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

本五年期計劃的主要目標在於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。希望達到三項目標: （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。在上一期（第三年）中共發表了5篇科技論文 (Nucleic Acids Research，影響係數: 8.0；PLoS Comput Biol，影響係數: 4.9；BMC Bioinformatics，影響係數: 3.0；Basic & Clinical Pharmacology & Toxicology，影響係數: 2.1；BMC Genomics，影響係數: 4.4)，並有2個美國專利申請中。 上一期中我們完成了以下事項:(一)蛋白磷酸化酵素-抑制劑-疾病家族地圖資料庫之建構。除可據此解釋蛋白磷酸化酵素抑制劑之選擇性與結合機制外，更可對對癌症等複雜疾病開發更有效的新型抑制劑（多標靶抑制劑）、克服耐藥性與舊藥新用。(二)以廣泛出現於蔬果之類黃酮為核心，在此資料庫的基礎下預測不同方向延伸之類黃酮對蛋白磷酸化酵素之選擇性，最後透過大規模之蛋白質磷酸化酵素－化合物之活性分析以驗證此資料庫。結果顯示大部分的類黃酮均如此資料庫的預測，只有JMY66在選擇性上有偏差，須進一步檢討修正。同時，也確認了包含扁柏雙黃酮 (JMY26)、槲皮素 (JMY27)、兒茶素 (JMY29)、黃芩素 (JMY51)、金雞菊查爾酮苷 (JMY91)在內的多種類黃酮為蛋白磷酸化酵素抑制劑，其選擇性從22%至56%不等，具潛力發展為癌症特效之多標靶抑制劑。並可由KIDFamMap預測其作用位向、結合機制與其選擇性。另外透過同源映射網絡推測JMY26有可能透過抑制IGF1R、KIT、RAF1與LCK等而影響細胞凋亡及透過抑制ABL1與LIMK1而影響軸突導向。(三)發現一個新穎的選擇性蛋白磷酸化酵素抑制劑作用袋點，如類黃酮ZINC12872097與迷迭香酸，均因作用於此袋點而深具選擇性，選擇性分別為6%與3%。且作用於該袋點之化合物(如:迷迭香酸)有機會克服傳統耐藥性突變的影響，我們特將這類的化合物命名為C型 (type C) 選擇性及抗耐藥性抑制劑。為了更進一步驗證此類抑制劑在臨床治療上的效果，已於八月時與淡水馬偕醫院合作申請【迷迭香酸治療肺癌病人與其病理機制探討】的臨床試驗，目前仍在審核中。(四)由單一的結構系統生物實驗室茁壯成涵蓋化學合成與新藥物傳輸系統設計、生物化學、動物實驗與臨床醫學等領域的成熟藥物設計與系統生物團隊，並積極與產業界合作:在與食工所的合作上，發現紅麴中含有化合物JMY103能抑制多種蛋白磷酸化酵素，且對EGFR野生型及抗藥型癌細胞皆具抑制能力，此初步結果將提升原有紅麴商品之新商機；在與傑昇生技的合作上，發現丹蔘含有化合物JMY102能有效抑制非小細胞肺癌的治療標靶EGFR與類風濕性關節炎的潛在治療標靶SYK，可望朝植物藥新藥努力；在與華聯生技的合作上，由細胞與動物實驗初步驗證JMY-P01為新的乳癌生物標記，結合ERBB2等已知生物標記可有效偵測乳癌細胞之生成及類型，將可結合華聯生技研發之晶片製造技術，開發癌症快篩檢驗試劑套組。 上述研究成果已達到計畫去年之預期目標，初步成果顯示相似化合物因具有相似交互作用與錨點而有類似的抑制效果的概念是可行的，並證實類黃酮可作為抑制劑之核心及藉由不同方向的延伸成為蛋白磷酸化酵素活化與否的開關。本年度的工作構想，主要分為三大部分：(一)將天然物由迷迭香酸與類黃酮擴展至藥用植物，建立草藥-天然物-作用蛋白質-疾病(症狀) 網路，使之成為中草藥藥理平台。(二)將蛋白磷酸化酵素-抑制劑-疾病家族地圖資料庫與同源映射網路結合，發展以疾病為主的多標靶蛋白磷酸化酵素抑制劑，並與華聯生技的強項-基因晶片結合，探討蛋白磷酸化酵素抑制劑與細胞行為的關聯，並從中尋找可作為疾病檢驗的潛在生物標記。(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。

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 (third year), we have published 5 scientific papers (Nucleic Acids Research, impact factor: 8.0; PLoS Comput Biol, impact factor: 4.9; BMC Bioinformatics, impact factor: 3.0; Basic & Clinical Pharmacology & Toxicology, impact factor: 2.1; BMC Genomics, impact factor: 4.4) and two applied U.S.A patents. During the period, we first constructed kinase-inhibitor-disease family map database (KIDFamMap). Though KIDFamMap database, we could elucidate the binding mechanisms and selectivity of kinase inhibitors. It also provides the opportunity to develop more effect inhibitors (multi-target drugs) for complex diseases (such as cancers), anti-drug resistance, new uses of existing drugs. Then, we focused on the flavonoid derivatives, which are a class of plant secondary metabolites and have over 8000 derivatives. These derivatives have the same core structure and different extended functional groups. Based on KIDFamMap and kinase-inhibitor family, the interactions and selectivity of flavonoid derivatives were predicted. The large-scale kinase-inhibitor profiling was used to verify the results. The experiments confirmed the predictions, excluding JMY66. The