Introduction: Enteroviruses are non-enveloped viruses of the Enterovirus genus of the Picornaviridae family. They cause human diseases ranging from respiratory diseases to more severe cases, including polio, encephalitis, myocarditis, and pancreatitis. To date, there are no approved direct-acting antiviral drugs for the treatment of enterovirus diseases, therefore search for new small molecules - inhibitors of the enterovirus life cycle is important.
Objective: to characterize the antiviral properties of new stable free radicals, verdazyls, and their precursors, leucoverdazyls. Leucoverdazyls have previously been shown to have antioxidant potential.
Materials and methods: leucoverdazyls and verdazyls were synthesized in the Laboratory of coordination compounds, Postovsky Institute of Organic Synthesis, Ural Branch of the Russian Academy of Sciences, Ekaterinburg, Russian Federation. The following strains and cell cultures were used: influenza A virus (strain A/Puerto Rico/8/1934 H1N1), Coxsackie virus B3 (CVB3, Nancy strain), Coxsackie virus B4 (CVB4, Powers strain), Coxsackie virus B5, herpes simplex virus type 1 (HSV1) and human adenovirus 5 (Ad5) obtained from the collection of the Pasteur Research Institute (St. Petersburg). The following cell cultures were used: MDCK (ATCC #CCL-34), Vero (ATCC #CCL-81), A549 (ATCC #CCL-185). Infectious activity of viruses was assessed by end point titration. The cytoprotective activity and cytotoxicity of the compounds were evaluated using MTT test. The antiviral activity of the compounds was evaluated in the viral yield reduction assay. The virucidal activity of the compounds was evaluated after incubation of the compounds in a cell-free system with Coxsackie B4 virus for 1 hour. To investigate the mechanism of action of the leader compound, a time-of-addition assay was performed.
Results:
Leucoverdazyls, unlike verdazyls, have cytoprotective activity when a permissive culture is infected with the Coxsackie B3 virus. The leading compound was identified: 1a, which demonstrated a high inhibitory ability against a wide panel of influenza B enteroviruses in micromolar range (IC50=5.48 M and 0.72 M for Coxsackie B3 and Coxsackie B4, respectively) and its activity was superior to pleconaril (IC50=15.2 and IC50=1.91). Nevertheless, pleconaril acted as a more powerful inhibitor than 1a towards Coxsackievirus B5. The compound showed only slight activity against influenza A (RNA virus), no activity against Ad5 and HSV1 (DNA viruses). 1a have no virucidal activity. The maximum decrease in the titers of viral progeny with the addition of 1a was observed in the early and middle stages of the life cycle of the Coxsackie virus.
Conclusion:
Leucoverdazyls are potent inhibitors of group B enteroviruses in vitro. Leucoverdazyl 1a doesnt belong to capsid binder class of inhibitors and has no virucidal activity against coxsackievirus. Further studies are needed to elucidate their precise mechanisms of action including assessment of its direct impact on intracellular ROS generation, resistant clone selection and mapping of resistance mutations. We plan to expand the library of leucoverdazyls through targeted chemical modifications in order to disclose its pharmacophore and improve their virus-inhibiting properties. Nevertheless, the results of the study can serve as a basis for future development of novel antivirals to use in monotherapy or in combinational treatment of enteroviral infections.
