Design of histidine-rich peptides with enhanced bioavailability and inhibitory activity against hepatitis C virus

Abstract

Recently, peptide drugs have evolved into mainstream therapeutics, representing a significant portion of the pharmaceutical market. However, their bioavailability remains to be improved compared with that of chemical drugs. Here, we screened and identified a new peptide, Ctry2459, from a scorpion venom peptide library that was proven to inhibit hepatitis C virus (HCV) infection via inactivating infectious viral particles. However, Ctry2459 cannot suppress established infection of HCV because of the poor cellular uptake and restriction of endosomes. Based on the molecular template of the Ctry2459 peptide, we designed two histidine-rich peptides (Ctry2459-H2 and Ctry2459-H3) with significantly enhanced cellular uptake and improved intracellular distribution. Moreover, the two mutated peptides, as well as the wild-type peptide Ctry2459, exhibited virucidal activities against HCV. In distinct contrast to the Ctry2459 peptide, the mutated peptides significantly suppressed the established HCV infection at the cellular level but demonstrated lower cytotoxic and hemolytic activities. Our work presents an effective design strategy for optimizing natural antiviral peptides and opens a new avenue for enhancing the bioavailability of peptide drugs.

Fig. 1

Screen of anti-HCV peptides from the scorpion venom peptide library. (A) Thirteen antimicrobial peptides were identified, and the mature peptides are indicated by red boxes. (B) The 13 peptides (20 μg/ml) were screened for the ability to inhibit HCV infection (JFH-1) in Huh7.5.1 cells. HCV RNA levels were calculated as a percentage (%) of the negative control. IFNα2a (100 IU/ml) was used as a positive control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Extracellular anti-HCV activity and viral inactivation of Ctry2459. (A, B, C) HCV RNA and core protein content in infected Huh7.5.1 cells decreased both time- and dose-dependently after treatment with indicated concentrations of the Ctry2459 peptide. (D, E) ApoE, HCV E2, HCV core protein, HCV RNA content and viral infectivity were determined by the virucidal assay.

Fig. 3

Time-dependent anti-HCV activities and cellular localization of Ctry2459. (A, B) The Ctry2459 peptide was added at a final concentration of 20 μg/ml under the following conditions: (i) preinoculation of cells: peptide was added to cells for 2 h at 37 °C followed by washing three times with growth medium before virus infection; (ii) preinoculation of virus: the peptide was added to the virus for 2 h at 37 °C, and then the mixture was used to infect cells for 4 h at 37 °C. The peptide–virus mixture was then removed, and then the cells were washed three times and replenished with growth medium without peptide. (iii) coinoculation: peptide was added to cells together with the virus for 4 h, at which time the cells were washed three times and replenished with growth medium without peptide; (iv) postinoculation: cells were infected for 4 h, at which point the virus was removed, and then the cells were washed as above and replenished with growth medium containing peptide throughout the experiment. After 48 h postinfection, HCV RNA and core protein levels were measured by real-time PCR and western blotting. (C) The cellular localization of the Ctry2459 peptide was determined by confocal microscopy after 48 h incubation.

Fig. 4

Effect of lysosomotropic agents on the cellular localization and anti-HCV activities of Ctry2459. (A) Effect of chloroquine (50 μm) and Ca²⁺ (3 mm) on the cellular uptake and intracellular distribution of the Ctry2459 peptide by confocal microscopy after 48 h incubation. (B) Effect of chloroquine (50 μm) and Ca²⁺ (3 mm) on the anti-HCV activity of the Ctry2459 peptide by real-time RT-PCR analysis. **P < 0.01.

Fig. 5

Design of histidine-rich peptides based on the molecular template of Ctry2459. (A) Sequence alignments of the WT, H2 and H3 peptides. The mutation sites are shown in green, and the histidine residues are shown in red. (B) Helical wheels of the three peptides showed the hydrophilic and hydrophobic faces. (C) CD spectra of the three peptides. Measurements were performed in the UV range of 250–190 nm at 25 °C in water on a Jasco-810 spectropolarimeter. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Extracellular anti-HCV activities and viral inactivation of Ctry2459-H2 and Ctry2459-H3. The HCV RNA content in infected Huh7.5.1 cells decreased both time- and dose-dependently after treatment with the indicated concentrations of H2 (A, C) and H3 (B, D). (E) The HCV RNA content and viral infectivity were determined using a viral inactivating assay.

Fig. 7

Intracellular anti-HCV activities and cellular localization of Ctry2459-H2 and Ctry2459-H3. (A, B) The cellular localization of Ctry2459-H2 and Ctry2459-H3 was determined by confocal microscopy after incubation for 1 h and 24 h (C) Ctry2459-H2 or Ctry2459-H3 was added 4 h, 24 h and 48 h postinfection, and then were incubated for another 48 h. At the indicated times, the HCV RNA content was determined by real-time RT-PCR.

Fig. 8

Effect of H⁺-ATPase inhibition on the intracellular distribution of Ctry2459-H2 and Ctry2459-H3. Bafilomycin A1 (150 nm) was added to cells together with Ctry2459-H2 and Ctry2459-H3, and then the cells were incubated for 1 h and 24 h. At the indicated time, the cellular localization was determined by confocal microscopy.

Fig. 9

Cytotoxic and hemolytic activities of Ctry2459, Ctry2459-H2 and Ctry2459-H3. Cytotoxic activities of Ctry2459, Ctry2459-H2 and Ctry2459-H3 against the Huh7.5.1 (A) and L02 (B) cell lines. The concentrations ranged from 0 to 500 μg/ml. (C) Hemolytic activity against human erythrocytes. The concentrations ranged from 0 to 1000 μg/ml.

Fig. 10

Schematic diagram illustrating a mechanism of the histidine-rich peptide action via the cellular uptake and endosomal escape pathways. The peptides were taken up by the cells via endocytosis and were restricted in endosomes. With the acidification of endosomes by H⁺-ATPase, common peptides would be digested by the enzyme, whereas Ctry2459-H2 and Ctry2459-H3 with endosomal escape properties can easily break through the endosomal barrier. The released peptides in the cytoplasm would be targeted to intracellular HCV particles and act as an antiviral compound.