PDI inhibition blocks the HIV early replicative cycle [7
] and HIV entry occur at 3 different steps: CD4 binding, the gp41 mediated membrane fusion process, and during uncoating events. Thus conformational changes, gp120 interacts with the enzyme PDI and the chemokine co-receptors form a PDI-CD4-gp120-chemokine complex also prevent HIV envelope mediated fusion to the host cell [8
DTNB and Bacitracin are PDI inhibitors that previously reported activity against HIV-1 T-tropic strains, which have CXCR4 co-receptor dependence (CXCR4) [8
]. DTNB is a sulfhydryl blocker that covalently reacts with the catalytic sites of PDI and abolishes its capacity for cleaving disulfides, whereas Bacitracin inhibits both its reductive and oxidative functions [14
]. In this paper we found that both compounds showed antiviral and virucidal activities in a dose-dependent manner. However T-tropic (HIVIIIB
) strains which has CXCR4 co-receptor were susceptible to both compounds and not to the M-tropic (HIV-BaL
) strain (Figure ) due probably at the increased of negative charge (T-tropic) in variable region (V3) of human immunodeficiency virus type 1 (HIV-1) envelope gp120 subunit participates in determination of viral infection coreceptor tropism [25
Furthermore, cell-based fusion assays [28
] were performed for these compounds, using HeLa-CD4-LTR-β-gal cells [29
] and H2/3 (Env-expressing) cells [21
], where co-culture expressing cells results in efficient cell fusion within 6-12 hours. We found that both Bacitracin and DTNB showed inhibition of cell-cell fusion in a dose-dependent manner when exposed to HeLa-CD4-LTR-β-gal cells (Figure ). These results are similar to those presented by Fenouillet et al
], where PDI inhibitors as well as anti-PDI antibodies, were used to inhibit cell-cell fusion assays. However exposure to HeLa-CD4-LTR-β-gal cells showed no significant difference on cell-cell fusion on either the 24 hour or the 30 minute exposure assay which is the time the process of conformational changes of gp120 after CD4 bonding takes place [30
]. Alternatively, long-term exposure (24 hours) of DTNB on HL2/3 (Env-expressing) cells and then mixing them with CD4 cells showed significant inhibition of cell-cell fusion as showed in Figure . This effect of Bacitracin and DTNB markedly on Env expressing cells has never been reported.
Also, our findings indicate that Bacitracin and DTNB act differently on HeLa-CD4-LTR-β-gal cells and HL2/3 (Env-expressing) cells, given that Bacitracin has more effect on HeLa-CD4-LTR-β-gal cells but less effect against HL2/3 (Env-expressing) cells fusion's activity, however DTNB has cell-cell fusion inhibition effect on HeLa-CD4-LTR-β-gal cells but predominantly on HL2/3 (Env-expressing) cells. This data confirmed that both compounds act on the HIV receptor dependent gp41 mediated fusion process per se through inhibition of a post-CD4 binding step that has antiviral effects and virucidal action [14
]. However our results with Bacitracin showed that has a better inhibition effect on the host CD4 cell acting only as fusion inhibitor on CD4 cells [20
] but also having an inhibition effect on env expressing cells.
In addition, to further determine the antiviral target of Bacitracin and DTNB, a time-of addition experiment was used to delimit the stage(s) of the viral life cycle that is blocked by these compounds separately, comparing it with several antiretroviral drugs as controls that point out different stages of the viral cycle [31
] (Figure ). Our findings suggest that Bacitracin inhibits HIV-1 infection at stage of viral entry or fusion (Figure ), however DTNB acts at a time period between and including viral entry and other later stages (Figure ). DTNB results shows that has probably an antiviral function affect on protease activity, this results are also according to Lebon [22
]. This is the first time, to our knowledge, that the activity of this compound has been associated to HIV-1 life cycle stages besides fusion. However studies has shown that down regulation of PDI using small interfering RNA had only a small effect on infection or cell fusion mediated by HIV-1, suggesting that other thiol active enzymes at the cell surfaces are involved in reduction of the HIV envelope glycoprotein, that is therefore an interesting result.
Lastly, in order to provide a barrier to infection by residual active virus on uninfected cells, we evaluated the long-term effectiveness of the compound, which is an important pharmacodynamic parameter tested for the development of a topical agent [37
], and is defined as the length of time that infection is suppressed following brief exposure to the antiviral agent. Ideally, a microbicide should remain effective for several hours after topical application. These compounds should be able to give cell protection for several hours after the removal of the extracellular drug [38
] Although Bacitracin did not show cell protection (Figure ), DTNB on the other hand, showed lasting cell protection (50% infection inhibition) even 10 hours after compound was washed and removed from the indicator cells (Figure ). These results are consistent with a time-of addition experiment that showed that DTNB act after viral entry suggesting that DTNB somehow bonds to target cell strongly and though DTNB's PDI bonding activity is not discarded, other cell sites could be participating. Previous reports showed DTNB only has effect when anti-PDI was present at the time of virus-cell interaction (Ryser et al., 1994). Our results demonstrated that exposure of uninfected cells to DTNB renders these cells refractory to subsequent HIV-1 infection, even in the absence of a continued extracellular presence of the drug. Thus, DTNB may serve to minimize the sexual transmission of HIV-1 from infected to noninfected individuals.
In summary, although both, Bacitracin and DTNB, are classified as fusion inhibitors of cell PDI, our findings suggest new data. The data presented here are novel in that they prove that both Bacitracin and DTNB (besides acting on cell PDI), are also virucidal agents against T-tropic HIV-1 infection, and DTNB acts not only at early viral cycle stages but also at late stages with long lasting effects on the CD4 cells.
Based on our results and the above requirements, DTNB could be considered as a leading compound to further studies to determine their potential use as therapeutic agents in HIV-1 infection, especially due to their virucidal and fusion inhibitor properties.