flavonoid derivatives, including hinokiflavone (JMY26), quercetin (JMY27), catechin (JMY29), baicalein (JMY51), marein (JMY91) were confirmed as kinase inhibitors (selectivity from 22% to 56%) and KIDFamMap also provided the binding models and mechanisms of these derivatives. It could be potential inhibitor for multiple targets for cancers. JMY26 may induce cancer cell apoptosis by inhibiting kinase IGF1R, KIT, RAF1 and LCK and affect axon guidance by inhibiting kinase ABL1 and LIMK1 through homologous mapping networks predictions. Moreover, we identified a novel selective binding pocket for kinase-inhibitor interactions and some inhibitors bind this pocket, including flavonoid derivative ZINC12872097 and rosmarinic acid. The kinase selectivity are 6% and 3% for ZINC12872097 and rosmarinic acid, respectively. The inhibitors bind the selective pocket can overcome the effects of drug resistant mutations and are called as Type C inhibitors. For testing the therapeutic effect of clinical trials of these Type C inhibitors, we cooperated with Mackay Memorial Hospital and applied the Institutional Review Board (IRB) of “Treatment of lung cancer with rosmarinic acid”. In addition, we cooperated with other laboratories to form a drug design and systems biology research team involved chemical synthesis, drug delivery, biochemistry, animal experiments, and clinical trials. We cooperated with The Food Industry Research and Development Institute (FIRDI) and found the compound (JMY103) in Monascus purpureus can target many protein kinases and inhibit both EGFR wild-type and EGFR with drug-resistant mutations. With Jason Life Tech Inc., compound (JMY102) of Salvia miltiorrhiza was found to target EGFR related to non-small-cell lung carcinoma (NSCLC) and SYK related to rheumatoid arthritis and can be potential herbal medicine. Cooperated with Phalanx Biotech, we have identified JMY-P01 as new biomarker of breast cancer and verified by cell and animal experiments. By combining known biomarkers, such as ERBB2, and it can be utilized to develop cancer diagnostic kits with Phalanx Biotech to detect breast cancer. Our results show that similar compounds with similar interactions will perform similar inhibitions and use flavonoid derivatives to explore the binding mechanisms and selectivity. The goals of this period are described as following: (1) we will focus on herbs and create herb-natural product-target-disease network and herb pharmacological platform. (2) Combining kinase-inhibitor-disease family map database and homologous mapping network to develop disease-driven kinase inhibitors and study the relationships between kinase inhibitors and cell-behaviors though gene chips by cooperating with Phalanx Biotech. (3) We propose a new kind of selective inhibitors (called Type S inhibitors) by extending broad-spectrum the inhibitors to kinase substrate binding pocket. We will develop highly selective Type S inhibitors and study kinase-inhibitor binding mechanisms.