人類透明質酸水解脢和其抑制劑之研究開發

Abstract

Hyaluronidase(Hyal)已被廣泛地應用在醫療上，但醫藥級Hyal 的供應卻時常短缺。 因此，如何獲得人類重組Hyal (recombinant human Hyal)已然成為一個重要且具價值的課 題。已有證據顯示Hyal 可能有抗癌的效應並且能抑制腫瘤轉移，不過，目前的結論卻 相當不一致。例如Hyal-1 和Hyal-2 在不同的腫瘤裡的具體功能似乎互相矛盾，且尚無 法解釋其究理。所以，更進一步研究hyaluronidase 酵素活性與其對生理的影響，甚或是 開發具選擇專一性和較強抑制能力的抑制劑，都是目前急迫的議題。此外，Hyal 抑制劑 除了可當作治療arthroses 的醫藥物使用外，亦可結合抗生素，用在抗細菌治療上，特別 是對可生產hyaluronate lyase 的細菌。雖然肝素 (heparin)、黃酮類化合物 (flavonoids) 和 全磺化(fully O-sulfonated)之glycosaminoglycans 已被發現在試管試驗中(in vitro) 可抑制 牛睪丸之hyaluronidase (BTH) 活性。但是這些化合物僅具有微弱的抑製作用，所以到目 前為止，並沒有較具潛力或選擇性較高的抑制劑被發現。L-ascorbic acid 6-hexadecanoate (Vcpal)是近年來被發現對BTH 具有有較佳的抑制效果的化合物，其IC50 為56 μM。我 們因此計畫使用Vcpal 作為本研究之內標的物(「threshold」 compound)，透過competitive binding technique 篩選出對哺乳動物Hyals 作用較強的抑制劑。這種competitive binding technique 技術的發展將結合螢光共振能轉移技術(FRET)。本研究亦將開發以FRET 技術 進行準確的酵素動力分析。此外，依據蜜蜂毒液中hyaluronidase 與HA 的四單元醣 (tetrasaccharide)碎片的蛋白質晶體結構，人類Hyal 的相似模組(homology models)可以利 用電腦模擬技術預測出來。這些蛋白模組將被實際應用於Hyal 抑制劑的篩選上。 計畫案將於三年內執行，具體的主題包括︰ 第1-第2 年 (1) 人類 Hyal-1 和Hyal-2 的基因重組和蛋白質表達 (2) 發展一種有效的方法分析 Hyal 的酵素活性 (3) 發展一以 FRET 為基礎、篩選Hyal 抑制劑的平台 (4) 電腦模擬(structure simulation)人類Hyal-1 和Hyal-2 的蛋白質結構 (5) 合成玻尿酸衍生物作為 FRET 酵素分析的基質(substrate) (6) 合成 Vcpal 的衍生物作為抑制劑的標的 第2-第3 年 (1) 篩選 Hyal 抑制劑 (2) 對篩選出的藥物做抑制效應的定量評估 (3) 用化學修飾法增進抑制劑的抑制效果 (4) Hyals 的酵素特性分析和基因突變的研究 (5) 哺乳動物 Hyals 的酵素動力與催化機制的探討

Hyaluronidase (Hyal) is widely used in medication. Owing to depleted supplies of pharmaceutical Hyal, to obtain the recombinant human Hyal becomes an important issue and valuable project. Evidences showed that Hyal may have intrinsic anticancer effects and can suppress tumor progression. However, the findings are rather inconsistent. The specific function of Hyal-1 and Hyal-2 in different tumors is still contradictory. To further investigate the relationship of enzyme activity and the physiological effects of hyaluronidase, selective and potent inhibitors are urgently required. Moreover Hyal inhibitors could be useful as drugs in the treatment of arthroses or, combined with antibiotics, in antibacterial therapy of hyaluronate lyase producing bacteria. Although heparin, flavonoids, and fully O-sulfonated glycosaminoglycans have been reported to inhibit bovine testicular hyaluronidase (BTH) in vitro, these compounds show only weak or partial inhibitory activity. No potent and selective hyaluronidase inhibitors are known to date. L-ascorbic acid 6-hexadecanoate (Vcpal) was recently examined as a better inhibitor of BTH with IC50 value of 56 μM. We plan to use Vcpal as a 「threshold」 compound to screen powerful inhibitors for mammalian Hyals by using competitive binding technique. The competitive binding technique will be developed in combining with the Fluorescence Resonance Energy Transfer (FRET) technique. The FRET technique will also be employed for accurate kinetic assay. Furthermore, the homology models of human Hyals will be constructed based on the crystal structure of bee venom hyaluronidase in complex with a HA tetrasaccharide fragment. These structures will be used as the templates for virtual screening of Hyal inhibitor. The project will be performed in 3 years with the following specific subjects: 1st - 2nd year (1) Cloning and expression of human Hyal-1 and Hyal-2 (2) Developing an effective method for Hyal assay (3) Developing the FRET-base platform for inhibitor screening (4) Structure simulation of human Hyal-1 and Hyal-2 (5) Synthesis of hyaluronic acid derivative for enzymatic assay by FRET (6) Synthesis of the derivatives of Vcpal as 「threshold」 inhibitor 2nd - 3rd year (1) Screening of inhibitor (2) Evaluation of inhibitory effect of screened drug (3) Improvement of the potency of inhibitor by chemical modification (4) Characterization and mutagenic study of Hyals (5) Mechanistic action of mammalian Hyals