New probes for matrix metalloproteinase 13

描述（由申请人提供）：骨关节炎（OA）是最常见的关节炎形式，其特征是关节软骨的破坏。关节或关节软骨的主要成分是II型胶原蛋白和各种蛋白聚糖，如聚集蛋白、硫酸软骨素和透明质酸。基质金属蛋白酶13 （Matrix metalloproteinase 13, MMP-13）已被证明是骨性关节炎患者关节软骨降解的主要胶原酶。开发基于MMP-13抑制剂的药物的多次尝试失败主要是由于剂量限制副作用统称为肌肉骨骼综合征（MSS）。虽然MSS的确切原因尚不清楚，但据信是由于候选药物对金属蛋白酶家族的其他代表缺乏选择性。鉴定蛋白酶二级结合位点（外源位点），即促进或调节蛋白酶活性的非活性位点区域，可用于蛋白酶家族中选择性抑制剂的设计。我们的实验室开发了三螺旋肽（THP）荧光共振能量转移（FRET）底物，用于高通量筛选（HTS）胶原溶解MMPs和鉴定假定的外源性结合化合物。利用HTS技术和FRET THP测定，我们先前鉴定了几种选择性的低微摩尔MMP-13抑制剂。一种抑制剂（化合物Q/4， PubChem CID 2047223）的性质表明其作用方式不是通过锌螯合。化合物Q/4具有比先前描述的MMP-13抑制剂明显更小的分子支架，但具有出色的选择性。我们希望进一步开发化合物Q/4及其类似物作为体内MMP-13探针。实现这一目标的具体目标如下：(1)x射线晶体学引导MMP-13外源探针的药物化学；(2) MMP-13外源探针的体内外实验。目前的工作将导致体内探针可以评估选择性抑制剂对OA细胞外蛋白水解活性的影响，并为新型MMP显像剂的开发奠定基础。此外，还将确定一种独特的MMP抑制模式。

DESCRIPTION (provided by applicant): Osteoarthritis (OA), the most common form of arthritis, is characterized by the destruction of articular cartilage. The main constituents of articular or joint cartilage are type II collagen and various proteoglycans, such as aggrecan, chondroitin sulfate, and hyaluronan. Matrix metalloproteinase 13 (MMP-13) has been shown to be main collagenase responsible for degradation of articular cartilage during OA. Multiple attempts to develop an MMP-13 inhibitor-based drug failed mostly due to the dose limiting side effects collectively known as musculoskeletal syndrome (MSS). While the exact cause of MSS is not known it is believed to be due to the lack of selectivity of drug candidates towards other representatives of metalloproteinase families. Identification of protease secondary binding sites (exosites), i.e. non-active site regions that facilitate or modulate protease activity, could be utilized for the design of selective inhibitors within protease families. Our laboratory has developed triple-helical peptide (THP) fluorescence resonance energy transfer (FRET) substrates for high throughput screening (HTS) of collagenolytic MMPs and identification of putative exosite-binding compounds. Using HTS techniques with FRET THP assays, we previously identified several selective low micromolar inhibitors of MMP-13. One inhibitor (compound Q/4, PubChem CID 2047223) exhibited properties suggesting that its mode of action was not via Zn chelation. Compound Q/4 possessed a significantly smaller molecular scaffold than previously described MMP-13 inhibitors, yet offered excellent selectivity. We wish to further develop compound Q/4 and its analogs as in vivo MMP-13 probes. The specific aims to achieve this goal are as follows: (1) X-ray crystallography-guided medicinal chemistry of MMP-13 exosite probes; and (2) in vitro and in vivo testing of MMP-13 exosite probes. The present work will lead to in vivo probes that can evaluate the effects of selective inhibitors on extracellular proteolytic activity in OA and lay the groundwork for the development of novel MMP imaging agents. In addition, a unique mode of MMP inhibition will be identified.