引發獲得性免疫缺陷綜合癥（AIDS）的人類免疫缺陷病毒（HIV）是一種逆轉錄病毒。過去的十餘年間，高效抗逆轉錄病毒治療療法（HARRT），在抗病毒感染方面取得了很大的成功。高效抗逆轉錄病毒治療療法是一種將多種抗逆轉錄病毒藥物複合的藥物聯用療法。然而，因為病毒的逆轉錄過程極易突變，導致HIV已經可以對大多數使用的抑製藥物產生抗藥性。因此，有越來越多的需要去尋找新型的抗病毒複製機理，例如將人體細胞蛋白作為載體，來達到克服病毒抗藥性的目的。 / HIV-1的複製離不開宿主細胞的剪接因子，例如SR蛋白。選擇性剪接因子ASF/SF2，一個典型的調控pre-RNA剪接的SR蛋白，在HIV-1的pre-mRNA剪接和複製中起到了很重要的調控作用。ASF/SF2和其他SR蛋白一樣，都被丝氨酸/苏氨酸蛋白激酶（SRPK）磷酸化，磷酸化位點位於C端的丝氨酸/苏氨酸結構域（RS domain）。SRPK通過磷酸化來調節ASF/SF2在細胞中的分佈。對於SRPK 和ASF/SF2複合物的結構學和功能學研究指出，ASF/SF2的docking motif和SRPK1的遠離活性位點的docking groove存在很強的相互作用。而這種相互作用是調節磷酸化過程關鍵。所以，在我們的研究過程中，我們希望通過阻斷2個蛋白的相互作用來干擾ASF/SF2的磷酸化，進而抑制其在HIV-1 pre-mRNA剪接過程中的活性。 / 我們採用以結構為基礎的藥物模擬篩選，來選擇潛在的抑制物，達到通過抑制物與docking groove的相互作用來阻斷ASF/SF2和SRPK1的相互作用，以達到抑制磷酸化的目的。我們使用的數據庫來自于ZINC數據庫（UCSF），包括天然產物數據庫和SPECS。我們採用AutoDock Vina 和AutoDock 4.2 二個模擬軟件來栓選數據庫中351473个化合物。并從中選出50個潛在的化合物用作之後的化學生物學測試。體外的激酶活性試驗顯示，6個化合物對ASF/SF2的磷酸化有抑製作用。 / 體外的HIV-1 pre-mRNA剪接實驗顯示，5個化合物在逆轉錄PCR（RT-PCR）中有一定得抑制效果。和DMSO對照組相比，在抑製劑作用下剪接產物的生成被抑制。HIV-1病毒合胞體感染實驗顯示，有一個化合物對病毒的感染起到了一定的抑制作用。 / 其他的測試實驗還在進行中，包括對SRPK1和抑制物複合物的結構研究，從而更好的研究抑制物的作用機理。以及，採用表面等離子共振波譜來進行動力學研究和其他關於化合物在病毒複製過程中的實驗測試。 / Human immunodeficient virus (HIV) is a retrovirus that cause acquired immunodeficiency syndrome (AIDS). Highly active antiretroviral therapy (HAART) is a treatment of HIV infection that uses combinations of antiretroviral drugs and has achieved great success in the past two decades. However, since the reverse transcription process of viral RNA is notoriously prone to error, HIV-1 can acquire resistance to nearly all known inhibitors and has started to develop resistance to HAART. Therefore, there is an ongoing search for new drugs with novel inhibitory mechanism such as targeting cellular proteins essential for HIV-1 replication to overcome drug resistance of the virus. / HIV-1 mRNA undergoes complex splicing and the expression of the integrated HIV-1 provirus is largely dependent on the host’s splicing machinery which assembly requires splicing factors such as serine-arginine rich proteins (SR proteins). Alternative splicing factor/splicing factor 2 (ASF/SF2), a prototypic SR protein that is essential for pre-mRNA splicing, has been shown to play critical roles during HIV-1 pre-mRNA splicing and replication. ASF/SF2, like other SR proteins, is phosphorylated by SR protein-specific kinases (SRPKs) at its C-terminal arginine/serine (RS) domain, which governs its localization and metabolism. Structural and functional studies of SRPK1 in complex with ASF/SF2 has revealed that a docking groove on SRPK1 that is distal to the active site interacts strongly with a docking motif and the RS domain of ASF/SF2, leading to high affinity binding as well as regulating the mechanism of phosphorylation. In this study, we propose that by blocking this interaction, we might interfere the phosphorylation of ASF/SF2 and inhibit its activity during splicing of HIV-1 pre-mRNA. / Structure-based in silico screening method is adopted to identify potential inhibitors that bind to the docking groove of SRPK1 to block the binding and phosphorylation of ASF/SF2. The compound libraries being used include the Natual Products Database and SPECS database from ZINC (UCSF). 