Human trefoil factor 3 (hTFF3) is a cysteine-rich peptide that is secreted in the gastrointestinal tract. TFF3 has three disulfide bonds stabilising a three loop trefoil structure (disordered loop 1, alpha helical loop 2, beta hairpin loop 3). TFF3 plays a key role in the restitution process of gastrointestinal wound-healing and holds promising therapeutic potential for the prevention and treatment of gastrointestinal disorders such as inflammatory bowel disease and irritated bowel syndrome. However, no TFF3 structure-activity relationship (SAR) studies have been carried out to date, and its pharmacophore and exact mechanism of action in gastrointestinal wound healing is poorly understood.
Here we present the first TFF3 SAR study with a focus on the three trefoil loops. Loop mimetics (three per loop) were synthesised using Fmoc solid phase peptide synthesis. These loop mimetics include either four artificial terminal cysteine residues or head to tail cyclisation by peptidyl linkers to stabilise the secondary structure. All three possible isomers (globular, ribbon, bead) were obtained in the two-disulfide bond constrained peptides. The stability of the loop mimetics was evaluated in simulated gastric and simulated intestinal fluid. Their bioactivity was assessed by scratch wound healing assay on HT29 cell and PAR2 binding activity. CD and NMR were carried out to characterise the loop mimetic design and confirm the presence of secondary structures. The developed loop mimetic designs are highly applicable to other disulfide-rich proteins and this work provides novel insights in the SAR of TFF3, new synthetic methodologies and thoughts for future optimisations.