由化學、造影、藥物設計探索雙亞硝基鐵錯合物及活性氮化合物的生理功能-由化學、造影、藥物設計探索雙亞硝基鐵錯合物及活性氮化合物的生理功能( I )

Abstract

雙亞硝基鐵化合物(DNIC)已經被證實是內生性一氧化氮的攜帶者。然而，其生理功能、生物 醫學的應用以及所誘發的訊號傳導性質仍是相當的不清楚。在前期的跨領域整合型計畫中,本研究 團隊已經證實可藉由不同的鍵結配位基來調控DNIC 釋放NO 的能力且DNIC 可進入細胞內釋放 NO。經由peptide-bound DNICs/RRE 研究中我們確認{Fe(NO)2}9 比較傾向與含有chelate-cysteine 的peptide 鍵結。藉由DNIC 與heme 模型(FeNCP) 的研究，我們也確認DNIC 上的一氧化氮可轉 移至代表血基質蛋白的異位紫質鐵錯合物上。同時本團隊也發展了針對生物分子以及金屬離子在 in vitro 及in vivo 的顯影技術，特別是發展了對於外生性及內生性的一氧化氮分子均有造影能力的 探針。在本研究計畫中我們將專注於DNIC 在化學、化學生物學及生物醫學應用上的探討。並結 合heme 的模擬研究，深入瞭解DNIC、NO、及NO 所衍生的活性氮分子的生化機能。我們將透 過合成水溶性DNIC、水溶性FeNCP 結合辨識胜肽(targeting peptide)及顯影分子進行細胞實驗。 除了DNIC 及FeNCP 生物活性及藥物動力學研究外，細胞實驗也將提出DNIC 如何調控細胞內鐵 離子的吸收以及其濃度的平衡的模型，並發展不同的合成策略來合成peptide-bound DNICs/RREs 用來研究在IscA 以及IscS 蛋白質存在下其鐵硫簇修復的機制。經由具專一性活性氮化物的MRI 或光學探針將可解析DNIC 與FeNCP 在生物體內所導致的活性氮化物濃度變化、動態傳遞路徑、 及所導致的生理反應。在生物醫學方面，利用鍵結特殊生物辨釋分子的水溶性DNICs/RREs 結合 分子造影技術，將可應用於非侵入性的心血管疾病臨床前的診療。另外，此些分子經由釋放NO 直接或間接造成癌細胞凋亡的可行性也將在此跨領域計畫中確認。

Dinitrosyl iron complexes (DNICs) have been established as a potential endogenous NO carrier. However, the biological functions and bio-medical applications of DNICs/RREs as well as the signaling pathways induced by NO released from DNICs/RREs are poorly understood. In the previous interdisciplinary project, Prof. Liaw’s group has demonstrated that the ability of NO releasing from DNICs can be modulated by the coordinated ligands. From cellular studies, DNIC has been confirmed to be delivered into cells and NO has been released inside the cells. Liaw's group also established the preference of forming {Fe(NO)2}9 DNIC motif from chelatable cysteine-containing peptides over monodentate cysteine-containing peptides. On the heme model studies, thoroughly understanding of nitrite reduction promoted by FeNCP have been achieved by Hung’s group. Corraboration studies using nitrite bound DNIC and iron N-confused porphyrin (FeNCP) also established that facile nitrite reduction and nitric oxide transfer from DNIC to heme model compound can occur. Meanwhile, Professor Wang's group has established the in vitro and in vivo molecular imaging techniques for biomolecules and metal ions. Wang's group also developed NO fluorescent probes which provide the capability of exogeneous and endogenous NO detection. Based on the solid results from the previous interdisciplinary project, we will focus on the chemical biology, and bio-medical applications of DNICs in this grant proposal. The DNIC chemistry will also connect with FeNCP heme model studies and further combine with newly developed NO fluorescent/MRI probes to decode the cellular responses associated with DNICs, FeNCP, and other related reactive nitrogen species (RNS). The objectives of this project will be (1) using de novo peptide-bound DNICs/RREs as the probes to understand how DNICs regulating the Fe uptake and cellular iron homeostasis, (2) combining MRI/optical RNS probes and the bio-related water-soluble DNICs/RREs and FeNCP containing targeting molecules to understand the roles of DNICs and FeNCP in stimulating the NO concentration changes and RNS production under physiological and physiopathological situations in cells and in animal model such as zebrafish and mice, (3) examining the toxicity and studying the pharmacokinetics to evaluate the potential of applying these molecules on in vivo preclinical diagnosis and therapy of thrombus or using as antitumor agents.