蛋白質間交互作用小分子抑制劑選擇性促使化學藥劑抗藥性或缺乏半胱胺酸蛋白酶-3之癌細胞死亡

Small-molecule inhibitors of protein-protein interactions selectively induce chemoresistant/caspase-3-deficient cancer cell death

陳世勳(Shih-Hsun Chen)

指導教授 ： 楊進木 ； 梁博煌

國立交通大學/生物科技學院/生物科技系所/博士(2014年)

https://doi.org/10.6842/NCTU.2014.00025

摘要

蛋白質與蛋白質間的交互作用對於生物體內的反應過程是不可或缺的，因此蛋白質複合體常常被認為是有用的藥物標的。雖然鎖定蛋白質間的作用介面對於藥物研發具有非常大的潛力，但研發可阻斷蛋白質間交互作用的小分子藥物是一項巨大的挑戰。根據我們之前的研究得知，對於臨床tubulin-binding試劑引起之抗藥性及過度表現CCT-β的腫瘤而言，β-tubulin:CCT-β蛋白質複合體可作為一個有效的藥物治療標的。而阻斷XIAP:CASP7蛋白質複合體的形成亦是一個有效及安全的新穎策略，可選擇性地促使缺乏或少量表現半胱氨酸蛋白酶-3之癌細胞死亡。因此，我們發展出一個創新的虛擬藥物篩選方法—multiple-sites (or modes) virtual screening with site-moiety maps—研發可同時佔據多個凹槽(pockets)或位置(modes)來破壞蛋白質間交互作用的小分子抑制劑。此外，我們發現三個在CCT-β與β-tubulin形成交互作用時扮演重要角色的關鍵胺基酸(K250, F252, and R293 in CCT-β)及小分子佔據I-Lys site促使XIAP與CASP7分離的異位調節機制(allosteric mechanism)。
我們利用一個小分子同時佔據多個凹槽(pockets)或位置(modes)的策略，成功地研發出第一個非共價鍵及高效促使HEK-293細胞凋亡(EC50= 4.6 M)的小分子抑制劑(R379603)。實驗的結果證實，R379603同時佔據在兩個全新且獨立位於β-tubulin:CCT-β交互作用介面的凹槽，進而阻斷β-tubulin與CCT-β間的交互作用而引起caspase-dependent的訊息傳遞。值得注意的是，相較於正常細胞及親代的乳癌細胞(parental MCF-7 cells)而言，R379603對於紫杉醇抗藥性的乳癌細胞(paclitaxel-resistant MCF-7 breast cancer cells)具有較高的敏感性。此外，我們也成功地研發出第一個可逆地佔據在I-Lys site並異位調控促使XIAP與CASP7分離的小分子抑制劑(643943)。643943 造成XIAP的釋出及活化半胱氨酸蛋白酶-7(caspase-7)的活性選擇性地誘發缺乏(deficient)或低表現(down-regulated)半胱氨酸蛋白酶-3(caspase-3)之癌細胞凋亡。另外，結合使用低濃度(≦5 μM)的643943即有效地使已產生化學藥物(如:紫杉醇)抗藥性的癌細胞恢復其對此藥物的敏感性。我們相信，未來對於研發具有潛力的小分子抑制劑或藥物，multiple-sites (or modes)的策略提供了一個新穎及有用的方法。

關鍵字

多部位結合 ； 藥物模擬篩選 ； 蛋白質-蛋白質交互作用 ； 抗藥性 ； 抗癌藥物 ； 細胞凋亡

並列摘要

Protein-protein interactions (PPIs) are essential for biological processes and thus often considered as potentially pharmaceutical drug targets. Targeting the interfaces between proteins has huge therapeutic potential, but discovering small-molecule drugs which block PPIs is an enormous challenge currently.
According to our previous studies, β-tubulin:CCT-β complex can serve as an effective chemotherapeutic target for treating clinical tubulin-binding agent-resistant or CCT-β-overexpressing tumors and disrupting XIAP:CASP7 complex represents an effective and safe novel strategy for selectively killing CASP3-deficent cancer cells. We have developed a novel multiple-sites (or modes) virtual screening strategy with site-moiety maps to discover small molecules which can be simultaneously fitted into multiple sites (or modes) to disrupt PPIs. Moreover, we identified three key residues in CCT-β, K250, F252 and R293, which play an important role in interacting with β-tubulin and binding of the small molecules to I-Lys site induces disassociation of XIAP:CASP7 through an allosteric mechanism.
Our method discovers the first potent and non-covalent inhibitor, R379603, efficiently induces apoptosis of HEK-293 cells with an EC50 at 4.6 M via targeting two previously uncharacterized and independent sites to block β-tubulin:CCT-β interface and triggering caspase-dependent signaling cascades. Notably, R379603 causes a more severe apoptosis of paclitaxel-resistant MCF-7 breast cancer cells in vitro and in cultured cells due to an increased level of CCT-β as compared with its parental MCF-7 cells. We also discovered a small molecule inhibitor (643943) which can reversibly bind to I-Lys site, an allosteric site away from the interface of the linker region between BIR1 and BIR2 domains of XIAP and CASP7 active site. This compound thus releases XIAP constraint and activated CASP7 to selectively kill CASP3 down-regulated (CASP3/DR) cancer cells. Besides, 643943 effectively sensitizes chemo-resistant cancer cells to chemotherapy at a low-micromolar concentration (≦5 μM). We believe that multiple-sites (or modes) strategy provides an alternative and useful method for discovering potential inhibitors and binding mechanisms for pharmaceutical targets.

並列關鍵字

Multiple sites ； Virtual screening ； Protein-protein interaction ； Drug-resistance ； Anti-cancer ； Cell apoptosis

參考文獻

1 Yang, J. M., Chen, Y. F., Shen, T. W., Kristal, B. S. & Hsu, D. F. Consensus scoring criteria for improving enrichment in virtual screening. Journal of chemical information and modeling 45, 1134-1146, doi:10.1021/ci050034w (2005).

2 Gigant, B. et al. Structural basis for the regulation of tubulin by vinblastine. Nature 435, 519-522, doi:10.1038/nature03566 (2005).

3 Lin, Y. F., Tsai, W. P., Liu, H. G. & Liang, P. H. Intracellular beta-tubulin/chaperonin containing TCP1-beta complex serves as a novel chemotherapeutic target against drug-resistant tumors. Cancer research 69, 6879-6888, doi:10.1158/0008-5472.CAN-08-4700 (2009).

4 Llorca, O. et al. Analysis of the interaction between the eukaryotic chaperonin CCT and its substrates actin and tubulin. Journal of structural biology 135, 205-218, doi:10.1006/jsbi.2001.4359 (2001).

5 Yang, J. M. & Chen, C. C. GEMDOCK: a generic evolutionary method for molecular docking. Proteins 55, 288-304, doi:10.1002/prot.20035 (2004).