分子間藥理作用介面家族應用在磷酸化酵素-藥物-疾病網絡與機制之研究

Abstract

利用所提出之新概念「分子間藥理作用介面家族(molecular pharma-interface family)」結合「區域官能基地圖(site-moiety map, SiMMap)」，以探討蛋白磷酸化酵素抑制劑之結合機制與選擇性是本五年期計劃的主要目標，並希望達成: （一）為上市藥物與中草藥尋找新用途（舊藥新用）。（二）發展多標靶藥物 (multi-target drugs)。（三）建立國人常見癌症之磷酸化酵素-藥物-疾病關聯性網絡資料庫，相信能藉此達到加速藥物發展及增進治療複雜疾病之效果。在前期（第四年）中我們已將相關成果發表於BMC Genomics (影響係數: 4.04) 與PLOS ONE (修訂中，影響係數: 3.53)，並於2014國際生物資訊研討會 (International Conference on Bioinformatics 2014)口頭報告兩篇論文，其中一篇獲得最佳論文獎，是國內少見的殊榮。另外，尚有一篇論文規劃投稿中及一個美國專利申請中。 前期中我們完成了以下事項:(一)提出「同源藥理(Homopharma)」概念，可用於舊藥新用與副作用預測，並據此擴大草藥-天然物-作用蛋白質-疾病(症狀) 網路。(二)成功從舊有作為抗生素的藥物中，發現可作為蛋白磷酸化酵素抑制劑的JMY104 [專利規劃中]，可專一性抑制表皮生長因子受體EGFR與第一型介白素受體之第四號相關激酶IRAK4，並擴大其使用範圍:抗發炎功效篩選的實驗証實，JMY104能有效抑制 (IC50 < 10 μM) NO及IL-6等發炎因子的產生；侵襲試驗 (invasion assay) 結果顯示，JMY104能有效抑制(IC50 ≈ 1.6 μM)惡性乳癌細胞MDA-MB-231的侵襲；細胞病變作用試驗 (cytopathic effect assay) 結果顯示，JMY104在高濃度時 (50 μM) 可抑制登革病毒 (DENV-2) 的複製。(三) 以廣效型蛋白磷酸化酵素抑制劑為基礎，藉由觸及下游蛋白受質辨識錨點而創造S型選擇性抑制劑(type S inhibitors)，成為繼以類黃酮為核心之後的另一類蛋白磷酸化酵素活化與否的開關[美國專利申請中]。已設計出18種S型選擇性抑制劑，並進行大規模蛋白磷酸化酵素抑制活性分析，結果顯示特定S型選擇性抑制劑可將選擇性由>90%降至<2%，証實此概念的可行性，並有助於釐清蛋白磷酸化酵素抑制劑的選擇性機制與探討人類為何需要高達518種蛋白磷酸化酵素。(四)在過去的學術研究與產學合作的基礎上，我們獲得了科技部『產學技術聯盟合作計畫 (產學小聯盟)』的肯定與補助，成立生技及醫藥服務平台於生技產品開發聯盟，提供五大服務: (1)中草藥開發；(2)新藥開發/舊藥新用；(3)藥物包覆及傳輸系統；(4)疾病檢驗暨中草藥功效分析基因晶片試劑套組；(5) 蛋白質酵素/細胞實驗。並成功舉辦「2014 生技及醫藥應用平台: 新藥開發-健康食品-醫藥檢測 產學聯盟研討會」，吸引152人次與20間廠商與會。與葡萄王生技、華聯生技、景凱生技、奎克生技等廠商簽約，共同合作開發產品。 上述研究成果已達到計畫去年之預期目標，初步成果顯示「同源藥理」的概念是可行的，並成功找出JMY104抗生素作為選擇性蛋白磷酸化酵素抑制劑，擴大其應用範圍至抗發炎、抗癌症轉移與抗登革等領域。而S型選擇性抑制劑的開發則有助於探討蛋白磷酸化酵素抑制劑的選擇性機制與人類為何需要高達518種蛋白磷酸化酵素。本年度的工作構想，主要分為三大部分：(一)將蛋白磷酸化酵素由人類擴展至常見致病菌株，以開發新型抗生素。(二)優化S型選擇性抑制劑，並找出其潛在醫療標的。(三)中草藥藥理平台是以系統生物學的方式為中草藥的療效提出科學化的解釋，而將其網路服務化，則使學者與一般民眾對中草藥的作用機理能有更進一步的認識，達到教育普及的目的。

The addressed issue of this project is to explore the binding mechanisms and kinase inhibitor selectivity by the novel concept, molecular pharma-interface family, and site-moiety map (SiMMap). There are three goal of this project including (1) new usages or targets of approved drugs and the Chinese herbs; (2) developments of multi-target drugs; (3) for popular diseases (such as cancers), to build the kinase-drug-disease network. It would be helpful to increase the drug developments and therapeutic effects of complex diseases. In previous period (fourth year), we have published 2 scientific papers (BMC Genomics, impact factor: 4.04; PLOS ONE, impact factor: 3.53), oral presented 2 papers at International Conference on Bioinformatics 2014, and one of them got the Best Paper Award. In addition, we have one submitted paper and one applied U.S.A patent. During the period, we first proposed the “Homopharma” concept for new uses of existing drugs and side-effect prediction. Moreover, we applied “Homopharma” to extend the current kinase-drug-disease network to herb-natural product-target protein-disease network. And we identified an antibiotics JMY104 as a protein kinase inhibitor. Through kinase profiling assays, JMY104 can selectively inhibit EGFR and IRAK4. For expending the indications of JMY104, we performed several assays that EGFR and IRAK4 involved in. For example, we discovered JMY104 can reduce (IC50 < 10 μM) the inflammatory factor production of NO and IL-6 by anti-inflammation assays; inhibit (IC50 ≈ 1.6 μM) MDA-MB-231 malignant breast tumor cell line invasion by invasion assays; disturb dengue virus infection through cytopathic effect assay. In addition, we developed selective type S inhibitors based on promiscuous protein kinase inhibitor staurosporine through targeting substrate recognition pocket for protein kinase switching. Through large scale kinase profiling for 18 developed type S inhibitors, we discovered the inhibitor selectivity can be significantly reduced from > 90% to < 2%. These results supported our hypothesis that substrate site is critical for inhibitor selectivity and were helpful for exploring inhibitor selectivity mechanisms and why human need 518 protein kinases. Furthermore, according to the academic and industrial results, we got the financial supports from “University-Industry Technology Alliance Program” of Ministry of Science and Technology. And we constructed the platform for integrating the resources between academia and industry for product development. In addition, we provides related services and consultation for industry, such as drug development, new uses for existing drugs and herbs, drug delivery, bio-marker identification, diagnostic kits, enzymatic assays and cell-based assays. Moreover, we successfully held the conference on drug development, health food, diagnosis at National Chiao Tung University and joined over 152 participants and 20 biotech companies. These results achieved the anticipated goals and showed that “Homopharma” is efficient for new uses for existing drugs. In addition, identified an antibiotics JMY104 as a selective EGFR and IRAK4 protein kinase inhibitor with anti-inflammation, anti-tumor metastasis and anti-dengue potentials. And type S inhibitors were helpful for kinase inhibitor selectivity mechanisms and exploring why human need 518 protein kinases. The goals of this year are described as following: (1) Develop novel antibiotics by targeting protein kinases in bacteria. (2) Identify new indications for type S inhibitors. (3) Provide the web services about “Herb-natural product-target protein-disease network.”