Evans Laboratory of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
J Am Chem Soc. 2011 Jun 29;133(25):9912-22. doi: 10.1021/ja203057z. Epub 2011 Jun 6.
A series of compounds that target reactive metal chelates to the HIV-1 Rev response element (RRE) mRNA have been synthesized. Dissociation constants and chemical reactivity toward HIV RRE RNA have been determined and evaluated in terms of reduction potential, coordination unsaturation, and overall charge associated with the metal-chelate-Rev complex. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were linked to a lysine side chain of a Rev-derived peptide by either EDC/NHS or isothiocyanate coupling. The resulting chelate-Rev (EDTA-Rev, DTPA-Rev, NTA-Rev, and DOTA-Rev) conjugates were used to form coordination complexes with Fe(2+), Co(2+), Ni(2+), and Cu(2+) such that the arginine-rich Rev peptide could mediate localization of the metal chelates to the Rev peptide's high-affinity mRNA binding partner, RRE stem loop IIB. Metal complexes of the extended peptides GGH-Rev and KGHK-Rev, which also contain N-terminal peptidic chelators (ATCUN motifs), were studied for comparison. A fluorescence titration assay revealed high-affinity RRE RNA binding by all 22 metal-chelate-Rev species, with K(D) values ranging from ~0.2 to 16 nM, indicating little to no loss of RNA affinity due to the coupling of the metal chelates to the Rev peptide. Dissociation constants for binding at a previously unobserved low-affinity site are also reported. Rates of RNA modification by each metal-chelate-Rev species were determined and varied from ~0.28 to 4.9 nM/min but were optimal for Cu(2+)-NTA-Rev. Metal-chelate reduction potentials were determined and varied from -228 to +1111 mV vs NHE under similar solution conditions, allowing direct comparison of reactivity with redox thermodynamics. Optimal activity was observed when the reduction potential for the metal center was poised between those of the two principal co-reagents for metal-promoted formation of reactive oxygen species: E°(ascorbate/ascorbyl radical) = -66 mV and E°(H(2)O(2)/hydroxyl radical) = 380 mV. Given the variety of oxidative activities of these metal complexes and their high-affinity binding to the targeted RRE mRNA following coupling to the Rev peptide, this class of metal-chelate-Rev derivatives constitutes a promising step toward development of multiple-turnover reagents for selective eradication of HIV-1 RRE mRNA.
已经合成了一系列靶向 HIV-1 Rev 反应元件（RRE）mRNA 的反应性金属螯合物的化合物。已经确定了离解常数和对 HIV RRE RNA 的化学反应性，并根据还原电位、配位不饱和以及与金属螯合物-Rev 配合物相关的总电荷进行了评估。乙二胺四乙酸（EDTA）、氮三乙酸（NTA）、二亚乙基三胺五乙酸（DTPA）和 1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸（DOTA）通过 EDC/NHS 或异硫氰酸酯偶联连接到 Rev 衍生肽的赖氨酸侧链上。所得的螯合物-Rev（EDTA-Rev、DTPA-Rev、NTA-Rev 和 DOTA-Rev）缀合物与 Fe(2+)、Co(2+)、Ni(2+)和 Cu(2+)形成配位复合物，使得富含精氨酸的 Rev 肽可以介导金属螯合物到 Rev 肽高亲和力 mRNA 结合伴侣 RRE 茎环 IIB 的定位。还研究了含有 N 端肽类螯合剂（ATCUN 基序）的扩展肽 GGH-Rev 和 KGHK-Rev 的金属配合物作为比较。荧光滴定分析表明，所有 22 种金属螯合物-Rev 物种均与 RRE RNA 具有高亲和力结合，K(D) 值范围为0.2 至 16 nM，表明由于金属螯合物与 Rev 肽的偶联，对 RNA 亲和力的几乎没有损失。还报道了在先前未观察到的低亲和力位点结合的解离常数。还确定了每种金属螯合物-Rev 物种的 RNA 修饰速率，范围为0.28 至 4.9 nM/min，但 Cu(2+)-NTA-Rev 的速率最佳。在相似的溶液条件下，测定了金属螯合物的还原电位，范围为-228 至+1111 mV 相对于 NHE，允许与氧化还原热力学直接比较反应性。当金属中心的还原电位位于金属促进形成活性氧物种的两种主要共试剂之间时，观察到最佳活性：E°(抗坏血酸/抗坏血基自由基) = -66 mV 和 E°(H2O2/羟基自由基) = 380 mV。鉴于这些金属配合物的多种氧化活性及其与 Rev 肽偶联后对靶向 RRE mRNA 的高亲和力结合，这类金属螯合物-Rev 衍生物构成了朝着开发用于选择性消除 HIV-1 RRE mRNA 的多轮试剂发展的有希望的一步。