351,473 compounds have been screened using the program Autodock Vina as well as Autodock 4.0. Until now 50 potential candidates of inhibitor have been selected for biochemical analyses. In vitro kinase assays showed that six compounds exhibit inhibitory activity against the phosphorylation of ASF/SF2. / To test the effect of the selected inhibitors on the splicing of HIV-1 mRNA, ex vivo splicing assay has been performed. Current results showed that the synthesis of splicing products extracted from drug-treated cells was less efficient when compared to untreated cells. Biological assays testing the inhibitory effects of the compounds on viral infection are currently underway. Our preliminary result suggested that one of the compounds could indeed inhibit HIV-1 viral infection. / Other biochemical and biological analyses including structural study of kinase-inhibitor complexes to understand the mode of inhibition; measurement of binding kinetics using surface plasmon resonance spectroscopy (SPR); and biological assays testing the inhibitory effects of the compounds on replication are underway. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yu, Xiyao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 95-107). / Abstracts also in Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgements --- p.V / TABLE OF CONTENTS --- p.VI / LIST OF FIGURES --- p.IX / LIST OF TABLES --- p.XI / Chapter Chapter I --- : Introduction --- p.1 / Chapter 1.1 --- HIV, HAART and HIV Drug Resistance --- p.2 / Chapter 1.2 --- HIV-1 alternative splicing mechanism --- p.9 / Chapter 1.3 --- SR Protein Family --- p.13 / Chapter 1.4 --- Functional roles of SR protein in HIV pre-mRNA splicing --- p.16 / Chapter 1.5 --- Phosphorylation States of SR Proteins --- p.18 / Chapter 1.6 --- SR protein Kinase --- p.20 / Chapter 1.7 --- Interaction between SRPK1 and ASF/SF2 --- p.23 / Chapter 1.8 --- IDC16 and SPRIN340 --- p.26 / Chapter 1.9 --- Structure-based drug screening --- p.27 / Chapter 1.10 --- AutoDock Suite --- p.29 / Chapter 1.11 --- Kinase-substrate interaction inhibitors --- p.30 / Chapter 1.12 --- Focus of study --- p.34 / Chapter Chapter II --- : Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Bacterial strain --- p.36 / Chapter 2.1.2 --- Antibodies --- p.36 / Chapter 2.1.3 --- Cell line --- p.36 / Chapter 2.1.4 --- Plasmid --- p.36 / Chapter 2.1.5 --- Reagents --- p.38 / Chapter 2.2 --- Expression and purification of Recombinant protein --- p.38 / Chapter 2.3 --- In silico screening of inhibitors --- p.44 / Chapter 2.4 --- Kinase Glo Assay --- p.45 / Chapter 2.5 --- In vitro kinase assay --- p.45 / Chapter 2.6 --- Cell Culture --- p.46 / Chapter 2.7 --- MTT Assay --- p.46 / Chapter 2.8 --- Immunocytochemistry --- p.47 / Chapter 2.9 --- Ex vivo splicing assay --- p.47 / Chapter 2.10 --- Surface plasmon resonance spectroscope --- p.48 / Chapter Chapter III --- : Results --- p.50 / Chapter 3.1 --- In silico screening of inhibitors --- p.51 / Chapter 3.2 --- Selected Compounds Inhibits SRPK1 in Vitro --- p.60 / Chapter 3.2.1 --- Protein purification --- p.60 / Chapter 3.2.2 --- Inhibits ASF/SF2 Phosphorylation by SRPK --- p.66 / Chapter 3.3 --- Surface Plasmon Resonance Binding Competition Assay --- p.76 / Chapter 3.4 --- Inhibitors Alters HIV-1 Alternative Splicing ex Vivo --- p.79 / Chapter 3.5 --- Cytotoxic effect of candidate compound on HeLa cells --- p.84 / Chapter 3.6 --- Nature compound alters ASF/SF2 localization --- p.86 / Chapter Chapter IV --- : Discussion and Conclusion --- p.89 / References --- p.95
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328701 |
| Date | January 2012 |
| Contributors | Yu, Xiyao., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xi, 107 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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