Optimizing delivery of an anti-cytomegalovirus inhibitory peptide using a cell-penetrating peptide

Cytomegalovirus (CMV) is a beta herpesvirus that persists quiescently in healthy individuals but causes severe disease in immunocompromised patients and congenitally infected newborns. Side effects posed by the currently available antivirals necessitate the development of new antivirals with improved safety profiles. This study aims to optimize the potency of an anti-CMV peptide (P10) mimicking conserved region 2 of tegument pp150, fused with an elastin-like polypeptide (ELP). Our previous study has demonstrated that ELP-P10 inhibits murine cytomegalovirus (MCMV) growth <i>in vitro</i> and <i>in vivo</i> with enhanced pharmacokinetic properties relative to the free peptide. To enhance the potency of ELP-P10 at a lower concentration, this study utilizes a cell-penetrating peptide, SynB1, to facilitate the delivery of ELP-P10 into the cells. SynB1 was added to the N terminus of ELP-P10 to generate SynB1-ELP-P10. Antiviral efficacy and cytotoxic effects of ELP-P10 and SynB1-ELP-P10 were studied in cell culture. Pharmacokinetics, biodistribution and antiviral efficacy were studied in a mouse model of CMV infection. While ELP-P10 maintained significant antiviral activity against human cytomegalovirus (HCMV) in cell culture at a higher concentration, SynB1-ELP-P10 shows potency against HCMV and MCMV at a threefold lower concentration compared to ELP-P10. SynB1-ELP-P10 had similar bioavailability after subcutaneous administration as ELP-P10, and SynB1 conjugation to ELP-P10 significantly enhanced its accumulation in the kidneys. Moreover, in an <i>in vivo</i> model of CMV infection, ELP-P10 and SynB1-ELP-P10 treatment led to a significant reduction in the viral titre compared to controls. In conclusion, the strategic modification of ELP-P10 with SynB1 potentiated CMV inhibition, allowing for the use of lower therapeutic doses and mitigating potential side effects.