Antiviral propierties of 5,5'-dithiobis-2-nitrobenzoic acid and bacitracin against T-tropic human immunodeficiency virus type 1

Abstract

Bacitracin and the membrane-impermeant thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) are agents known to inhibit protein disulfide isomerase (PDI), a cell-surface protein critical in HIV-1 entry therefore they are fusion inhibitors (FI). Here we investigated the possibility that Bacitracin and or DTNB might have other antiviral activities besides FI. By means of residual activity assays, we found that both compounds showed antiviral activity only to viruses T-tropic HIV-1 strain. Cell-based fusion assays showed inhibition on HeLa-CD4-LTR-β-gal (CD4) and HL2/3 cells treated with Bacitracin, and DTNB with the latest compound we observed fusion inhibition on both cells but strikingly in HL2/3 cells (expressing Env) indicating a possible activity on both, the cell membrane and the viral envelope. A time-of-addition experiment showed that both compounds act on HIV entry inhibition but DTNB also acts at late stages of the viral cycle. Lastly, we also found evidence of long-lasting host cell protection in vitro by DTNB, an important pharmacodynamic parameter for a topical microbicide against virus infection, hours after the extracellular drug was removed; this protection was not rendered by Bacitracin. These drugs proved to be leading compounds for further studies against HIV showing antiviral characteristics of interest.

Figure 1

Residual Activity on HIV-1 strains. A, B) HIV-1_IIIB and C, D) HIV-1_BaL cell-free viruses were exposed to serial dilutions of A, C) Bacitracin and B, D) DTNB for 5 minutes. The viruses were then ultracentrifuged, washed three times and added to HeLa-CD4-LTR-β-gal cells After 24 hours, β-gal activity was measured. Percentage values are relative to the positive control (no compound pretreatment).

Figure 2

Inhibition of Env/CD4-mediated membrane fusion. β-gal activity was measured after CD4 and Env cells were co-cultured when exposed to A) Bacitracin, B) DNTB, C) UC781 and D) T-20, in different circumstances: (red) CD4 cells were exposed to the compound and co-cultured with Env cells for 24 hours. (green) Env cells were exposed to the compound and co-cultured with CD4 cells for 24 hours. (purple) CD4 cells were exposed to the compound for 30 minutes, washed, and co-cultured with Env cells for 24 hours. (blue) Env cells were exposed to the compound for 30 minutes, washed, and co-cultured with CD4 cells for 24 hours. Percentage values are relative to the positive control (no treatment). The data represent the means ± standard deviations from three separate experiments, each of which was carried out in duplicate.

Figure 3

Time of Intervention in HIV-1 Life Cycle. HeLa/CD4-LTR-β-gal cells were infected with HIV-1_IIIB cell-free virus before A) Bacitracin (3.5 mM), B) DTNB (6 mM), C) T-20 (100 μM), D) UC781 (70 nM), E) 118-D-24 (120 μM) and F) Amprenavir (0.1 mM), were added upon HIV-1 inoculation (time zero) or at various time points post-inoculation and β-gal activity was measured following 24 hr of incubation. Percentage values are relative to the positive control (no treatment). The data represent the means ± standard deviations from three separate experiments, each of which was carried out in duplicate.

Figure 4

Cell Protection Against HIV-1 Infection. β-gal activity was measured after HeLa/CD4-LTR-β-gal cells were exposed to A) Bacitracin (5.3 mM) and B) DTNB (12.6 mM) for 30 minutes, washed and exposed to HIV-1_IIIB cell-free virus at various time points post-treatment. Percentage values are relative to the positive control (no treatment). The data represent the means ± standard deviations from three separate experiments, each of which was carried out in duplicate.