Novel broad spectrum virucidal molecules against enveloped viruses

Abstract

Viral infections are an important cause of death worldwide. Unfortunately, there is still a lack of antiviral drugs or vaccines for a large number of viruses, and this represents a remarkable challenge particularly for emerging and re-emerging viruses. For this reason, the identification of broad spectrum antiviral compounds provides a valuable opportunity for developing efficient antiviral therapies. Here we report on a class of rhodanine and thiobarbituric derivatives displaying a broad spectrum antiviral activity against seven different enveloped viruses including an HSV-2 acyclovir resistant strain with favorable selectivity indexes. Due to their selective action on enveloped viruses and to their lipid oxidation ability, we hypothesize a mechanism on the viral envelope that affects the fluidity of the lipid bilayer, thus compromising the efficiency of virus-cell fusion and preventing viral entry.

Fig 1. Synthesis of substituted rhodanine derivatives 9 a–e: i. Pd(PPh₃)₂Cl₂, Na₂CO₃, DMF/EtOH, RT, 1h; ii. 1N NaOH (aq), MeOH/THF, reflux; iii. DME, Et₃N, MW (300 W), 90°C, 10 min. iv. aldehyde 4 or 6, DME, MW (300 W), 110°C, 5 min.

Fig 2

Synthesis of monosubstituted thiobarbituric compounds 15a and 15b: i. a) NH₄SCN, (CH₃)₂CO, 60°C MW, min, b) opportune amine (CH₃)₂CO, 60°C MW, ii. NH₂NH₂; iii. Na⁰, EtOH, reflux, iv. HCl, EtOH, 70°C.

Fig 3. Antiviral activity against HSV-2.

*EC₅₀ half maximal effective concentration; °EC₉₀ 90% effective concentration; #C.I. confidence interval; †CC₅₀ half maximal cytotoxic concentration; ‡SI selectivity index; n.a. not assessable. 9d n = 5, other compounds n = 2.

Fig 4. Time of addition assays.

Compound 9d was added on cells 2 h before infection (PRE-TREATMENT CELLS) and subsequently the inocula were removed and cells were infected or compound and virus were added simultaneously on cells (DURING INFECTION) or after 1 h pre-incubation between the virus and the compound (PRE-TREATMENT VIRUS). Cells were fixed and stained 24 h later and plaques were counted. The percentage of infection was calculated comparing treated wells to wells treated with equal volumes of solvent. n = 3.

Fig 5. Viral yield reduction.

Cells were infected with HSV-2 (MOI 0.01) and after the removal of inoculum they were treated with increasing concentrations of compound. At 24 hpi when the untreated wells showed extensive cytopathology, supernatants and cells were harvested and subsequently titrated with plaque assay. The percentage of infection was calculated comparing viral titer in treated wells and wells treated with equal volumes of solvent. n = 4.

Fig 6. Virucidal activity.

A) 10⁵ pfu of HSV-2 were incubated for 0, 15’, 30’, 45’ or 60’ at 37°C with 10 μM, 5 μM or 1 μM of compound and subsequently titrated on Vero cells. B) 10⁵ pfu of HSV-2 were incubated with different doses of compounds 9d for 1h and subsequently titrated on Vero cells. Viral titers were calculated at dilutions of compounds at which, in plaquing efficiency assay, did not show any inhibitory activity. The percentage of infection was calculated comparing viral titer in treated wells and wells treated with equal volumes of solvent. Results show the mean and SD or square roots of the sum of squares, n = 6 for 60’ and n = 2 for other conditions.

Fig 7. Binding and entry assays.

(A-B-C) HSV-2 (MOI 10) was incubated for 1 h at 37°C with 10 μM of compound and subsequently added for 2 h on Vero cells at 4°C. Subsequently cells were subjected to qPCR (A) or immunostaining (B) or IF (C). (D-E) HSV-2 (MOI 10) was incubated for 1 h at 37°C with 10 μM of compound and subsequently added for 1 h on Vero cells at 4°C and then shifted at 37°C for 1.5 h (D) or 4 h (E) subsequently cells were fixed and subjected to ICC (D) or IF (E). UT = untreated ** p<0.001 *** p<0.0005 **** p<0.0001. n = 3.

Fig 8. In vitro determination of lipid peroxidation capability.

MDA (micromoles/mg) produced from the linoleic acid peroxidation after exposure to compound 9d in the presence and in the absence of (±)-α-tocopherol. ** p<0.01 NaCl 0.9% and DMSO are blank samples prepared without the addition of 9d and (±)-α-tocopherol. n = 3.