Modulation of the Fibrillation Kinetics and Morphology of a Therapeutic Peptide by Cucurbit[7]uril

Abstract

Fibrillation is a challenge commonly encountered in the formulation and development of therapeutic peptides. Cucurbit[7]­urils (CB[7]), a group of water soluble macrocycles, have been reported to suppress fibrillation in insulin and human calcitonin through association with Phe and Tyr residues which drive fibril formation. Here, we report the effect of CB[7] on the fibrillation behavior of the HIV fusion inhibitor enfuvirtide (ENF) that contains N-terminal Tyr and C-terminal Phe residues. Thioflavin T fluorescence, CD spectroscopy, and transmission electron microscopy were used to monitor fibrillation behavior. Fibrillation onset showed a strong pH dependency, with pH 6.5 identified as the condition most suitable to monitor the effects of CB[7]. Binding of CB[7] to wild-type ENF was measured by isothermal titration calorimetry and was consistent with a single site (K a = 2.4 × 105 M–1). A weaker interaction (K a = 2.8 × 103 M–1) was observed for an ENF mutant with the C-terminal Phe substituted for Ala (ENFm), suggesting that Phe was the specific site for CB[7] recognition. The onset of ENF fibrillation onset was delayed, rather than fully suppressed, in the presence of CB[7]. The ENFm mutant showed a greater delay in fibrillation onset but with no observable effect on fibrillation kinetics in the presence of CB[7]. Interestingly, ENF/CB[7] and ENFm fibrils exhibited comparable morphologies, differing from those observed for ENF alone. The results indicate that CB[7] is capable of modulating fibrillation onset and the resulting ENF fibrils by specifically binding to the C-terminal Phe residue. The work reinforces the potential of CB[7] as an inhibitor of fibrillation and highlights its role in determining fibril morphologies.

Antiviral Protein–Protein Interaction Inhibitors

“…The problem of fibrillation as a common issue in the development of therapeutic peptides has been addressed recently, and cucurbit[7]­urils (CB[7]), a group of water-soluble macrocycles, was reported. These compounds are capable of modulating fibrillation behavior of the HIV fusion inhibitor enfuvirtide by specifically binding to the C-terminal Phe residue . The second-generation peptide, the fusion inhibitor T1249, exhibited enhanced antiviral potency, but its clinical development was discontinued owing to the drug formulation problem .…”

“…These compounds are capable of modulating fibrillation behavior of the HIV fusion inhibitor enfuvirtide by specifically binding to the C-terminal Phe residue. 46 The second-generation peptide, the fusion inhibitor T1249, exhibited enhanced antiviral potency, but its clinical development was discontinued owing to the drug formulation problem. 47 Sifuvirtide (SFT) is a third-generation peptide-based HIV-1 fusion inhibitor approved for phase III clinical trials in China.…”