發展及應用以Ro48-8071為基礎的新型式螢光性探針並針對氧化鯊烯環化酵素進行抑制作用的研究

Abstract

氧化鯊烯環化酵素(OSC, E.C.5.4.99.-)將受質氧化鯊烯經由一連串環化/重組的反應催化形成膽固醇的前驅物-羊毛硬脂醇。此家族的酵素在固醇類和三萜類產物的生合成途徑中扮演著特別的角色，並且對於降膽固醇藥物以及抗真菌劑的研發都很有幫助。本論文已成功的從牛肝中，經由一連串的純化步驟，並利用SDS-PAGE電泳分析，得到分子量約為80 kDa純的氧化鯊烯環化酵素。並且利用Ro48-8071這一個很有效的氧化鯊烯環化酵素抑制劑，將其與實驗中所選的五個具螢光性質的化合物：4-(4,5-diphenyl-lH-imidazol- 2-yl)-phenylboronic acid(DPA)、naphthalene-1-boronic acid(NA-1)、naphthalene-2-boronic acid(NA-2)、biphenyl-3-boronic acid(BP-3) 和biphenyl-4-boronic acid(BP-4)，利用Suzuki偶合的方法進行結合，期望發展成具有抑制效果的螢光探針，經由質譜儀與核磁共振儀的分析後，確定已成功的合成出目標物。另外，由抑制酵素活性的實驗中發現，Ro4-NA1、Ro4-NA2和Ro4-BP4約在100μM的濃度時有明顯的抑制活性，而Ro4-BP3約在100μM的濃度時有相較於Ro4-NA1、Ro4-NA2和Ro4-BP4具有稍差的抑制活性。而Ro4-DPA抑制活性最差，在濃度100μM時尚無明顯的抑制，由此結果推測，可能為經修飾的螢光化合物對於酵素有較大的立體障礙，所以抑制的效果沒有未經過修飾的Ro48-8071來的有效；因此並利用入塢作用(Docking)的實驗方式加以探討經螢光探針修飾的Ro48-8071在牛肝中的氧化鯊烯環化酵素活性區中的立體空間關係以及此修飾對於抑制能力的影響。另外，在螢光光譜的測量實驗發現，氧化鯊烯環化酵素與Ro4-DPA做混合與沒有做混合的對照組在螢光放射光譜上沒有改變，但是在經過去鹽管柱（desalting column）層析法將蛋白質與沒有作用的螢光探針做分離，發現在有蛋白質出現的部分有螢光的現象，初步推測有可能是因為DPA的結構沒有進入酵素的活性區中，使得螢光的強度沒有被削弱，之後還要再做進一步的確認。 本論文希望最後能利用這些帶有螢光特性的Ro48-8071衍生抑制劑能提供氧化鯊烯環化酵素在結構與功能性上的探討，或能更進一步提供在蛋白質體學上的應用，以及對於降膽固醇藥物篩選上有所幫助。

Oxidosqualene cyclases (OSC, E.C.5.4.99.-) catalyze the conversion of the linear 2,3-oxidosqualene into the fused ring compounds such as lanosterol, the precursor of cholesterol, via a series of cyclization and rearrangement. Due to its intrinsic important role in the biosynthetic pathway of steroids and triterpenoids, OSC has been an attractive target for the development of antifungal and hypocholesterolemic drugs. Accordingly, bovine liver OSC had been successfully purified via three chromatographic columns in our laboratory and exhibited a clear single band with molecular weight about 80 kDa from the analysis of SDS-PAGE. Moreover, OSC in different mammalian sources has been effectively inhibited by a potent inhibitor, Ro48-8071. But the different orientation of this inhibitor was examined from either crystal structure or photoaffinity labeling experiments. In order to better understand the inhibiting mechanism as well as to solve the exact inhibitor binding site, several Ro48-8071-based fluorescent probes were developed. Those fluorescence derivatives including 4-(4,5-diphenyl-lH-imidazol-2-yl)-phenylboronic acid (DPA), biphenyl-3- boronic acid (BP-3), biphenyl-4-boronic acid (BP-4), naphthalene-1-boronic acid (NA-1) and naphthalene-2-boronic acid (NA-2) were synthesized successfully from Ro48-8071 using the palladium-catalyzed Suzuki coupling reactions and confirmed by GC/MS and nuclear magnetic resonance (NMR) spectroscopy analysis. According to the inhibition experiment of all newly synthesized fluorescence probes, it was found that Ro4-NA1, Ro4-NA2 and Ro4-BP4 showed apparent inhibition activity at the concentration of 100μM. Ro4-BP3 had also obvious inhibition activity at the concentration up to 100μM but less than Ro4-NA1, Ro4-NA2 and Ro4-BP4. Among those fluorescent probes, Ro4-DPA showed the worst inhibition even though at the concentration of 100μM. The results of inhibition experiment indicated that the fluorescent modification of Ro48-8071 dramatically reduces the inhibition activity. The steric effect or orientation changes way occur among these modified inhibitors within the enzymatic active site. In terms of the molecular docking experiment, the interactions between the fluorescent modification of Ro48-8071 probes and the active site of bovine liver OSC as well as the orientation of probes have dramatically changed. Hence, the lower-inhibition activity via fluorescent modification could be reasonably explained via the three-dimensional protein-ligand docked complex models. On the other hand, from the fluorescent spectra examination, there is no obvious difference between the bovine liver OSC-Ro4-DPA complex and the native Ro4-DPA molecule. However, after Hi-Trap desalting column purification, the fraction with OSC also exhibited slight fluorescent intensity, the result showed that the DPA group might interact but not integrate within the protein active site. In the future, further improved newly site-specific fluorescent probes will be developed and applied to the field of proteomics for effectively screening of OSC drugs.