A Search and Optimization of SRPK1 Inhibitors to Target Various Diseases

• Other Authors: Zeng, Chuyue (author.)
• Format: Others
• Language: English; Chinese
• Published: 2016
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292171

絲氨酸／精氨酸富含蛋白（SR 蛋白）是一類必需的剪接因子蛋白，屬於非核內小分子核糖核蛋白（snRNP）剪接因子家族，參與調節真核細胞 mRNA 的特定以及選擇性剪接。SR 蛋白 C 端含有富含絲氨酸／精氨酸的結構域，其不同的磷酸化狀態與 SR 蛋白細胞內功能緊密相關。現已發現，一類 SR 蛋白激酶（SRPKs）家族主要負責 SR 蛋白的磷酸化修飾，并調節其亞細胞定位以及生理活性。 === SRPK1 是一種典型的 SR 蛋白激酶，參與調節 SR 蛋白的入核以及核內分佈過程，因此對剪接因子的胞內運輸以及剪接功能起到重要的作用。現發現，在多種實體瘤中，SRPK1 具有不正常的蛋白表達水平。SRPK1 表達過量與表達不足均被證實與多種腫瘤發生相關。有研究顯示，抑制SRPK1 蛋白的表達可導致 SRSF1（一種 SR 蛋白）磷酸化水平下降，進而影響一種關鍵的血管新生因子（VEGF）的選擇性剪接，抑制促血管生成 mRNA 異構體（VEGF165）的生成，促進抗血管生成 mRNA 異構體（VEGF165b）的生成，從而抑制腫瘤生長。由此推斷，SRPK1 可作為一種潛在的抗癌靶點。 === 對於 SRPK1 和 SRSF1 複合物的結構學和功能學研究指出， SRPK1 具有一個遠離活性位點的對接槽（docking groove）。該 docking groove 對于 SRPK1 對底物的高親和性結合以及磷酸化過程具有關鍵作用。當對該 docking groove 中的氨基酸進行突變，或引入可與對接槽結合的特異性多肽時，SRPK1 磷酸化底物的功能便會受到影響，導致 SRSF1 不正常的磷酸化水平以及亞細胞分佈。因此，該 docking groove 可作為一個很好的靶點，用於篩選 SRSF1 磷酸化的抑制劑。 === 因此，我們首先採用計算機模擬輔助藥物篩選手段，用 SRPK1 的 docking groove 為靶點來篩選具有抑制 SRPK1 活性潛能的小分子。目前我們已篩選出一種小分子可有效抑制 SRPK1 體外以及細胞內的磷酸化活性。體外的剪接實驗顯示，通過抑制 SR 蛋白磷酸化，該小分子可影響 HIV-1 mRNA 的正常剪接。此外，根據 SRPK1 與其不同底物的複合物結構，我們設計了一些多肽分子，以期靶向結合 docking groove。 目前，我們成功發現一條多肽分子可抑制 SRPK1 的磷酸化活性以及與 SRSF1 的結合。體外剪接實驗及類血管生成實驗顯示，該多肽可抑制 VEGF 促血管剪接異構體的生成并可抑制 HUVEC 細胞形成類血管結構。對於該多肽的化學修飾顯著提高其抑制效率。綜上所述，我的研究證明 SRPK1 的 docking groove 是一個潛在的藥物治療靶點，可通過調節重要基因的選擇性剪接來靶向治療多種腫瘤相關疾病。 === Serine/arginine-rich proteins (SR proteins) constitute a family of non-snRNP splicing factor that are essential for both constitutive splicing and selection of the alternative splice sites during alternative splicing in eukaryotic mRNA. Cellular functions of SR proteins are tightly governed by the phosphorylation state of their C-terminal serine/arginine rich domain (RS domains). SR protein kinases (SRPKs) have been identified as a major family of kinases that phosphorylates SR proteins, altering their cellular distribution and activities. === The prototypic member of SRPK family, SRPK1, shows pivotal role in the regulation of splicing factor trafficking and subsequently splicing by inducing nuclear entry and redistribution of SR proteins inside the nucleus. Abnormal expression levels of SRPK1 have been implicated in multiple types of solid tumor. Either overexpression or downregulation of SRPK1 have been found to associate with different tumorigenesis. Previous studies showed that repression of SRPK1 leads to decreased SRSF1 (a prototypic SR protein) phosphorylation and subsequently inhibits the pro-angiogenic (VEGF165) splicing isoform of the key angiogenesis factor VEGF while upregulating its anti-angiogenic (VEGF165b) isoform. This suggests that SRPK1 may serve as a potential anti-cancer target. === Structural and biochemical studies of SRPK1 and SRSF1 unveil that docking interactions between the substrate and a docking groove on SRPK1 that is distal to its active site is critical for their high affinity binding and regulates the mode of phosphorylation. Interference of this interaction by mutagenesis or peptides results in aberrant phosphorylation and subcellular localization of SRSF1. Thus this docking groove serves as a good target to screen for inhibitors that block the phosphorylation of SRSF1. === Therefore, based on the structures of SRPK1:substrate complexes (PDB ID = 1WBP and 3BEG), we conducted structure-based in silico screening to screen for small molecule inhibitors that bind to the docking groove of SRPK1. One hit compound has been identified to show inhibitory activities toward SRPK1 and phosphorylation of SR protein both in vitro and ex vivo. The inhibited phosphorylation of SR protein successfully altered its function in HIV-1 pre-mRNA alternative splicing. In addition, using structure-based approach we designed peptides that might bind to SRPK1 docking groove and interfere SR protein phosphorylation. One peptide has been identified to show inhibition on SRPK1 phosphorylation activity and binding affinity towards SRSF1, as a result regulated the generation of VEGF pro- and anti- angiogenic isoforms and inhibited tube formation by HUVEC cells. Chemical modifications of this peptide show improvements in inhibitory activity, which provides new lead on peptide drug development against SRPK1. Altogether, my studies suggest that the docking groove of SRPK1 can be a potential therapeutic target for various cancers and diseases through the regulation of alternative splicing of important genes. === Zeng, Chuyue. === Thesis Ph.D. Chinese University of Hong Kong 2016. === Includes bibliographical references (leaves ). === Abstracts also in Chinese. === Title from PDF title page (viewed on …). === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only.