A novel approach utilizing spirocyclic thiopyrimidinone compounds against herpes simplex virus with underlying antiviral mechanisms of action

Abstract

In recent decades, viral outbreaks have significantly threatened global health, with herpes simplex virus type 2 (HSV-2) being one of the most prevalent infections. This study evaluated novel spiropyrimidine derivatives as potential antiviral agents against HSV-2, building on previous research that examined spirocyclic thiopyrimidinone derivatives against human coronavirus 229E (hCoV-229E). Among the eleven synthesized compounds, spiropyrimidinone derivative 3 demonstrated promising antiviral activity, with a selectivity index of 11.2. The drug mechanism of infection studies indicated that compound 3 primarily inhibits HSV-2 at the viral adsorption stage, achieving approximately 83% inhibition and reducing viral multiplication by 34%. Its efficacy is linked to its diketone moiety, which is known for its ability to enhance antiviral effects. Furthermore, the effect of compound 3 on viral inhibition is reflected in the level of caspase-3 protein expression, revealing that the apoptotic pathway is modulated. Docking studies revealed multiple interactions with herpes virus entry mediator (HVEM), indicating its potential as an entry inhibitor. These findings confirm that compound 3 could be a potential candidate for further development in HSV-2 antiviral therapy.

Keywords: Antiviral activity; Herpes simplex virus; Mechanism of action; Spirocyclic thiopyrimidinones.

Fig. 1

Antiherpes drugs containing pyrimidine moieties

Fig. 2

Synthesis of spirocyclic thiopyrimidinones 2–6

Fig. 3

The plots of the CC₅₀ and IC₅₀ values are presented in Table 1 for the most promising compounds (2, 3, and 5c)

Fig. 4

Mechanism of action of drug candidates 2, 3, and 5c in HSV-2 inhibition. The histograms show the different percentages of HSV-2 inhibition resulting from three different mechanisms of the viral infection life cycle: adsorption, replication, and virucidal. The histogram data are expressed as the mean ± S.D. of three independent experiments of viral inhibition mechanisms of action against cell control. Groups were statistically analyzed by f tests (ANOVAs) followed by post hoc Duncan tests. Groups with different letters are considered statistically significant at p < 0.05

Fig. 5

Microscopy images of the morphological changes in HSV-2-infected Vero cells (MOI = 1) treated with compound 3 via different viral infection mechanisms. A) Herpes virus control, B) infected Vero cells treated with 3 in the replication mechanism, C) infected Vero cells treated with 3 in the adsorption mechanism, and D) normal Vero cell control

Fig. 6

Western blot analysis for cleaved Caspase-3 protein expression in HSV-2-infected Vero cells (MOI = 1) treated with compounds 2, 3, and 5c compared with the HSV-2 virus control (infected Vero cells without any treatment (VC)). A) The protein expression level of β-actin at 1 h, 3 h, 6 h, 12 h, and 24 h postinfection. B) The protein expression level of Caspase-3 in infected HSV-2 virus control and infected Vero cells treated with 2, 3, or 5c. The measured band intensities of all the compound-treated cells and the virus control were plotted to compare the differences in the protein expression levels of Caspase-3. A t-test was used to compare the protein expression levels of caspase-3 in untreated and treated cells. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.005. However, ns is not significant

Fig. 7

A) The best ranked binding mode of 3 in HVEM. B) The best ranked binding mode of 3 (magenta) in the binding pocket of HVEM (PDB ID 1JMA). Hydrogen bonding interactions, hydrophobic interactions and π‒π interactions are colored yellow, gray, and green, respectively