Chemical Synthesis of TFF3 Reveals Novel Mechanistic Insights and a Gut-Stable Metabolite

TFF3 regulates essential
gastro- and neuroprotective functions,
but its molecular mode of action remains poorly understood. Synthetic
intractability and lack of reliable bioassays and validated receptors
are bottlenecks for mechanistic and structure–activity relationship
studies. Here, we report the chemical synthesis of TFF3 and its homodimer via native chemical ligation followed by oxidative folding.
Correct folding was confirmed by NMR and circular dichroism, and TFF3
and its homodimer were not cytotoxic or hemolytic. TFF3, its homodimer,
and the trefoil domain (TFF310‑50) were susceptible
to gastrointestinal degradation, revealing a gut-stable metabolite
(TFF37‑54; t1/2 >
24
h) that retained its trefoil structure and antiapoptotic bioactivity.
We tried to validate the putative TFF3 receptors CXCR4 and LINGO2,
but neither TFF3 nor its homodimer displayed any activity up to 10
μM. The discovery of a gut-stable bioactive metabolite and reliable
synthetic accessibility to TFF3 and its analogues are cornerstones
for future molecular probe development and structure–activity
relationship studies.