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. 2005 Mar 1;33(4):1249-56.
doi: 10.1093/nar/gki268. Print 2005.

Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones

Scott R Budihas  1 , Inna Gorshkova, Sergei Gaidamakov, Antony Wamiru, Marion K Bona, Michael A Parniak, Robert J Crouch, James B McMahon, John A Beutler, Stuart F J Le Grice
Affiliations

Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones

Scott R Budihas et al. Nucleic Acids Res. .

Abstract

High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives beta-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). beta-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 microM, while the IC50 for Escherichia coli and human RNases H was 50 microM and 5.7 microM, respectively. In contrast, the related tropolone analog beta-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC50 = 1.5 microM and 40 microM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both beta-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 microM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between beta-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted.

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Figures

Figure 1
Figure 1
Structures of tropolone and its derivatives.
Figure 2
Figure 2
(A) Inhibition of HIV-1 RT-catalyzed polypurine tract processing by tropolone and its derivatives. Left, schematic representation of the RNase H assay. Right, RNase H activity in the presence of 50 μM tropolone derivative. Lane C, no inhibitor; lane 1, no inhibitor, +DMSO; lane 2, β-thujaplicin; lane 3, α-thujaplicin; lane 4, β-thujaplicininol; lane 5, γ-thujaplicin; lane 6, nootkatin; lane 7, manicol; and lane 8, tropolone. PPT hydrolysis products have been indicated. (B) DNA-dependent DNA polymerase activity of HIV-1 RT in the presence of tropolone and hydroxylated derivatives. DNA polymerase activity was determined at a single inhibitor concentration of 50 μM. Lane notations are as in (A).
Figure 3
Figure 3
Selectivity of RNase H inhibition. Dose–response curves for RNase I inhibition by β-thujaplicinol (A, C and E) and manicol (B, D and F) are presented. (A and B) HIV-1 RT; (C and D), HIV-2 RT; (E and F), human RNase H. IC50 determinations are the results of triplicate assays.
Figure 4
Figure 4
Analysis of inhibitor intercalation by surface plasmon resonance. In (A), binding of ethidium bromide to an immobilized 12mer RNA/DNA hybrid was performed as a control. Sensorgrams a–f illustrate the response to successive injection of the intercalator at concentrations of 10, 5, 1.0, 0.5, 0.25 and 0.1 μM, respectively. (B) Combined sensorgrams following injection of β-thujaplicin, α-thujaplicin and β-thujaplicinol over the same concentration range as in (A).
Figure 5
Figure 5
Michaelis–Menten plot for the determination of the Ki for β-thujaplicinol for HIV-1 RT-associated RNase H. A Ki of 0.20 ± 0.07 μM was determined from non-linear curve fitting. Inhibitor concentrations were 0.024 μM (open circle), 0.049 μM (filled triangle), 0.098 μM (open triangle), 0.20 μM (filled square), 0.39 μM (open square), 0.78 μM (filled diamond), 1.56 μM (open diamond), RNase H activity in the presence of 5% DMSO (filled circle).
Figure 6
Figure 6
Yonetani–Theorell plot for the inhibition of HIV-1 RT in the presence of the NNRTI calanolide A and β-thujaplicinol. The inverse of the rate of RNase H cleavage was plotted as a function of β-thujaplicinol concentration at calanolide A concentrations of 12.5 μM (open square), 0.78 μM (filled square), 0.39 μM (open circle) and DMSO (filled circle). The convergent best-fit lines indicate mutually exclusive binding sites for calanolide A and β-thujaplicinol.
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