Biophysical characterization of zinc ejection from HIV nucleocapsid protein by anti-HIV 2,2'-dithiobis[benzamides] and benzisothiazolones.
HIV nucleocapsid protein (NCp7) has been suggested as a possible target for 2,2'-dithiobis-[benzamide] and benzisothiazolone agents that inhibit viral replication in infected cells (Rice et al. Science 1995, 270, 1194-1197). The solution behavior of these compounds and the mechanistic events leading to removal of Zn from HIV nucleocapsid protein in vitro has been studied by electrospray ionization mass spectrometry, 500 MHz one- and two-dimensional nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy. We demonstrate that (1) Zn ejection is accompanied by formation of covalent complexes formed between the 2,2'-dithiobis[benzamide] monomers and Cys residues of Zn-depleted NCp7, (2) the rate of Zn ejection is faster for the C-terminal Zn finger and slower for the N-terminal finger, (3) Zn ejection results in a loss of structural integrity of the NCp7 protein, and (4) there is no appreciable interaction between a nonreactive isostere of the lead 2,2'-dithiobis[benzamide] and NCp7 in buffered aqueous solution. These findings are discussed in terms of the mechanism of action of Zn ejection by aromatic 2,2'-dithiobis[benzamides].
Loo
J A
JA
Parke-Davis Pharmaceutical Research, Division of Warner Lambert Company, Ann Arbor, Michigan 48105, USA.
Holler
T P
TP
Sanchez
J
J
Gogliotti
R
R
Maloney
L
L
Reily
M D
MD
D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000737:chemistry / Q000494:pharmacology*; D001549:Benzamides / Q000737:chemistry / Q000494:pharmacology*; D002213:Capsid / Q000187:drug effects*; D036022:Capsid Proteins; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry / Q000494:pharmacology*; D015683:Gene Products, gag / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012996:Solutions; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology*; D014764:Viral Proteins; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
C101893:2,2'-dithiobis(N-(1-carboxy-2-methylbutyl)benzamide); D019380:Anti-HIV Agents; D001549:Benzamides; D036022:Capsid Proteins; D004220:Disulfides; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D012996:Solutions; D013844:Thiazoles; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C101894:N-(1-carboxy-2-methylbutyl)benzisothiazolone; C015699:1,2-benzisothiazoline-3-one