螢光發報之原位適配接合反應應用於異戊二烯焦磷酸合成酶抑制劑的開發

類異戊二烯族廣泛存在於生物系統，例如：以四異戊二烯焦磷酸 (C20-GGPP) 進行蛋白質異戊二烯化的訊息傳遞過程，在腫瘤細胞觀察到異常的表現。十一異戊二烯焦磷酸 (C55-UPP) 在細菌細胞壁生合成中作為肽聚醣單體的載體。因此，發展異戊二烯焦磷酸合成酶抑制劑，具有高度生醫應用價值。在本論文中，我們利用螢光訊號輸出的原位適配接合反應，針對異戊二烯焦磷酸合成酶發展了有效的抑制劑篩選方法。　　我們設計並合成了數個疏水性的3-疊氮香豆素分子片段及親水性的苯炔分子片段，分別對應目標酵素活性中心結構的疏水性通道和具有極性殘基的催化區域。利用靶標導向合成的策略，希望酵素自行篩選出合適的分子片段，並促使3-疊氮香豆素和親水性片段炔基之間的疊氮—炔環化加成反應。加成後接合產物中香豆素的螢光不再由疊氮基所抑止，螢光因而增強。如此由螢光訊號的輸出判斷酵素是否能找到合適的抑制劑 (分子片段接合產物)。　　搭配先前建立的MANT-GPP螢光探針 (證實可作為UPPs的受質及抑制劑，與UPPs結合後在425 nm的螢光放射會增強)，十個疏水性分子片段對UPPs的結合能力利用螢光競爭實驗加以評估 (先預混合MANT-GPP及UPPs再加入分子片段)。滴定的結果顯示片段的結合能力由碳鏈取代基的長度所主導。因此，在後續的原位適配接合實驗中，我們著重三個不同長短異戊二烯取代的分子片段：Pre-hAzC、Ger-hAzC、Far-hAzC。另外也選用具有結合潛力的三個焦磷酸根、雙膦酸根親水性片段：pPP、mPP和mBP。上述分子片段的排列組合與三種目標酵素 (UPPs、OPPs和hGGPPs) 分別作用三天，再以螢光分析儀量測個別組合的螢光強度。我們發現在27 (3 x 3 x 3) 種可能的組合中，hGGPPs與Pre-hAzC/mBP作用的組合有最大的螢光增強幅度，可達到4.5倍。為了確認原位適配接合實驗中生成產物的組態，我們合成了Pre-hAzC和mBP可能的環化加成產物Pre-1,4-mBP和Pre-1,5-mBP並進行生物活性測試。結果顯示在100 uM的濃度下，Pre-1,4-mBP可以抑制hGGPPs 84%的活性，然而Pre-1,5-mBP抑制效果則不到15%，顯示Pre-1,4-mBP為主要抑制hGGPPs的化合物。最後，我們也利用電腦虛擬的嵌合實驗，理論對實驗結果相契合。

關鍵字

螢光；原位適配接合化學；異戊二烯焦磷酸；抑制劑；篩選

並列摘要

Isoprenoids are widespread in the biological systems. For examples, abnormal cellular signal transduction via protein prenylation with C20-GGPP is observed in some tumor cells, and the C55-UPP serves as a lipid carrier in the bacterial cell wall biosynthesis. Therefore, developing inhibitors for prenyl pyrophosphate synthases has great implications in the biomedical applications. In this thesis, we have developed an effective screening method based on the fluorogenic in situ click reactions to fish out inhibitors for a series of prenyl pyrophosphate synthases more effecitvely. We designed and synthesized several hydrophobic 3-azidocoumarins and hydrophilic arylalkynes, to complement the hydrophobic tunnel and the polar catalytic region of the target enzymes. Utilizing the strategy of in-situ target-guided synthesis, we are hoping that the enzymes select the proper fragments themselves and further promote the azide-alkyne cycloaddition between the hydrophobic fragments having 3-azidocoumarin and the hydrophilic ones with alkyne functionality. Once the cycloadduct formed, the fluorescence of azicocoumarin is no longer suppressed by the azido group and the resultant fluorescence enhancement is a good indication that the target enzyme promotes the click reaction itself. The binding ability of 10 hydrophobic fragments to the active sites of UPPs was evaluated by fluorescence competition experiments by first incubating the enzyme with MANT-GPP, which was proven to be the substrate and inhibitor for UPPs and could give fluorescence increase at 425 nm once bounded. The titration results revealed that the binding ability of the hydrophobic fragments could attributed to the length of substituted hydrocarbon tails. Hence, we only employed three different lengths of prenyl substituent (denoted as Pre-hAzC, Ger-hAzC, Far-hAzC) and three hydrophilic arylalkynes (denoted as pPP, mPP, mBP) with potent pyrophosphate or bisphosphonate groups were chosen for further in situ click experiments. These fragments (3 x 3) were arranged in a spatial combinatorial fashion and each combination was incubated with three target enzymes (UPPs, OPPs or hGGPPs) for 3 days, respectively. The fluorescence was then measured by the fluorescence plate reader for each combination. These results showed that the combination of Pre-hAzC/mBP incubated with hGGPPs gave the best fluorescence with a 4.5-fold fluorescence enhancement among 27 (3 x 3 x 3) possible combinations. To confirm and verify which is the isomer of Pre-hAzC/mBP generated by the in situ click reactions, the possible cycloadducts of Pre-hAzC and mBP, denoted as Pre-1,4-mBP and Pre-1,5-mBP, were synthesized and tested their biological activity. The inhibition results revealed that the former suppresses 84% of hGGPPs activity at 100 uM concentration, the latter only inhibits 15% of hGGPPs activity. Moreover, the molecular modeling results of docking are consistent with the biological inhibition data.

並列關鍵字

fluorogenic ； in situ click ； prenyl pyrophosphate ； inhibitor ； screening

參考文獻

45. 劉維民. 設計與合成十一異戊二烯焦磷酸合成酶的抑制劑及其生物活性探討. 臺灣大學, 2007.

44. 李育瑾. 以法呢焦磷酸酯螢光類似物探測十一異戊二烯焦磷酸合成酶的活性中心和反應構形變化. 臺灣大學, 2004.

49. Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Berozzi, C. R. J. Am. Chem. Soc. 2008, 130, 11486-11493.

40. Ko, T.-P.; Chen, Y.-K.; Robinson, H.; Tsai, P.-C.; Gao, Y.-G.; Chen, A. P.-C.; Wang, A. H.-J.; Liang, P.-H. J. Biol. Chem. 2001, 276, 47474-47482.

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螢光發報之原位適配接合反應應用於異戊二烯焦磷酸合成酶抑制劑的開發

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