP-1生长因子活性的受体。在绿色茶中的主要多酚--EGCG的化学预防生物活性中，发现了几种MMPs的抑制。将调查哪些MMPs被EGCG抑制。将研究EGCG抑制MMP的新机制及其结合MMP的结构模式。用于心血管和肺部病症的当代药物靶标是MMP-12。MMP-12对血管和肺的弹性蛋白组分以及对弹性蛋白酶的弹性蛋白保留抑制剂（称为α 1-抗胰蛋白酶）具有显著的高活性。MMP-12对弹性蛋白和α 1-抗胰蛋白酶活性的序列决定因素尚不清楚，将进行探讨。MMP-12中与α 1-抗胰蛋白酶相互作用的位点将通过NMR作图。弹性蛋白酶特异性将从MMP-12中工程化出来，然后转移到MMP-3中。发现赋予MMP-12独特特异性的特征应有助于设计更具选择性的抑制剂，并可能提示其他MMP特异性的决定因素。

DESCRIPTION (provided by applicant): 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.