Hydroxylated tropolones inhibit hepatitis B virus replication by blocking viral ribonuclease H activity

Abstract

Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC50) being 2.3 μM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC50) being 0.34 μM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50]=25 to 79 μM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection.

FIG 1

Primary screening of hydroxytropolones against HBV RNaseH. (A) Oligonucleotide-directed RNA cleavage assay. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and detected by autoradiography. Gray line, ³²P-labeled RNA; black line, DNA oligonucleotide; S, substrate; P1, product 1; P2, product 2. (B) Examples of primary screening assays against HBV genotype C and D RNaseH enzymes. DMSO, vehicle control; +, complementary oligo, −, inclusion of a non-complementary DNA oligonucleotide as a specificity control.

FIG 2

IC₅₀s for hydroxylated tropolone inhibitors of HBV RNaseH. IC₅₀s were measured using the oligonucleotide-directed RNA cleavage assay. (A) IC₅₀ curves for compound 106 against genotype C and D HBV RNaseHs. (B) IC₅₀ curves for compound 110 against genotype C and D HBV RNaseHs. The IC₅₀ curves are from representative assays, and the IC₅₀s are the averages ± 1 standard deviation from two or three assays.

FIG 3

Inhibition of HBV replication by hydroxylated tropolones. The inhibition of replication by hydroxylated tropolones was measured against an HBV genotype D isolate in HepDES19 cells. (A) Principle underlying the strand-preferential quantitative PCR (qPCR) assay. Plus-polarity DNA is measured by amplifying HBV DNA across the gap in the minus-polarity DNA strand. Minus-polarity DNA strands are measured by amplifying sequences downstream of the 3′ end of the vast majority of plus-polarity DNA strands in viral capsids. (B to D) Select EC₅₀ curves. EC₅₀s were calculated based on the decline of the plus-polarity DNA strand. The curves and the EC₅₀s are the means ± 1 standard deviation from two or three independent experiments, each conducted in duplicate.

FIG 4

Representative cytotoxicity measurements. Representative MTT cytotoxicity assay results are shown. The curves are from representative experiments conducted in triplicate, and the CC₅₀ values are the averages ± 1 standard deviation from two or three independent experiments, each conducted in triplicate.

FIG 5

Preliminary SAR for hydroxylated tropolones against HBV RNaseH.