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 reve rse transcriptases (RT) [i.e., of human immunodeficiency virus (HIV-1) , equine infectious anemia virus, and murine leukemia virus] and cellu lar DNA polymerases (i.e., of DNA polymerases alpha and beta and Esche richia coli DNA polymerase I). A thorough investigation of the mode of inhibition was conducted with HIV-1 RT-associated DNA polymerase acti vity. The inhibition is unaffected by the nature of template-primer us ed. The inhibitory active site of toxiusol is attributable to the pola r moieties at the benzene ring. The presence of either sulfate groups in the natural lead compound or hydroxyl groups in the corresponding h ydroquinone is critical, because both compounds are equally effective at low micromolar concentrations. Conversely, the presence of acetyl g roups in the same position in the derivative toxiusol diacetate lowers significantly or abolishes the inhibitory activity. Toxiusol binds th e HIV-1 RT irreversibly and in a noncompetitive way with high affinity (K-i = 1.2 mu M), 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 mu M). Still , the compound binds irreversibly, probably through its hydrophobic st ructure skeleton. Toxiusol diacetate loses its ability to inhibit the first step in the DNA polymerization process (that is, the formation o f the DNA-enzyme complex as measured by a gel retardation assay), whic h contributes to its poor inhibitory capacity. On the other hand, toxi usol has been demonstrated to effectively block the binding of HIV-1 R T to its template-primer. This general mechanism of inhibition is Like ly to be typical of a universal inhibitor of DNA polymerases such as t oxiusol.