| 摘要: | 經由鈣池調控鈣離子通道(SOC)進入細胞(SOCE)的鈣離子 在生理學和病理學中有著重要的作用，例如免疫反應和癌症進程。參與在鈣池調控鈣離子通道激活過程的兩個主要分子，包含位於內質網膜上的鈣離子感測器，STIM1和鈣離子通道的主要亞基 Orai1。目前已有幾種鈣池調控鈣離子通道抑製劑被開發，然而礙於其細胞毒性，用途僅限於基礎研究。因此，開發具臨床應用潛力的鈣池調控鈣離子通道抑製劑更顯其必要性。本研究採用“老藥新用”和“新藥開發”兩種藥物開發策略來探索具潛力的鈣池調控鈣離子通道抑製劑。同時，也結合影像及螢光共振能量轉移（FRET）技術，建立高通量藥物篩選平台。
首先，通過鈣離子影像和全細胞膜片鉗技術從候選藥物中篩選出具有鈣池調控鈣離子通道抑制能力的2種非類固醇抗發炎藥（阿斯匹靈和舒林酸）和2種新化合物（MPT0M004和MPT0M021）。接著利用即時FRET-TIRF影像系統驗證藥物對鈣池調控鈣離子通道的抑制作用；同時以WST-8細胞生長試劑和LDH細胞毒性試劑檢查藥物對細胞存活的影響。此外，利用插入式細胞培養皿運動試驗檢驗藥物對細胞爬行的影響，結果顯示阿斯匹靈和舒林酸可能通過不同的機制抑制鈣池調控鈣離子內流：阿斯匹靈干擾STIM1-Orai1的交互作用，而舒林酸抑制 STIM1的易位。此外，由以上的實驗中也發現MPT0M004和MPT0M021皆具有成為鈣池調控鈣離子通道抑製劑的潛力，但MPT0M021表現出比MPT0M004高的細胞毒性。接著，本研究建立一個結合FRET的高內涵影像篩選系統平台，並利用已知的配體-受體對 (AMF-AMFR) 及其抑製劑 (E4P) 進行驗證。初步結果顯示此平台具有潛力成為一種新型的高通量藥物篩選系統。
在本研究中，2種潛在的非類固醇抗發炎藥（阿斯匹靈和舒林酸）和2種新化合物（MPT0M004和MPT0M021）被證實具有抑制 SOC 活性的能力。此外，由即時生物影像系統結果提出阿斯匹靈和舒林酸對鈣池調控鈣離子通道的不同抑制機制。最後，本研究建構一個利用FRET螢光技術的高內涵影像篩選系統的新高通量藥物篩選平台。更重要的是，在本研究中發現的具潛力的鈣池調控鈣離子通道抑製劑不僅可作為癌症治療的新策略外，也具有治療與鈣池調控鈣離子通道相關疾病的潛力。
Ca2+ entry from the store-operated Ca2+ channel (SOCE) plays an important role in physiology and pathology, such as immune response and cancer process. Two key molecules are involved in SOC activation, STIM1, a Ca2+ sensor located on the ER membrane and Orai1, a major subunit of the Ca2+ channel. Recently, several SOC inhibitors have been developed and used in basic research, but not clinically due to cytotoxicity. Therefore, it is imperative to develop potential SOC inhibitors. In this study, two drug development strategies, "drug repurposing" and "new drug development" were used to explore potential SOC inhibitors. In addition, a high-throughput drug screening platform combined with FRET had also been established.
First, a total of 2 NSAIDs (aspirin and sulindac) and 2 new compounds (MPT0M004 and MPT0M021) were screened from the candidate drugs with SOC inhibitory ability by Ca2+ imaging and whole-cell patch clamp. The real-time FRET-TIRF imaging system was used to further validate their SOC inhibitory effect. The WST-8 cell proliferation assay and LDH cytotoxicity assay were used to examine the effects on cell survival. In addition, a transwell motility assay was used to investigate the effects on cell motility. The results suggested that Aspirin and Sulindac might inhibit SOCE through different mechanisms: Aspirin interrupts STIM1-Orai1 interaction and Sulindac suppresses STIM1 translocation. Moreover, both MPT0M004 and MPT0M021 had the potential to inhibit SOC, but the MPT0M021 exhibits higher cytotoxicity than MPT0M004. Next, a drug screening platform was established with high-content systems containing FRET technique and examined with known ligand-receptor pairs (AMF-AMFR) and its inhibitors (E4P). The results demonstrated the platform had the potential to be a novel high-throughput drug screening system.
In this study, 2 potential NSAIDs (Aspirin and Sulindac) and 2 new compounds (MPT0M004 and MPT0M021) were verified to have the ability to inhibit SOC activity. Furthermore, differential inhibition mechanisms of Aspirin and Sulindac were proposed by the real-time bioimaging system. At last, a new high throughput drug screening platform with a high content system based on FRET was constructed. More importantly, the potential SOC inhibitors identified in this study could provide additional therapeutics for cancers and SOC-related diseases. |
