Evans Laboratory of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
J Am Chem Soc. 2012 Feb 22;134(7):3396-410. doi: 10.1021/ja208791f. Epub 2012 Feb 10.
A series of compounds that target reactive transition-metal chelates to somatic angiotensin converting enzyme (sACE-1) have been synthesized. Half-maximal inhibitory concentrations (IC(50)) and rate constants for both inactivation and cleavage of full-length sACE-1 have been determined and evaluated in terms of metal chelate size, charge, reduction potential, coordination unsaturation, and coreactant selectivity. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and tripeptide GGH were linked to the lysine side chain of lisinopril by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride/N-hydroxysuccinimide coupling. The resulting amide-linked chelate-lisinopril (EDTA-lisinopril, NTA-lisinopril, DOTA-lisinopril, and GGH-lisinopril) conjugates were used to form coordination complexes with iron, cobalt, nickel, and copper, such that lisinopril could mediate localization of the reactive metal chelates to sACE-1. ACE activity was assayed by monitoring cleavage of the fluorogenic substrate Mca-RPPGFSAFK(Dnp)-OH, a derivative of bradykinin, following preincubation with metal chelate-lisinopril compounds. Concentration-dependent inhibition of sACE-1 by metal chelate-lisinopril complexes revealed IC(50) values ranging from 44 to 4500 nM for Ni-NTA-lisinopril and Ni-DOTA-lisinopril, respectively, versus 1.9 nM for lisinopril. Stronger inhibition was correlated with smaller size and lower negative charge of the attached metal chelates. Time-dependent inactivation of sACE-1 by metal chelate-lisinopril complexes revealed a remarkable range of catalytic activities, with second-order rate constants as high as 150,000 M(-1) min(-1) (Cu-GGH-lisinopril), while catalyst-mediated cleavage of sACE-1 typically occurred at much lower rates, indicating that inactivation arose primarily from side chain modification. Optimal inactivation of sACE-1 was observed when the reduction potential for the metal center was poised near 1000 mV, reflecting the difficulty of protein oxidation. This class of metal chelate-lisinopril complexes possesses a range of high-affinity binding to ACE, introduces the advantage of irreversible catalytic turnover, and marks an important step toward the development of multiple-turnover drugs for selective inactivation of sACE-1.
已经合成了一系列靶向活性过渡金属螯合物的化合物，用于躯体血管紧张素转换酶（sACE-1）。已经确定了半最大抑制浓度（IC（50））和全长 sACE-1 的失活和切割的速率常数，并根据金属螯合物的大小、电荷、还原电位、配位不饱和性和共反应物选择性进行了评估。乙二胺四乙酸（EDTA）、氮三乙酸（NTA）、1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸（DOTA）和三肽 GGH 通过 1-乙基-3-[3-（二甲基氨基）丙基]碳二亚胺盐酸盐/N-羟基琥珀酰亚胺偶联连接到赖脯酸的赖氨酸侧链上。由此得到的酰胺连接的螯合赖脯酸（EDTA-赖脯酸、NTA-赖脯酸、DOTA-赖脯酸和 GGH-赖脯酸）缀合物与铁、钴、镍和铜形成配位复合物，使得赖脯酸可以介导活性金属螯合物定位到 sACE-1。ACE 活性通过监测荧光底物 Mca-RPPGFSAFK（Dnp）-OH 的切割来测定，Mca-RPPGFSAFK（Dnp）-OH 是缓激肽的衍生物。金属螯合赖脯酸化合物预孵育后，sACE-1 的浓度依赖性抑制揭示了 Ni-NTA-赖脯酸和 Ni-DOTA-赖脯酸的 IC（50）值分别为 44 至 4500 nM，而赖脯酸的 IC（50）值为 1.9 nM。更强的抑制与附着的金属螯合物的更小尺寸和更低负电荷相关。金属螯合赖脯酸化合物对 sACE-1 的时程失活揭示了显著的催化活性范围，二阶速率常数高达 150,000 M（-1）min（-1）（Cu-GGH-赖脯酸），而催化剂介导的 sACE-1 切割通常以低得多的速率发生，表明失活主要来自侧链修饰。当金属中心的还原电位定位于 1000 mV 左右时，sACE-1 的最佳失活得到观察，这反映了蛋白质氧化的难度。这类金属螯合赖脯酸复合物对 ACE 具有高亲和力结合，引入了不可逆催化周转的优势，并标志着朝着开发用于选择性失活 sACE-1 的多轮药物的重要一步。
