The Molecular Basis of Heat-Stable Enterotoxin for Vaccine Development and Cancer Cell Detection.

Heat-stable enterotoxin (ST) produced by Enterotoxigenic is responsible for causing acute diarrhea in infants in developing countries. However, the chemical synthesis of ST peptides with the native conformation and the correct intra-molecular disulfide bonds is a major hurdle for vaccine development. To address this issue, we herein report on the design and preparation of ST analogs and a convenient chemical method for obtaining ST molecules with the correct conformation. To develop an ST vaccine, we focused on a structure in a type II β-turn in the ST molecule and introduced a D-Lys residue as a conjugation site for carrier proteins. In addition, the -Glu-Leu- sequence in the ST molecule was replaced with a -Asp-Val- sequence to decrease the toxic activity of the peptide to make it more amenable for use in vaccinations. To solve several issues associated with the synthesis of ST, such as the formation of non-native disulfide isomers, the native disulfide pairings were regioselectively formed in a stepwise manner. A native form or topological isomer of the designed ST peptide, which possesses a right-handed or a left-handed spiral structure, respectively, were synthesized in high synthetic yields. The conformation of the synthetic ST peptide was also confirmed by CD and NMR spectroscopy. To further utilize the designed ST peptide, it was labeled with fluorescein for fluorescent detection, since recent studies have also focused on the use of ST for detecting cancer cells, such as Caco-2 and T84. The labeled ST peptide was able to specifically and efficiently detect 293T cells expressing the recombinant ST receptor (GC-C) protein and Caco-2 cells. The findings reported here provide an outline of the molecular basis for using ST for vaccine development and in the detection of cancer cells.