GA-Hecate antiviral properties on HCV whole cycle represent a new antiviral class and open the door for the development of broad spectrum antivirals

In recent years, synthetic peptides have been considered promising targets for drug development that possess low side-effects, are cost-effective and are susceptible to rational design. Hecate was initially described as a potent bacterial inhibitor and subsequently as an anticancer drug with functions related to its lipid interaction property. Viruses, such as hepatitis C virus (HCV), have a lipid-dependent life cycle and could be affected by Hecate in many ways. Here, we assessed modifications on Hecate’s N-terminus region and its effects on HCV and hepatotoxicity. Gallic acid-conjugated Hecate was the most efficient Hecate-derivative, presenting high potential as an antiviral and inhibiting between 50 to 99% of all major steps within the HCV infectious cycle. However, the most promising aspect was GA-Hecate’s mechanism of action, which was associated with a balanced lipid interaction with the viral envelope and lipid droplets, as well as dsRNA intercalation, allowing for the possibility to affect other ssRNA viruses and those with a lipid-dependent cycle.

The ability of GA-Hecate inducing cell death by necrosis and apoptosis was assessed in 48 and 72h post-treatment (p.t) at concentrations: 1.25; 2.5; 5; 10; 20 and 40 μM. For necrosis evaluation, cells were stained using Propidium Iodide at 1M, which was incubated with the cells 15 minutes prior fixation. Subsequently, cells were fixed using paraformaldehyde 4% in PBS (PFA 4%) for 15 minutes and nuclei stained using DAPI 1 μg/mL for 5 minutes. For apoptosis evaluation, cells were fixed using PFA 4% during 15 minutes, permeabilized using Triton X-100 (Sigma) 0.1% in PBS and stained using anti-Active Caspase 3 antibody (host: Rabbit) (1:200 in BSA 1% in PBS) (Abcam 13847) for 2h. Then cells were rinsed using PBS and stained using anti-rabbit AlexaFluor 594 (1:500 in BSA 1% PBS) (Thermo Scientific A21207) during 2h. Nuclei were stained using DAPI for 5 minutes. For both assays, cells were counted in 5 randomly chosen fields in duplicate.

Circular Dichroism
Circular dichroism (CD) spectra were obtained between 190 and 260 nm with a JASCO J-815 CD spectrophotometer (Japan) on nitrogen flush in 1 mm path-length quartz cuvettes at room temperature. All CD spectra were acquired using an average of 8 scans, with a resolution of 0.2 nm. To investigate the conformational changes by the membrane environments, the data were obtained in vesicles containing POPC:POPS (9:1), and POPC:Cholesterol (9:1). CD spectra were typically recorded as an average of the eight scans that were obtained in millidegrees and converted to molar ellipticity [θ] in deg·cm 2 ·dmol -1 . The peptide concentration was 10 μmol·L -1 , and the proportion of lipid/peptide was 100 (L/P = 100).

Effect of peptides on dsRNA intercalation assay
To assess GA-Hecate's dsRNA intercalation capacity, we performed an assay adapted

Cell viability
The peptide showed an inhibition time and dose dependent. The maximum effect with lower cell death was reached at 48h ( Figure S1). The maximum toxicity is reduced at 72h and is reached at 10 µM. An increased ability to inhibit the virus in lower concentrations is reached at 72h post-infection and the lower used concentration (1.25 µM) inhibited about 50% of the viral replication and presented no cell toxicity. IC50 and CC50 of GA-Hecate at this time point were 3.8 µM and 19 µM respectively (SI: 5).
In order to guarantee that the virus inhibition effect was not related exclusively to cell death trigger, all concentrations were evaluated to necrosis and apoptosis induction.

Necrosis and Apoptosis
At 40 μM, GA-Hecate induced 50% of necrosis 48h p.t. However, at 72h p.t this percentage is reduced to 10%, considering that the cells which were in necrosis process at 48h are already dead at 72h p.t. and the increase in the cell death presented by MTT assays was attributed to the end of the necrosis process at 72h ( Figure S2 and S3). The 20% of death found at 20 μM was also attributed to necrosis, since there was no significate apoptosis induction in any time point for this concentration and the percentage of necrosis at this concentration is about 20% over the time ( Figure S2 and S3). The peptide showed nonselective properties at high concentrations. However in low concentrations there was selectivity for virus interaction over cell interaction as depicted in the figure S4.

GA-Hecate effect on HCV genotype 1b subgenomic Replicon
GA-Hecate presented a reduced ability to inhibit genotype 1b replication when compared to the other tested HCV genotypes and reached a maximum inhibition of 65% in the maximum safe concentration.

GA-Hecate intercalation property in dsRNA
To further evaluate the influence of charge on peptide effect and considering the property of some peptides to intercalate into dsRNA, we also evaluated this capacity for GA-Hecate (charge +9) and compared it with Hecate (charge +10) and Lys-Hecate (charge +11). All tested peptides were able to intercalate in HCV dsRNA ( Figure S8).

GA-Hecate and Hecate structuration in negatively charged and uncharged membranes
CD spectroscopy of Hecate and GA-Hecate peptides in lipid vesicles showed that the addition of GA at the N-terminal position decreased the α-helix percentage of the peptide in negatively charged membranes. In uncharged membranes, Hecate does not show a clear secondary structure formation (Fig. S1). However, GA-Hecate showed a mixture of random coil and α-helix structures (Fig. S2). Figure S1.    Figure S8. dsRNA intercalation assay. The assay shows the most positive peptide (Lys-Hecate), the least positive peptide with the steric effect (GA-Hecate) and the Hecate general structure effects on dsRNA intercalation. Water was used as a negative control for dsRNA intercalation and doxorubicin 100 μM was used as a positive control.