香豆素衍生物BPRHIV001藉由Akt途徑抑制第一型人類免疫缺乏病毒Tat蛋白質調節之轉錄活性

Inhibition of HIV-1 Tat-mediated transcription by a coumarin derivative BPRHIV001 through Akt pathway

林必涵(Pi-Han Lin)

指導教授 ： 張淑媛

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.00888

摘要

第一型人類免疫缺乏病毒(Human immunodeficiency virus type 1, HIV-1)的Tat調節蛋白質是病毒轉錄RNA所必需。因此，Tat功能受影響時，病毒的複製將會被抑制。先前我們設計一個可偵測Tat功能的細胞篩選平台，來篩選抑制HIV-1的新興化合物。經此平台篩選了292個香豆素衍生物，其中BPRHIV001在奈莫耳濃度（nanomolar concentration）即具有抑制Tat轉錄活化的活性。進一步實驗發現，BPRHIV001不是透過影響Tat的生成以及Tat轉錄活化相關蛋白質複合物- Tat/P-TEFb組成，來抑制其對RNA第二型聚合酶（RNAPII）的活化作用。因此我們想探究BPRHIV001是否透過調控Tat轉譯後的修飾來影響其功能。結果發現，加入BPRHIV001後，細胞內可以乙醯化Tat並且幫助其離開TAR RNA的p300蛋白質量明顯減少，但mRNA量並沒有受到影響。因為磷酸化的Akt可穩定p300蛋白質，減緩其衰減，我們接著發現Akt及其活化分子PDPK1的磷酸化都在BPRHIV001的存在下降低許多。我們進行蛋白質嵌合分析(Docking analysis)來探討BPRHIV001與PDPK1之間的關係，結果顯示BPRHIV001可能藉由變構效應(allosteric effect)與PDPK1交互作用進而抑制PDPK1的磷酸化使其活性降低。最後in vitro實驗發現，BPRHIV001可有效抑制HIV-1的複製，其50%有效濃度（50% inhibitory concentration；IC50）為1.3 nM。BPRHIV001也可與現行使用的反轉錄酶抑制劑azidothymidine (AZT) 以及efavirenz (EFV)有強烈的加成效應。
綜觀以上結果，BPRHIV001可能透過干擾PI3K/Akt 途徑，來抑制HIV-1 Tat蛋白質所調控的轉錄活化活性。由於其與現行使用的抗病毒藥物具有良好的加成作用，我們認為BPRHIV001可望發展成為抗HIV-1感染的新興治療藥物的前導化合物。

關鍵字

人類免疫缺乏病毒 ； 人類免疫缺乏病毒調節蛋白-Tat ； BPRHIV001 ； p300 ； PI3K/Akt 途徑

並列摘要

The HIV-1-encoded RNA-binding protein Tat is known to play an essential role in viral gene expression. In the search of novel compounds in inhibiting Tat transactivity, BPRHIV001 was identified after screening of 292 coumarin-derivatives. BPRHIV001 was shown to significantly inhibit Tat-mediated transactivation at nanomolar range. BPRHIV001 is likely to exert its effects at the stage after initiation of RNAPII elongation since Tat protein expression and the assembly of Tat/P-TEFb complex which is essential for processivity of RNAPII elongation complex, remained unchanged. Next, the level of p300 was determined since acetylation of Tat by p300/CBP could facilitate the dissociation of Tat from TAR and subsequent recycling of Tat. The p300 protein level was reduced in the presence of BPRHIV001, while the p300 mRNA level was unaffected. The reduced p300 protein level was possibly resulted from reduced stability of p300 since the level of phosphorylated Akt, which was shown to be closely related with p300 stability, decreased in the presence of BPRHIV001. A concordant reduction of phosphorylated PDPK1, a well-known Akt activator was also observed. Furthermore, the docking analysis revealed that the reduced PDPK1 phosphorylation is likely resulted from the allosteric effect of interaction between BPRHIV001 and PDPK1. Finally, BPRHIV001 was shown to effectively inhibit HIV-1 replication in vitro and the 50% inhibitory concentration (IC50) of BPRHIV001 against HIV-1 was 1.3 nM. BPRHIV001 also had strong synergistic effect with current reverse transcriptase inhibitor azidothymidine (AZT) and efavirenz (EFV).
Above all, BPRHIV001 was shown to have strong inhibitory effect on HIV-1 Tat-mediated transactivation, possibly through repressing the PI3K/Akt pathway.With strong synergistic effects with current reverse transcriptase inhibitors, BPRHIV001 has the potential to become a promising lead compound for the development of novel therapeutic agent against HIV-1 infection.

並列關鍵字

HIV ； Tat ； BPRHIV001 ； p300 ； PI3K/Akt pathway

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