2013-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647304摘要：鑑於三合一療法的有效性及解決當前HIV-1 抗藥性病毒的問題，本實驗室於先前報導一個能藉由影響HIV-1 Tat 的轉錄活化而抑制HIV-1 病毒複製之香豆素衍生物，BPRHIV001。我們初步的結果顯示BPRHIV001 可藉由降低Akt及PDPK1 的磷酸化來調控p300，進而影響Tat 的轉錄活性。我們利用docking分析推測出PDPK1 磷酸化的降低，很可能是BPRHIV001 和PDPK1 作用後產生之異位調節所造成。然而， PDPK1 是否為BPRHIV001 唯一的抑制目標，及是否有其它蛋白質的參與，都須進一步的實驗驗證。於此三年提案中，我們首先利用具生物素標幟的BPRHIV001 進行免疫沉澱來鑑別與BPRHIV001 交互作用的蛋白質。接著再利用體外結合試驗和介質表面感應技術來確認BPRHIV001 與鑑定蛋白質之間確實有直接結合。其次，我們將利用體外結合試驗來進一步確認docking 分析之假設，以決定PDPK1 或鑑定蛋白質與BPRHIV001 結合的重要關鍵區域及位點。最後，我們將進行實驗以確定PDPK1 和PI3K/Akt 訊息傳遞路徑在HIV 病毒複製中的影響，並評估其在HIV所感染之巨噬細胞存活週期中所扮演之角色。此外， 我們近期也發現BPRHIV001 可在低濃度下抑制癌細胞生長。因此，我們將評估BPRHIV001作為抗癌藥物的潛力，並探討PI3K/Akt 訊息傳遞和PDPK1 對於BPRHIV001抑制癌細胞的機制。我們的實驗結果不僅可鑑定BPRHIV001 的作用標的，而且還能說明其如何參與PI3K/Akt 訊息傳遞路徑及其在HIV 病毒感染和癌症治療上的可能應用。<br> Abstract: Due to superior therapeutic outcome of combinatorial therapy in HIV-1infection and the emergence of drug-resistance for current drug classes,previously, our laboratory identified a coumarin derivative, BPRHIV001, whichexhibits inhibitory effects on HIV replication through interfering with Tat-mediatedtransactivation. Our preliminary results implicate that BPRHIV001 may interferewith Tat-mediated transactivation through regulation of p300 protein level byreducing Akt phosphorylation and a decrease of phosphorylated PDPK1, awell-known Akt activator. Further docking analysis hypothesizes that the reducedPDPK1 phosphorylation is likely resulted from the allosteric effect of interactionbetween BPRHIV001 and PDPK1. Nevertheless, whether BPRHIV001 inhibitsPDPK1 phosphorylation through direct binding and the potential involvement ofother unidentified factors remain to be validated. In the present three-yearproposal, we will first use immunoprecipitation and biotin-labeled BPRHIV001 toidentify the interacting proteins of BPRHIV001. The direct binding betweenBPRHIV001 and the candidate proteins will then be validated by in vitro bindingassay and the surface plasmon resonance binding assay. Second, to validate thehypothesis of docking analysis, an in vitro binding assay will be performed todetermine the regions and residues critical for binding of PDPK1 and /or theidentified protein(s) to BPRHIV001. The specificity and synergistic effects ofBPRHIV001 as compared to other PDPK1 inhibitors will also be determined.Finally, experiments will be conducted to determine the influences of PDPK1 andthe identified proteins in the PI3K/Akt pathway and HIV replication, and tosubsequently evaluate their role in the life-span of HIV-infected macrophage, arecognized in vivo HIV reservoir. In addition, we recently found that BPRHIV001could also inhibit cancer cell proliferation at nanomolar range. Therefore, we willevaluate the potential of BPRHIV001 as an anti-cancer drug and explore theinvolvement of the PI3K/Akt signaling and PDPK1 in the inhibitory effects ofBPRHIV001 in cancer cells. Our experimental results will not only elucidate thegenuine target of BPRHIV001, but also shed light on its involvement in thePI3K/Akt pathway and the potential application in HIV and cancer therapy.