Mechanism of Inhibition of HIV Reverse Transcriptase by Toxiusol, a Novel General Inhibitor of Retroviral and Cellular DNA Polymerases

Toxiusol, a natural product isolated from the Red Sea sponge Toxiclona toxius, has been shown to be a potent inhibitor of various viral reverse transcriptases (RT) [i.e., of human immunodeficiency virus (HIV-1), equine infectious anemia virus, and murine leukemia virus] and cellular DNA polymerases (i.e., of DNA polymerases α and β and Escherichia coli DNA polymerase I). A thorough investigation of the mode of inhibition was conducted with HIV-1 RT-associated DNA polymerase activity. The inhibition is unaffected by the nature of template-primer used. The inhibitory active site of toxiusol is attributable to the polar moieties at the benzene ring. The presence of either sulfate groups in the natural lead compound or hydroxyl groups in the corresponding hydroquinone is critical, because both compounds are equally effective at low micromolar concentrations. Conversely, the presence of acetyl groups in the same position in the derivative toxiusol diacetate lowers significantly or abolishes the inhibitory activity. Toxiusol binds the HIV-1 RT irreversibly and in a noncompetitive way with high affinity (K_i = 1.2 /rM), probably through polar groups. The replacement with acetyl moieties in the analog toxiusol diacetate hampers the binding of the inhibitor to the enzyme (K_i increases to about 26 µM). Still, the compound binds irreversibly, probably through its hydrophobic structure skeleton. Toxiusol diacetate loses its ability to inhibit the first step in the DNA polymerization process (that is, the formation of the DNA-enzyme complex as measured by a gel retardation assay), which contributes to its poor inhibitory capacity. On the other hand, toxiusol has been demonstrated to effectively block the binding of HIV-1 RT to its templateprimer. This general mechanism of inhibition is likely to be typical of a universal inhibitor of DNA polymerases such as toxiusol.