利用奈米金粒子建立快篩玻尿酸水解酶抑制劑之平台

Abstract

玻尿酸大量存在於人體細胞外基質(extracellular matrices)中，其結構為醣胺多醣體中最簡單的一種，但在人體內扮演的功能卻是不容小覷，除了提供潤滑、保濕的作用外，文獻也顯示出不同分子量大小的玻尿酸在癌細胞的生長、血管新生及發炎反應上都有其重要性。而不同片段大小的玻尿酸是由於玻尿酸水解酶的水解作用產生的，因此對於玻尿酸水解酶的研究也逐漸重要起來，其中在人的玻尿酸水解酶中，研究最深的為玻尿酸水解酶-1(human hyaluronidase-1)，有文獻顯示出，human hyaluronidase-1與癌症表現有很大的相關性，在不同腫瘤模型中，各有抑制腫瘤生長或是促進腫瘤生長的作用。因此，進一步研究玻尿酸水解酶的酵素活性及其對生理的影響，或者是開發具選擇專一性和較強抑制玻尿酸水解酶的抑制劑，都是目前重要的課題之一。 傳統檢測玻尿酸水解酶的活性方法有許多，本研究使用DNS 測量玻尿 酸水解酶的活性。但在篩選抑制劑上，需要能夠有快速檢測、操作簡單及容易製備的特性，可是目前檢測玻尿酸水解酶活性的眾多方法中卻無法達到此目的，因此我們建立了一個快篩玻尿酸水解酶抑制劑之平台。其中選用的玻尿酸水解酶為牛睪丸上的玻尿酸水解酵素(Bovine PH20)。我們可藉著簡單的化學反應將玻尿酸修飾於奈米金的表面，當目標藥物與BovinePH20作用並進而造成奈米金粒子間的距離縮短甚至聚集的情形後，只需檢測奈米金粒子吸收波長的改變或是肉眼觀察奈米金粒子的顏色變化，就能達到快速篩選抑制劑的目的。利用這個新方法我們也成功篩選出了4種有效抑制Bovine PH20 的抑制劑，同時也使用DNS法驗證新方法的可行性。另外，基於human hyaluronidase-1於癌症上的相關性，我們也希望尋找出能抑制human hyaluronidase-1的藥物。但由於human hyaluronidase-1取得 困難，所以我們選用與之結構及序列相似度高達80%的bovine hyal-1，希望藉著尋找出對bovine hya-1有抑制的藥物，能延伸到對human hyaluronidase-1抑制研究上。

Hyaluronan is a polymer-like polysaccharide that is a ubiquitous component of the extracellular matrices of vertebrates. It is uniformly repetitive, linear glycosaminoglycan composed of disaccharides of glucuronic acid and N-acetylglucosamine. Despite its relatively simple chemical composition, hyaluronan fulfils several distinct molecular functions. Some studies have shown that low molecular weight HA is leading to human inflammation, angiogenesis and tumor movement. Hyaluronidases (HAases) belongs a class of enzymes that predominantly degrade hyaluronan. Among the six mammalian HAases, HYAL-1 is the major tumor-derived HAases and is expressed by a variety of tumor cells. The function of HYAL-1 can be a tumor promoter and suppressor. As a result, finding a potential and selective inhibitor is an important issue. The assay of the activities of such enzymes involves tedious process as for the polysaccharide and the product are commonly optically invisible. By integrating the application of gold nanoparticle (AuNP), an original and novel enzymatic assay system for polysaccharide degradation was developed. Using this assay, we successfully discovered several of inhibitors for HAases . More importantly, this newly developed assay system can be employed as a convenient and effective platform for high-throughput screening of inhibitor for HAases. Owing to the difficulty of expression of HYAL-1, we used bovine hyal-1, which is highly identity to human HYAL-1, to make a model for the research on human HYAL-1. We expect that, by searching the inhibitors from bovine hyal-1, can apply to the study of inhibition to human HYAL-1.