The in vitro ejection of zinc from human immunodeficiency virus (HIV) type 1 nucleocapsid protein by disulfide benzamides with cellular anti-HIV activity.

Abstract

Several disulfide benzamides have been shown to possess wide-spectrum antiretroviral activity in cell culture at low micromolar to submicromolar concentrations, inhibiting human immunodeficiency virus (HIV) type 1 (HIV-1) clinical and drug-resistant strains along with HIV-2 and simian immunodeficiency virus [Rice, W. G., Supko, J. G., Malspeis, L., Buckheit, R. W., Jr., Clanton, D., Bu, M., Graham, L., Schaeffer, C. A., Turpin, J. A., Domagala, J., Gogliotti, R., Bader, J. P., Halliday, S. M., Coren, L., Sowder, R. C., II, Arthur, L. O. & Henderson, L. E. (1995) Science 270, 1194-1197]. Rice and coworkers have proposed that the compounds act by "attacking" the two zinc fingers of HIV nucleocapsid protein. Shown here is evidence that low micromolar concentrations of the anti-HIV disulfide benzamides eject zinc from HIV nucleocapsid protein (NCp7) in vitro, as monitored by the zinc-specific fluorescent probe N-(6-methoxy-8-quinoyl)-p-toluenesulfonamide (TSQ). Structurally similar disulfide benzamides that do not inhibit HIV-1 in culture do not eject zinc, nor do analogs of the antiviral compounds with the disulfide replaced with a methylene sulfide. The kinetics of NCp7 zinc ejection by disulfide benzamides were found to be nonsaturable and biexponential, with the rate of ejection from the C-terminal zinc finger 7-fold faster than that from the N-terminal. The antiviral compounds were found to inhibit the zinc-dependent binding of NCp7 to HIV psi RNA, as studied by gel-shift assays, and the data correlated well with the zinc ejection data. Anti-HIV disulfide benzamides specifically eject NCp7 zinc and abolish the protein's ability to bind psi RNA in vitro, providing evidence for a possible antiretroviral mechanism of action of these compounds. Congeners of this class are under advanced preclinical evaluation as a potential chemotherapy for acquired immunodeficiency syndrome.

Inactivation of HIV-1 Nucleocapsid Protein P7 by Pyridinioalkanoyl Thioesters

“…The lower plateau values obtained at low compound concentration may be the consequence of the requirement for activation of the compound, which for unknown reasons is less efficient at low concentrations. We note, however, that similarly reduced plateau values have been reported for inactivation of NCp7 by lower concentrations of the unrelated disulfide benzamides, which do not require activation (15).…”

“…First, it is interesting to note that only two molecules of the antiviral compound are required to completely inactivate one molecule of NCp7 as opposed to three molecules of disulfide based inhibitors (15,20,21) due to the lack of reactivity toward the aminoterminal zinc finger. Second, both the need for activation and the initial formation of a noncovalent complex preceding transacylation are indicative of the lower reactivity of thioester compounds compared with disulfide agents.…”

“…Ejection from NCp7 by the Pyridinioalkanoyl Thioesters-Since the PATEs analyzed in this study had a very high absorbance at 280 nm, the commonly used method of monitoring the rate of zinc ejection by following the decrease in tryptophan fluorescence (excitation at 280 nm) was not feasible (12,15). Instead, a method of monitoring zinc ejection directly using NPG was adopted.…”

Reactivity of the HIV-1 nucleocapsid protein p7 zinc finger domains from the perspective of density-functional theory

“…Observed reaction rates of recombinant HIV-1 NCp7 (55 residues) were obtained from fluorescence measurements of W 37 . Upon reaction, W 37 fluorescence is diminished by increased internal quenching due to a NCp7 conformational change (8,14). Time course data were obtained with a Shimadzu RF5000 spectrofluorimeter at 25…”

Microdialysis sampling of the isothiazolone, PD-161374, and its thiol and disulfide metabolites

“…Among the compounds selected by the National Cancer Institute HIV Screening Program are several isothiazolones and disulfide benzamides [1][2][3][4]. These compounds have been shown to possess anti-HIV activity at low micromolar concentrations.…”