抑制劑－磷酸化酵素－生化途徑－疾病交互作用網絡應用於癌症機制探討與新型抑制劑開發

Abstract

本計畫研究領域為“1-3 Development of novel therapeutics (small molecule drugs) in cancer development, progression and metastasis”，我們將探討蛋白質激酶(Protein kinase)及與其交互作用的 蛋白質、新型激酶抑制劑，以及其在癌症生化途徑中所扮演的角色。蛋白質激酶在生物體中扮演 關鍵的角色，由於常在許多疾病中過度表現，因此常作為治療疾病之藥物標靶。在先前執行的國 衛院計畫中，我們提出“分子間藥理作用介面家族(Molecular Pharma-interface Family)”以及“蛋白質 激酶-抑制劑-疾病家族地圖(Kinase-Inhibitor-Disease Family Map; KIDFamMap)”的創新概念，用來 探討激酶-分子結合機制以及開發激酶抑制劑，根據這些結果我們已發表了6 篇論文(致謝欄中載明國衛院經費補助)在相關重要的期刊上，如Nucleic Acids Research、Scientific Reports, PLoS Computational Biology、BMC Genomics、及PLoS ONE 等，並有2 項申請中的專利，另有一項正 與馬偕紀念醫院人體試驗委員會申請中的臨床試驗。 本計畫將延伸先前計畫來達到兩項目的：(1)建立抑制劑－蛋白質激酶－生化途徑－疾病交互 作用網絡；(2)發展新型激酶抑制劑(Type-C 及Type-S)。本計畫的這二項目標，是針對目前癌症 標靶藥物產生的抗藥性(如gefitinib 對EGFR)及專一性不足造成副作用的急迫性議題，提出突破性 的策略。針對特定疾病(如癌症)我們提出NetPharma 的概念，結合計算系統生物及HomoPharama 建立抑制劑－蛋白質激酶－生化途徑－疾病交互作用網絡的多標靶藥物(目標ㄧ) ，此網絡囊括蛋 白質激酶與其他蛋白質之交互作用、和抑制劑間之關聯性，以及其在癌症生化途徑中所扮演的角 色。另外，我們利用前期國衛院計畫的成果(KIDFamMap)，分析約300 種蛋白質激酶-抑制劑家 族，發現蛋白質激酶的選擇性結合部位，發展新型激酶抑制劑(Type-C 及Type-S) (目標二)。Type- C 抑制劑結合於C 端鉸鏈部位附近的特定位點，而Type-S 抑制劑則同時結合於ATP 結合位置及 蛋白質受質識別位點，由於這些位點距離ATP 結合位置遠，因此新型抑制劑具有較高選擇性。目 前人類共有約518 蛋白質激酶，每一激酶依特定生物功能及結合特性，催化特定蛋白質 (substrate)，我們發展的Type-S 抑制劑就是依此特性針對特定激酶設計的高專ㄧ性小分子抑制 劑。我們已經累積了豐富經驗及優勢來達到計畫目的：1) 我們是全球第一個提出分子介面家族的 團隊；2)提出區域官能基地圖(site-moiety map)，透過官能基偏好(moiety preference)以及結合位的 物化特性來研究蛋白質的結合機制，並應用於藥物設計；3)提出了全面性的分子交互作用網絡來 探討蛋白質交互作用；4)設計了知名且廣為所用的分子嵌合軟體GEMDOCK，可用於探討結合機 制及設計藥物。 在初步結果中，我們已建立了與肺癌及乳癌相關激酶(如EGFR、ALK、KIT 和AURKA)及與 其交互作用之蛋白質網絡。我們將透過分析446 份正常組織以及711 份腫瘤組織之微陣列晶片資 料來探索更多與癌症相關之激酶，以期建構出更全面之疾病交互作用網絡。此外，我們初步建立 了肺癌、淋巴癌和乳癌三組激酶NetPharma，並且合成了3 個Type-C 抑制劑及9 個type-S 抑制 劑，實驗結果顯示，Type-C 及Type-S 抑制劑具有較高之選擇性及較不受抗藥性影響之特性。如 Type-C 抑制劑中的 RA-D1 能同時抑制野生型及抗藥型EGFR，並能降低乳癌細胞的侵襲能力； Type-S 抑制劑中的STU-Trp(Boc) 在40 個受測激酶中僅抑制1 個，具有高選擇性； STU-Glu (Type-S)由於能同時抑制多個參與肺癌生化途徑的激酶，包含INSR、KDR 及FGFR1 (IC50 < 500 nM)，而具有治療肺癌之潛力。我們相信藉由抑制劑－蛋白質激酶－生化途徑－疾病交互作用網 絡，能協助我們針對特定疾病生化途徑上的多個標靶激酶設計出高專一性之Type-C 及Type-S 抑 制劑。我們正致力於合成開發出具有高活性之Type-C 及Type-S 抑制劑，將具有應用至臨床研究 之潛力。我們相信此網絡及新型激酶抑制劑對於基本機制的了解及在癌症治療中克服抗藥性及選 擇性提供有效的解決方案。

