MODE OF INHIBITION OF HIV REVERSE-TRANSCRIPTASE BY 2-HEXAPRENYL-HYDROQUINONE, A NOVEL GENERAL INHIBITOR OF RNA-DIRECTED AND DNA-DIRECTED DNA-POLYMERASES

A natural compound from the Red Sea sponge Ircinia sp., 2-hexaprenylhy droquinone (HPH), has been shown to be a general inhibitor of retrovir al reverse transcriptases (from HIV-1, HIV-2 and murine leukaemia viru s) as well as of cellular DNA polymerases (Escherichia coli DNA polyme rase I, and DNA polymerases alpha and beta). The pattern of inhibition was found to be similar for all DNA polymerases tested. Thus the mode of inhibition was studied in detail for HIV-I reverse transcriptase. HPH is a non-competitive inhibitor and binds the enzyme irreversibly w ith high affinity (K-1 = 0.62 mu M). The polar hydroxy groups have bee n shown to be of key importance. A methylated derivative, mHPH, which is devoid of these polar moieties, showed a significantly decreased ca pacity to inhibit all DNA polymerases tested. Like the natural product , mHPH binds the enzyme independently at an allosteric site, but with reduced affinity (K-1 = 7.4 mu M). We show that HPH does not interfere with the first step of the polymerization process, i.e. the physical formation of the reverse-transcriptase-DNA complex. Consequently, we s uggest that the natural inhibitor interferes with the subsequent steps of the overall reaction. Since HPH seems not to affect the affinity o f dNTP for the enzyme (the K-m is unchanged under conditions where the HPH concentration is increased), we speculate that its inhibitory cap acity is derived from its effect on the nucleotidyl-transfer catalytic reaction. We suggest that such a mechanism of inhibition is typical o f an inhibitor whose mode of inhibition should be common to all RNA- a nd DNA-directed polymerases.