ABSTRACT
PURPOSE: Acyclovir is most commonly used for treating ocular Herpes Keratitis, a leading cause of infectious blindness. However, emerging resistance to Acyclovir resulting from mutations in the thymidine kinase gene of Herpes Simplex Virus -1 (HSV-1), has prompted the need for new therapeutics directed against a different viral protein. One novel target is the HSV-1 Processivity Factor which is essential for tethering HSV-1 Polymerase to the viral genome to enable long-chain DNA synthesis. METHODS: A series of peptides, based on the crystal structure of the C-terminus of HSV-1 Polymerase, were constructed with hydrocarbon staples to retain their alpha-helical conformation. The stapled peptides were tested for blocking both HSV-1 DNA synthesis and infection. The most effective peptide was further optimized by replacing its negative N-terminus with two hydrophobic valine residues. This di-valine stapled peptide was tested for inhibiting HSV-1 infection of human primary corneal epithelial cells. RESULTS: The stapled peptides blocked HSV-1 DNA synthesis and HSV-1 infection. The unstapled control peptide had no inhibitory effects. Specificity of the stapled peptides was confirmed by their inabilities to block infection by an unrelated virus. Significantly, the optimized di-valine stapled peptide effectively blocked HSV-1 infection in human primary corneal epithelial cells with selectivity index of 11.6. CONCLUSIONS: Hydrocarbon stapled peptides that simulate the α-helix from the C-terminus of HSV-1 DNA polymerase can specifically block DNA synthesis and infection of HSV-1 in human primary corneal epithelial cells. These stapled peptides provide a foundation for developing a topical therapeutic for treating human ocular Herpes Keratitis.
Subject(s)
Herpesvirus 1, Human , Keratitis, Herpetic , DNA , Epithelial Cells , Herpesvirus 1, Human/genetics , Humans , Keratitis, Herpetic/drug therapy , Peptides/pharmacologyABSTRACT
The respiratory syncytial virus fusion (RSV-F) protein is a primary target for vaccine and drug development against respiratory infection and pediatric pneumonia. The RSV-F core forms a trimer-of-hairpins (TOH) motif in postfusion conformation, which is characterized by a six-helix bundle (6HB) where the three N-terminal HRn helices define a central coiled-coil, while three C-terminal HRc helices pack on the coiled-coil surface in an antiparallel manner. Here, one tightly packed HRn-HRc helix-helix interaction is stripped from the 6HB, which represents the minimum unit of RSV-F TOH motif. The helix-helix interaction sequence can be truncated to derive a core binding region (CBR) that covers intense nonbonded interactions across the interaction interface. Dynamics simulation and energetics analysis reveal that the CBR HRc peptide has a large flexibility and intrinsic disorder in unbound free state, which would incur a considerable entropy penalty upon its binding to CBR NRn peptide. Two strategies are described to constrain the HRc peptide conformation. First, the four non-interfacial residues of HRc peptide are artificially substituted with new amino acid combinations of high helical propensity and, second, the helical conformation of wild-type and mutant HRc peptides is stabilized by adding an all-hydrocarbon bridge across two spatially vicinal, non-interfacial residues 503 (i) and 507 (i + 4). Free energy calculation and fluorescence-based assay confirm that the substitution and stapling can effectively improve the binding affinity of CBR HRn-HRc interaction.
Subject(s)
Hydrocarbons/chemistry , Respiratory Syncytial Virus, Human/metabolism , Viral Fusion Proteins/chemistry , Amino Acid Motifs , Child , Crystallography, X-Ray , Humans , Molecular Dynamics Simulation , Mutagenesis , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Maps , Thermodynamics , Viral Fusion Proteins/genetics , Viral Fusion Proteins/metabolismABSTRACT
OBJECTIVE: New rationally designed i,i+7-hydrocarbon-stapled peptides that target both HIV-1 assembly and entry have been shown to have antiviral activity against HIV-1 subtypes circulating in Europe and North America. Here, we aimed to evaluate the antiviral activity of these peptides against HIV-1 subtypes predominantly circulating in China. METHODS: The antiviral activity of three i,i+7-hydrocarbon-stapled peptides, NYAD-36, NYAD-67, and NYAD-66, against primary HIV-1 CRF07_BC and CRF01_AE isolates was evaluated in peripheral blood mononuclear cells (PBMCs). The activity against the CRF07_BC and CRF01_AE Env-pseudotyped viruses was analyzed in TZM-bl cells. RESULTS: We found that all the stapled peptides were effective in inhibiting infection by all the primary HIV-1 isolates tested, with 50% inhibitory concentration toward viral replication (IC50) in the low micromolar range. NYAD-36 and NYAD-67 showed better antiviral activity than NYAD-66 did. We further evaluated the sensitivity of CRF01_AE and CRF07_BC Env-pseudotyped viruses to these stapled peptides in a single-cycle virus infectivity assay. As observed with the primary isolates, the IC50s were in the low micromolar range, and NYAD-66 was less effective than NYAD-36 and NYAD-67. CONCLUSION: Hydrocarbon-stapled peptides appear to have broad antiviral activity against the predominant HIV-1 viruses in China. This finding may provide the impetus to the rational design of peptides for future antiviral therapy.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Infections/epidemiology , HIV Infections/virology , HIV-1/drug effects , Peptides, Cyclic/pharmacology , Amino Acid Sequence , Anti-HIV Agents/chemistry , China/epidemiology , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/metabolism , HIV-1/genetics , Humans , Peptides, Cyclic/administration & dosage , PhylogenyABSTRACT
<p><b>OBJECTIVE</b>New rationally designed i,i+7-hydrocarbon-stapled peptides that target both HIV-1 assembly and entry have been shown to have antiviral activity against HIV-1 subtypes circulating in Europe and North America. Here, we aimed to evaluate the antiviral activity of these peptides against HIV-1 subtypes predominantly circulating in China.</p><p><b>METHODS</b>The antiviral activity of three i,i+7-hydrocarbon-stapled peptides, NYAD-36, NYAD-67, and NYAD-66, against primary HIV-1 CRF07_BC and CRF01_AE isolates was evaluated in peripheral blood mononuclear cells (PBMCs). The activity against the CRF07_BC and CRF01_AE Env-pseudotyped viruses was analyzed in TZM-bl cells.</p><p><b>RESULTS</b>We found that all the stapled peptides were effective in inhibiting infection by all the primary HIV-1 isolates tested, with 50% inhibitory concentration toward viral replication (IC50) in the low micromolar range. NYAD-36 and NYAD-67 showed better antiviral activity than NYAD-66 did. We further evaluated the sensitivity of CRF01_AE and CRF07_BC Env-pseudotyped viruses to these stapled peptides in a single-cycle virus infectivity assay. As observed with the primary isolates, the IC50s were in the low micromolar range, and NYAD-66 was less effective than NYAD-36 and NYAD-67.</p><p><b>CONCLUSION</b>Hydrocarbon-stapled peptides appear to have broad antiviral activity against the predominant HIV-1 viruses in China. This finding may provide the impetus to the rational design of peptides for future antiviral therapy.</p>