Hathout Y, Fabris D, Han M S, Sowder R C, Henderson L E, Fenselau C
Structural Biochemistry Center, University of Maryland Baltimore County, 21250, USA.
Drug Metab Dispos. 1996 Dec;24(12):1395-400.
Oxidants targeted toward inactivation of the nucleocapsid zinc finger protein are under development as antiviral agents, especially for use against human immunodeficiency virus. In the present study, electrospray ionization-mass spectrometry is used to follow in situ the progress of the reactions of 2,2'-dithiodipyridine and disulfiram with recombinant nucleocapsid protein p7 (Ncp7) from human immunodeficiency virus-1 at pH 7.4. Both reagents react with the two zinc fingers in the protein, resulting in the ejection of two zinc ions and the formation of oxidized apo-Ncp7 with three intramolecular disulfide bonds. The ejection of zinc by 2,2'-dithiodipyridine occurs in two steps. Alkylation of unreacted cysteine residues with N-ethylmaleimide after a 2-min reaction with 2,2'-dithiodipyridine reveals that the carboxyl-terminal zinc finger is disrupted first. Cys-49, Cys-36, and, to a lesser extent, Cys-39 are all shown to be target residues for initial electrophilic attack. In the reaction of disulfiram with Ncp7, ejection of the two zinc ions also occurs in two steps; however, the fully oxidized apo-Ncp7 is formed more rapidly. Thus, after a 40-min reaction, 45% of native Ncp7 is oxidized by 2,2'-dithiodipyridine, whereas 75% is oxidized by disulfiram.
旨在使核衣壳锌指蛋白失活的氧化剂正作为抗病毒药物进行研发，特别是用于对抗人类免疫缺陷病毒。在本研究中，采用电喷雾电离质谱法原位跟踪2,2'-二硫代二吡啶和双硫仑与来自人类免疫缺陷病毒1型的重组核衣壳蛋白p7（Ncp7）在pH 7.4条件下的反应进程。两种试剂均与该蛋白中的两个锌指发生反应，导致两个锌离子被逐出，并形成具有三个分子内二硫键的氧化脱辅基Ncp7。2,2'-二硫代二吡啶逐出锌的过程分两步进行。在与2,2'-二硫代二吡啶反应2分钟后，用N-乙基马来酰亚胺对未反应的半胱氨酸残基进行烷基化，结果表明羧基末端的锌指首先被破坏。Cys-49、Cys-36以及程度稍轻的Cys-39均被证明是初始亲电攻击的目标残基。在双硫仑与Ncp7的反应中，两个锌离子的逐出也分两步进行；然而，完全氧化的脱辅基Ncp7形成得更快。因此，在反应40分钟后，2,2'-二硫代二吡啶将45%的天然Ncp7氧化，而双硫仑则将75%的天然Ncp7氧化。