The primary focus of our project will be "1-3 Development of novel therapeutics (small molecule drugs) in cancer development, progression and metastasis". We will focus on protein kinases to discover their interacting proteins, inhibitors, and their playing roles in cancer pathways. Protein kinases regulate various biological processes. Their overexpressions in many cancers have led them to be considered as promising drug targets. In our previous NHRI project, we have proposed novel concepts "molecular pharma-interface family" and "Kinase-Inhibitor-Disease Family Map (KIDFamMap)" to study molecular binding mechanisms and discover kinase inhibitors. Based on these results, we have published 6 papers with acknowledgements for the support of NHRI on some journals, such as Nucleic Acids Research, Scientific Reports, PLoS Computational Biology, BMC Genomics、and PLoS ONE. One paper won the Best Paper Award at International Conference on Bioinformatics. In addition, we are applying for two USA patents. One of our lead compounds is currently applying for an IRB approval at Mackay Memorial Hospital (馬偕醫院) for clinical trials. In this project, we will focus on two issues: (1) Establishing the inhibitor-kinase-pathway-disease network; and (2) Developing new types (type-C and type-S) of kinase inhibitors. Currently, there are two major issues in designing kinase inhibitors for cancer treatment: low selectivity and drug resistance. For example, the anti-cancer drug gefitinib has 64 kinase targets in 356 tested kinases, which results in side effects, such as diarrhae or live damage. In addition, gefitinib loses its efficacy for T790M/L858R mutant EGFR. Here, we propose a new concept, namely "NetPharma", to address these two issues. This concept will be used to establish the inhibitor-kinase-pathway-disease network by combining systems biology approaches and HomoPharama (Aim 1). The network will consist of relationships between protein kinases, their interacting proteins, new types of kinase inhibitors, and their roles in cancer pathways. In addition, we will apply and extend KIDFamMap to analyze ~300 protein-inhibitor families for the development of type-C and type-S kinase inhibitors (Aim 2). Type-C inhibitors bind a specific pocket located near the C-terminal hinge region of kinases, while type-S inhibitors occupy the protein substrate recognition site. The new type inhibitors have higher selectivity, because these two regions are located far from the conserved ATP-binding site. Protein kinases regulate biological functions by selectively interacting with physico-chemical properties of protein substrates. Type-S inhibitors are designed by attaching moieties that mimic physico-chemical properties of protein substrates of a protein kinase; therefore, type-S inhibitors have high selectivity. We have accumulated extensive experiences and advantages to address the two issues: 1) We are the first team that propose molecular-interface families; 2) We have established the statistical biophysics methods to infer site-moiety maps (hotspots) of proteins for protein-ligand binding mechanisms and drug discovery; 3) We have proposed the comprehensive molecular interaction networks to study interactomes; and 4) We have developed GEMDOCK, which is a world-wide used docking programs. In our preliminary results, we have established a network containing cancer-relevant kinases (e.g., EGFR and AURKA) and their interacting proteins (substrates) for cancers. We will improve the scope of this network by analyzing microarray data of 446 normal and 711 tumor tissue samples, which can lead to identify cancer-relevant kinases and explore the mechanisms. We have identified three NetPharma kinase groups for lung cancer, lymphoma, and breast cancer and synthesized 3 type-C inhibitors and 9 type-S inhibitors. Our kinase profiling results showed that these inhibitors are selective for certain kinases and are less susceptible to drug resistance. For example, a type-C inhibitor, RA-D1, can maintain activities for drug-resistant EGFR and decrease the invasion of breast cancer cells. STU-Trp(Boc), a type-S inhibitor, only inhibits 1 kinase out of 40 kinases tested. STU-Glu (type-S) has potential for treating lung cancer because it inhibits multiple kinases involved in lung cancer pathways, such as INSR, KDR, and FGFR1 (IC50 < 500 nM). By using the inhibitor-kinase-pathway-disease network, we can design type-C and type- S inhibitors that specifically target multiple kinases that participate in a specific disease pathway. We are working to discover and synthesize type-C and type-S kinase inhibitors with high potency. We believe that the network and the new types of kinase inhibitors are useful to understand basic mechanism and overcome drug resistance and kinase selectivity during cancer treatments.