探討抑製劑的結構改變如何影響CASP7:XIAP 之間的相互作用並選擇性地殺死CASP3表現量低的癌細胞與具有抗藥性癌細胞

Abstract

先前我們發現I-Lys可藉由iodomethyl ketone來烷基化caspase-7 (CASP7)上的Cys246，從而阻止CASP7與X-linked inhibitor of apoptosis protein (XIAP) 的蛋白-蛋白交互作用(PPI)並釋放活化的CASP7，進而有選擇性地殺死具CASP7:XIAP蛋白複體累積的癌細胞，其通常為CASP3表現量低(CASP3/DR)的癌細胞 [1]. 因CASP3表現量減低使得細胞凋亡也減少，從而讓癌細胞產生抗藥性。I-Lys提供了一個有效且安全的治療方法，因正常細胞無CASP7:XIAP蛋白質複體。除不可逆I-Lys，之前我們實驗室的博士畢業生陳世勳透過電腦模擬從Sigma化合物庫找到一個可逆的抑制劑，643943。本篇論文旨在測試I-Lys及643943的類似物對於毒殺MCF-7乳癌細胞的結構-功能關係及引發癌細胞凋亡的機制。我們發現較弱的離去基會減弱I-Lys類似物的活性而保護基影響不大。643943與 Staurosporine (STS) 的結合對於殺死具有Taxol抗性的MCF-7乳癌細胞(7TR)有加成作用。我們發現643943的類似物-0909所引發的細胞凋亡機制與643943不同，乃通過MAPK來調控細胞凋亡，並且活化許多caspases，而643943 主要活化CASP7。我們的研究提供了一個對於CASP3表現量低和抗藥性癌細胞的治療方法。

Previously, we demonstrated that disruption of protein-protein interaction (PPI) of caspase-7 (CASP7) and X-linked inhibitor of apoptosis protein (XIAP) by using I-Lys with an iodomethyl ketone warhead to alkylate Cys246 of CASP7, selectively killed cancer cells with accumulation of CASP7:XIAP complexes, which are caspase-3 down-regulated (CASP3/DR) [1]. CASP3/DR frequently confers resistance to cancer therapy due to reduced apoptotic machinery. This represents an effective and safe strategy for chemotherapy because the CASP7:XIAP complexes are not accumulated in normal cells. Unlike the I-Lys irreversible PPI inhibitor, through compute virtual screening, a reversible PPI inhibitor, 643943, a compound from Sigma compound bank was discovered by Chen, S. H. et al. In this thesis, I aimed to determine the structure-activity relationship of the I-Lys and 643943 analogues and the induced apoptotic mechanisms. We found that in I-Lys analogues, the weaker leaving group reduced the activity, but C-terminal protecting groups did not affect so much. We also found synergistic effect of 643943 combining STS in killing 7-TR, a Taxol-resistant MCF-7 cells. The analogue 0909 adopts different mechanism to induce apoptosis in MCF-7 cells through MAPK family to activate various caspases, unlike 643943 that mainly activated CASP7. Our studies demonstrated a promising therapeutic strategy against CASP3/DR and multidrug resistant cancers.