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 (Ka= 2.4 × 105M–1). A weaker interaction
(Ka= 2.8 × 103M–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.