Synthetic α-Hydroxytropolones Inhibit Replication of Wild-Type and Acyclovir-Resistant Herpes Simplex Viruses

Abstract

Herpes simplex virus 1 (HSV-1) and HSV-2 remain major human pathogens despite the development of anti-HSV therapeutics as some of the first antiviral drugs. Current therapies are incompletely effective and frequently drive the evolution of drug-resistant mutants. We recently determined that certain natural troponoid compounds such as β-thujaplicinol readily suppress HSV-1 and HSV-2 replication. Here, we screened 26 synthetic α-hydroxytropolones with the goals of determining a preliminary structure-activity relationship for the α-hydroxytropolone pharmacophore and providing a starting point for future optimization studies. Twenty-five compounds inhibited HSV-1 and HSV-2 replication at 50 μM, and 10 compounds inhibited HSV-1 and HSV-2 at 5 μM, with similar inhibition patterns and potencies against both viruses being observed. The two most powerful inhibitors shared a common biphenyl side chain, were capable of inhibiting HSV-1 and HSV-2 with a 50% effective concentration (EC50) of 81 to 210 nM, and also strongly inhibited acyclovir-resistant mutants. Moderate to low cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50] of 50 to >100 μM). Therapeutic indexes ranged from >170 to >1,200. These data indicate that troponoids and specifically α-hydroxytropolones are a promising lead scaffold for development as anti-HSV drugs provided that toxicity can be further minimized. Troponoid drugs are envisioned to be employed alone or in combination with existing nucleos(t)ide analogs to suppress HSV replication far enough to prevent viral shedding and to limit the development of or treat nucleos(t)ide analog-resistant mutants.

FIG 1

Screen of selected α-hydroxytropolones at low concentrations. Vero cells were infected with a primary clinical isolate of HSV-1 or HSV-2 at an MOI of 0.1 in the presence of each compound or the diluent control and incubated for 24 h. Cultures were then collected, and the virus titer was determined by a plaque assay. The log₁₀ reduction in plaque counts for each compound at 0.5 μM or 0.17 μM compared to the diluent control is shown. Values are the averages of data from duplicate samples ± ranges from one experiment.

FIG 2

EC₅₀s for α-hydroxytropolone inhibitors of HSV-2. EC₅₀s were determined over a range of 12 to 14 concentrations. EC₅₀ curves are shown for compounds 114, 115, 118, and 146 against HSV-2. The EC₅₀ curves are from representative assays, and the EC₅₀s values in Table 1 are the averages ± 1 standard deviation from two or three independent assays.

FIG 3

α-Hydroxytropolones inhibit an ACV-resistant mutant of HSV-2. ACV or the indicated α-hydroxytropolone compound was added at 50 μM (A) or 5 μM (B) to cultures infected with wild-type HSV-2 or a TK-deficient mutant of the same strain. Log₁₀ suppression was determined relative to the diluent control. Data are the averages of data from duplicate samples ± ranges from one of two independent experiments at each concentration.

FIG 4

ACV and compound 118 synergistically inhibit HSV-2 replication and DNA accumulation. ACV and compound 118 were diluted in 3-fold steps and added alone or together in constant ratios to Vero cell monolayers infected with HSV-2 at an MOI of 0.1. After 24 h, the cultures were collected and divided into two aliquots. Shown are a representative volume plot of viral titers for combinations of ACV and/or compound 118 against HSV-2, as determined by a plaque assay (A), and isobolograms for qPCR for HSV-2 genomic DNA (B) and viral titer (C) data, as analyzed by the Chou-Talalay method. In panels B and C, the x axis indicates the effective concentration in this experiment for compound 118 alone, and the y axis indicates the effective concentration for ACV alone. The colored lines represent efficacy expected from mixing compound 118 and ACV at various proportions if the effects of the compounds are additive. The area below the lines indicates synergism, and the area above indicates antagonism. EC₉₀, EC₇₅, and EC₅₀ values were calculated in this experiment from the combinations of compound 118 and ACV.

FIG 5

Preliminary SAR for α-hydroxytropolones against HSV-1 and HSV-2. (A) Anti-HSV data from lead natural-product α-hydroxytropolones from previous studies (33). Also shown are tropolone natural-product analogs that were inactive against HSV-1 and HSV-2 at 5 μM. n.d., not determined. (B) Qualitative comparison of HSV-1 suppression (>3 log₁₀ units) by closely related synthetic α-hydroxytropolones at 50 μM (+), 5 μM (++), and 0.5 μM (+++), highlighting a trend of increasing potency with larger substituents. (C) EC₅₀ and CC₅₀ values of synthetic α-hydroxytropolones, reported as averages of data from 2 runs.