藉由同源藥理及激酶-抑制劑-疾病家族設計激酶抑制劑

Abstract

蛋白激酶在調節細胞功能中扮演了很重要的角色，所以當激酶活性異常時，可能導致增殖性疾病，例如：癌症、腫瘤血管生成等。雖然現在已有三萬多個激酶抑制劑被開發出來，往往因為抑制劑效果不佳或副作用過大，而無法通過臨床試驗。截至2016年6月為止，已經有33個激酶抑制劑通過美國食品暨藥物管理局臨床試驗，原因在於人類的518個激酶都具有演化上高度保留的三磷酸腺苷結合位置，並且目前對於激酶的結合機制了解有限。在此研究中，為了克服現階段激酶抑制劑效果不佳或副作用過大，我們提出一個設計抑制劑的模型。此模型結合同源藥理概念及激酶-抑制劑-疾病家族資料庫來設計抑制劑，由於激酶-抑制劑-疾病家族資料庫利用數個錨點來呈現蛋白質-抑制劑間的物化特性及喜好的官能基，並考量激酶在疾病中扮演的角色，有助於提高臨床通過率。而目前也尚未有研究提出以大規模激酶活性測試資料、組學數據結合結構上的分析來設計抑制劑。 我們利用同源藥理發現JMY11與AURKA、CSNKA1及GSK3B為同一個同源藥理家族，基於同一個家族中，化合物結構相似，蛋白質結合環境相似，交互作用力相似，再加上JMY11符合激酶-抑制劑-疾病家族裡2bmc的多個錨點，我們認為JMY11為潛在抑制劑，經激酶活性試驗證實JMY11確實對激酶AURKA有抑制效果。在64個測定的蛋白激酶中，JMY11除了抑制了AURKA也有效抑制了另外12個激酶的活性。並經由實驗室先前分析21種癌症腫瘤的基因晶片發現JMY11有機會藉由抑制AURKA的活性來治療卵巢癌、子宮頸癌、乳癌、淋巴癌及肝癌，其中根據中華民國衛生福利部統計，在所有癌症種類中，男女死亡率第二高的癌症，均為肝癌。因此，我們進一步將JMY11測試在肝癌細胞株，並以公開資料庫輔助分析JMY11影響的基因，了解JMY11可能藉由抑制特定激酶，降低激酶下游基因表現量來抑制癌細胞增生或轉移等。以AURKA 激酶為例，AURKA 激酶參與了細胞週期調控，在肝癌病患中AURKA 激酶在腫瘤的表現量為正組織的3.5倍以上，分析基因表現測試結果得知JMY11對肝癌細胞株AURKA 激酶的表現量有下降的趨勢，透過文獻我們知道當AURKA 激酶過度磷酸化，會導致下游的MTDH及MMP9過度表現，進而促進癌細胞的轉移；若能藉由JMY11來抑制AURKA 激酶的磷酸化，將可能可以抑制癌細胞的轉移。另外，現今研究已知一部分的肝癌病患是罹患肝炎，導致肝硬化，進一步惡化成肝癌，而肝炎病毒代謝產物HBx會導致YWHAB的表現量升高，YWHAB的表現量升高會抑制細胞凋亡，由基因表現測試結果我們知道JMY11能使YWHAB的表現量下降。若能藉由JMY11降低YWHAB的表現量，將可能減少癌細胞逃離凋亡。 在此研究中，我們提出了一個可以用來設計激酶抑制劑的模型，並且成功發現了抑制劑JMY11。此外，由基因表現結果分析得知在肝癌中，JMY11可能藉由抑制特定激酶，調控特定路徑來抑制癌細胞轉移。在未來，我們將透過細胞移動實驗及侵襲實驗來驗證JMY11確實能夠透過抑制特定激酶來調控激酶下游基因，達到抑制癌細胞遷移的目的。

Protein kinase (PK) plays a very important role in modulating cellular functions. The abnormal kinase activity often causes proliferative diseases, such as cancer and tumor angiogenesis. Though more than 30,000 kinase inhibitors have been developed so far, the clinical trials with these inhibitors failed often due to their poor inhibitory effects or overly strong side effects. By June 2016, the U.S. Food and Drug Administration had approved the safety and effectiveness of 33 kinase inhibitors through clinical trials. The 518 human protein kinases share an evolutionary conserved ATP-binding site. But the binding mechanism of kinases still remains to be understood in greater depth. This study proposed an inhibitor-designing model to cope with the poor effects or overly strong side effects of kinase inhibitors. This model designed inhibitors by combining the concept of Homopharma and the KIDFamMap (kinase-inhibitor-disease family maps) database. The KIDFamMap database shows the kinase-inhibitor physicochemical properties and moiety preferences by several statistical anchors and evaluates the role of kinases in diseases, which thus improves the pass rate in clinical trials. Up till now, a few studies have proposed designing inhibitors with substantial kinase activity testing data, omics data and structural analysis. By using Homopharma, we identified that JMY11 can inhibit AURKA, CSNKA1 and GSK3B which are in the same Homopharma family. Generally, the compounds have similar structure, the protein binding environment is similar and the interactive force is similar in the same family. In addition, JMY11 matched multiple anchors of AURKA (PDB code 2BMC) in the kinase-inhibitor-disease family. Based on these results, this study considered JMY11 as a potential inhibitor for AURKA kinase-inhibitor family. Furthermore, we tested JMY11 on 64 selected kinases from 518 human protein kinases. Among these 64 tested protein kinases, JMY11 effectively inhibited the activity of AURKA and 12 other kinases. According to the previous laboratory analysis on the gene chips of 21 cancers and tumors, there was a chance that JMY11 could treat the ovarian cancer, cervical cancer, breast cancer, lymph cancer and liver cancer by inhibiting the activity of AURKA. As shown by Ministry of Health and Welfare of Taiwan, among all kinds of cancers, the liver cancer mortality rate was the second highest both for men and women. Therefore, this study further tested the effect of JMY11 on the human hepatoma cell line and analyzed the genes influenced by JMY11 with the help of open databases. As learned from the test and analysis, JMY11 might inhibit the cancer cell proliferation or metastasis by inhibiting a specific kinase and reducing its downstream gene expression. For example, AURKA participated in the cell-cycle regulation. For the patients of liver cancer, the expression of AURKA on the cancer was over 3.5 times more than that of the positive tissue. The analysis on the gene expression test revealed that JMY11 reduced the expression of AURKA in the human hepatoma cell line. The literature showed that the AURKA hyper-phosphorylation would result in the over-expression of the downstream MTDH and MMP9 and thus promote the metastasis of cancer cells. If JMY11 could be used to inhibit the AURKA phosphorylation, the metastasis of cancer cells would be probably controlled. Furthermore, according to current research, some liver cancer patients first suffered from hepatitis which later led to cirrhosis and further deteriorated into liver cancer. The hepatitis virus metabolite HBx could increase the expression of YWHAB and the increased YWHAB expression would inhibit the cell apoptosis. The gene expression test demonstrated that JMY11 was able to decrease the YWHAB expression. If JMY11 was used to reduce the YWHAB expression, the cancer cell apoptosis would not be inhibited. This study applied a model that could be used to design kinase inhibitors and successfully discovered the inhibitor JMY11. Moreover, as shown by the gene expression analysis, JMY11 might control the liver cancer cell metastasis by inhibiting specific kinases and regulating specific pathways. Future studies will conduct the cell migration test, the cell invasion test and the cytotoxicity test to verify that JMY11 can really modulate the downstream genes by inhibiting specific kinases and finally control the cancer cell metastasis and kill cancer cells.