Pyridine N-oxide derivatives inhibit viral transactivation by interfering with NF-κB binding

Pyridine N-oxide derivatives represent a new class of anti-HIV compounds for which some members exclusively inhibit HIV-1 RT, whereas other members act, additionally or alternatively, at a post-integrational event in the replicative cycle of HIV. A prototype pyridine N-oxide derivative, JPL-32, inhibited tumor necrosis factor alpha (TNF-α)-induced HIV-1 expression in latently HIV-1-infected OM-10.1 and U1 cells, which could be reversed by the addition of N-acetyl-l-cysteine (NAC). The reversal of the antiviral activity of JPL-32 by NAC suggested the possible role of a redox-sensitive factor as target of inhibition. Indeed, when nuclear extracts of TNF-α-stimulated OM-10.1 and U1 cells cultured in the presence of JPL-32 were analyzed by an electrophoretic mobility shift assay (EMSA), a dose-dependent inhibition of DNA binding of nuclear NF-κB was observed, which could be reversed by the addition of NAC. JPL-32 did not inhibit the release and subsequent degradation of IκBα, nor did JPL-32 affect the nuclear translocation of NF-κB. EMSA revealed that the inhibition of the NF-κB DNA binding activity by JPL-32 could be reversed by the addition of reducing agents such as dithiothreitol or β-mercaptoethanol. Moreover, JPL-32 was able to directly oxidize the thiol groups on the purified p50 subunit of recombinant NF-κB. The oxidative modification of the thiol groups on NF-κB by JPL-32 could be ascribed to the intracellular pro-oxidant effect of JPL-32. Consequently, JPL-32 was able to increase the intracellular glutathione (GSH) levels and to induce apoptosis in a dose-dependent way.