The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to global public health and the economy. The enzymatic product of cholesterol 25-hydroxylase (CH25H), 25-Hydroxycholesterol (25-HC), was reported to have potent anti-SARS-CoV-2 activity. Here, we found that the combination of 25-HC with EK1 peptide, a pan-coronavirus (CoV) fusion inhibitor, showed a synergistic antiviral activity. We then used the method of 25-HC modification to design and synthesize a series of 25-HC-modified peptides and found that a 25-HC-modified EK1 peptide (EK1P4HC) was highly effective against infections caused by SARS-CoV-2, its variants of concern (VOCs), and other human CoVs, such as HCoV-OC43 and HCoV-229E. EK1P4HC could protect newborn mice from lethal HCoV-OC43 infection, suggesting that conjugation of 25-HC with a peptide-based viral inhibitor was a feasible and universal strategy to improve its antiviral activity.

To develop new HIV-1 fusion inhibitors with improved antiviral activities and resistance profiles, we designed two categories of artificial peptides, each containing four heptad repeats (m4HR) conjugated with a pocket-specific small molecule (pssm) or pssm and cholesterol (chol), designated pssm–m4HR or pssm–m4HR–chol, respectively, and tested their anti-HIV-1 activity. We synthesized the artificial peptides and conjugated these peptides with pssm and chol using a standard solid-phase Fmoc protocol and a chemoselective thioether conjugation method, respectively. We tested the inhibitory activities of the peptide conjugates against HIV-1 Env-mediated cell–cell fusion and infection by laboratory-adapted and primary HIV-1 isolates, and enfuvirtide-resistant HIV-1 strains using cell–cell fusion and p24 production assays, respectively. We assessed their cytotoxicity towards MT-2 cells using the XTT assay. We found that pssm–m4HR conjugates exhibited promising inhibitory activity against HIV-1 Env-mediated cell–cell fusion and laboratory-adapted HIV-1 replication with IC₅₀ values at the low micromolar level, whereas the pssm–m4HR–chol conjugates exhibited dramatically increased anti-HIV-1 activities with IC₅₀ values at the low nanomolar level. Some of the pssm–m4HR–chol conjugates (e.g. 5a and 5b) showed highly potent antiviral activity against infection by primary HIV-1 isolates and enfuvirtide-resistant HIV-1 strains. All the conjugates displayed no or low cytotoxicity towards MT-2 cells. The result of a prime/wash assay indicated pssm–m4HR–chol conjugates were strongly anchored to the membrane and sustained a potent inhibitory effect after washing. These results suggest this scaffold design is a promising strategy for developing novel peptide conjugates with improved antiviral activity against a broad spectrum of HIV-1 strains, including those highly resistant to enfuvirtide.

Specific interactions were introduced between an artificial heptad repeat peptide template and HIV-1 gp41 for fusion inhibitor design, using a structure based rational design strategy. The designed peptides are nonhomologous with naturally occurring peptide and protein sequences, specifically interact with HIV-1 gp41, and show strong anti-HIV activity.