Yuxian He

The SARS-CoV-2 fusion-inhibitory lipopeptide IPB29 features a unique design incorporating the α-helix-promoting EAAAK sequence as a rigid linker between the peptide backbone and the conjugated lipid, exhibiting greatly improved antiviral activity compared to lipopeptides with conventional flexible linkers. Here, we characterized its mechanism of action and uncovered several critical insights. First, IPB29 remained the most potent inhibitor of emerging Omicron subvariants compared to IPB24 and IPB28, which share the same peptide sequence but employ flexible polyethylene glycol or glycine-serine repeat linker. Notably, this enhanced potency was abolished in Vero E6 cells stably expressing TMPRSS2, where viral entry occurs via the plasma membrane fusion pathway. Visualization using FITC-labeled lipopeptides demonstrated that IPB29 binds to and enters target cells more efficiently. Subsequently, we generated two unconjugated template peptides, P43 (containing EAAAK) and P37 (lacking EAAAK). P43 exhibited superior inhibitory activity compared to P37 in Huh-7 and 293T/ACE2 cells, but not in Vero E6-TMPRSS2 cells. Both peptides showed comparable efficacy in blocking S protein-driven cell-cell fusion. Although P43 displayed enhanced α-helicity relative to P37, both peptides exhibited similar target-binding affinities. Instead, the cellular uptake of P43 was markedly more efficient, further supporting that the improved antiviral effect is attributable to the inhibition of the viral endosomal fusion pathway. Collectively, our studies provide molecular insights into the antiviral mechanism of IPB29 and propose the short EAAAK sequence as a generalizable strategy for enhancing the efficacy of peptide-based therapeutics. IMPORTANCE: The development of effective therapeutics against SARS-CoV-2 remains an urgent priority. IPB29, a potent broad-spectrum lipopeptide fusion inhibitor currently in clinical trials, requires mechanistic elucidation. Here, we demonstrate that the C-terminal EAAAK motif in IPB29—originally designed as a rigid linker connecting the peptide backbone to the lipid moiety—is critical for its antiviral activity and functions via the endosomal fusion pathway. These findings reveal a generalizable strategy for optimizing antiviral drug design.

Shiftless (SFL) is a broad-spectrum inhibitor of programmed −1 ribosomal frameshift (−1 PRF) and exhibits various antiviral activities. Here, we characterized human SFL structurally and biochemically. The 2.0-angstrom resolution crystal structure of SFL reveals a boat-like module comprising an N-terminal helical bundle and three zinc fingers at the C terminus. A hyperphosphorylation loop (HPL) buried between the helical bundle and the zinc finger 1 harbors four phosphorylated residues (p-S249, p-T250 p-T253, and p-S256), which are important to protein folding. SFL forms monomers in solution and binds the HIV-1 −1 PRF sequence with nanomolar affinity ( K _D = 5.7 nanomolar). Disruption of HPL phosphorylation decreased the RNA binding affinity and undermined the SFL-mediated −1 PRF inhibition of various viruses. Proximity-dependent biotinylation identified three cellular Ser/Thr kinases—EEF2K, NEK9, and PBK—that phosphorylate SFL in cells. These findings shed light on the mechanisms underlying −1 PRF regulation by SFL and provide insights into the role of SFL in virus inhibition.

YKYY017 is a SARS-CoV-2 membrane fusion inhibitor. We report efficacy and safety of inhaled YKYY017 for COVID-19 patients with mild to moderate illness from a phase 2 trial (ChiCTR2300075467). 239 patients aged 18-75 years with mostly mild COVID-19 were randomly allocated to receive aerosol inhalation of 10 or 20 mg YKYY017 or placebo once daily. The primary endpoint is the change in SARS-CoV-2 viral load from baseline to Day 4. The mean (±SE) differences in viral load change from baseline were −0.48 ± 0.27 log₁₀ copies/mL (95% CI, −1.01 to 0.06) for the 20 mg group and −0.27 ± 0.27 log₁₀ copies/mL (95% CI, −0.79 to 0.26) for the 10 mg group, compared to the placebo group. Viral load changes at visits other than Day 4 did not differ significantly from placebo in either the 10 or 20 mg YKYY017 groups. The time to sustained symptom recovery was shorter in the 20 mg YKYY017 group (median 117.53, 95%CI 95.33 to 141.45 hours) than in the placebo group (median 143.00, 95%CI 139.17 to 186.87 hours; HR 1.552, 95%CI 1.089 to 2.214, p = 0.0151), whereas the 10 mg YKYY017 group showed a similar but not statistically significant trend compared to placebo (p = 0.0833). The time to sustained symptom alleviation was shorter in both the 20 and 10 mg YKYY017 groups than in the placebo group. The adverse events were mostly mild to moderate. The primary outcome was not met. Following a supplementary phase 1b trial, we are planning another phase 2/3 trial using a twice-daily 20 mg YKYY017 regimen to further assess efficacy and safety.

PRO 140, a humanized anti-HIV monoclonal antibody targeting the cell coreceptor CCR5, is currently under clinical trials, but it only affects CCR5-tropic viruses. In this study, we have engineered two tandem fusion proteins (2P23-PRO140SC and 2P23-PRO140-Fc) with bifunctional activity by adding short fusion-inhibitory peptide 2P23 to the single-chain fragment variable (scFv) of PRO 140 (PRO140SC) with or without the Fc domain of human IgG4. We first demonstrated that 2P23-PRO140SC and 2P23-PRO140-Fc could efficiently bind to the cell membranes through CCR5 anchoring, which did not affect the expression level of CCR5 on the cell surface. We then verified that the addition of 2P23 peptide to PRO140SC enabled a very potent activity against CXCR4-tropic HIV-1 isolates. As expected, the bispecific fusion proteins exhibited highly potent activities in inhibiting divergent HIV-1 subtypes and viral mutants that were resistant to the fusion inhibitors 2P23 and T20, and they displayed relatively low in vitro cytotoxicity. Furthermore, both the fusion proteins had robust in vivo anti-HIV activities in rats, with 2P23-PRO140-Fc much better than 2P23-PRO140SC. In conclusion, our studies have provided bispecific HIV-1 inhibitors that overcome the drawbacks of PRO 140 antibody and offered novel tools for studying the mechanisms of HIV-1 infection. IMPORTANCE: Given that HIV-1 evolves with high variability and drug resistance, the development of novel antivirals is important. CCR5-directed antibody PRO 140 is currently under clinical trials, but it only inhibits CCR5-tropic HIV-1 isolates. The designed fusion proteins by adding a minimum fusion-inhibitory peptide to PRO 140 enable dramatically increased activities in inhibiting both CCR5-tropic and CXCR4-tropic viruses, thus offering novel antiviral agents with a bispecific functionality that can overcome the drawbacks of PRO 140 antibody.