Abstract
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.
Highlights
The trefoil factor family (TFF) comprises three disulfide-rich peptides (TFF1, TFF2, TFF3) that are abundantly secreted in the gastrointestinal tract where they regulate gut homeostasis by promoting gut protection and repair.[1−5] They are expressed in mucosal tissues outside the gut, including in the respiratory tract, urinary tract, uterus, eyes, and salivary glands, where they have similar mucosal repair and protective functions
We switched to a two-fragment native chemical ligation (NCL) approach using the Fmoc-SPPS (9-fluorenylmethyloxycarbonyl-solid-phase peptide synthesis)-compatible hydrazide strategy (Figure 1).[50]
TFF37‐54 was active in the anti-apoptosis assay, equivalent to TFF3 and TFF3 homodimer (Figure 4E). These results suggest that the N- and C-terminal extended domain residues in TFF37‐54 provide some steric protection against intestinal proteases and that the pharmacophore sits within the trefoil domain
Summary
The trefoil factor family (TFF) comprises three disulfide-rich peptides (TFF1, TFF2, TFF3) that are abundantly secreted in the gastrointestinal tract where they regulate gut homeostasis by promoting gut protection and repair.[1−5] They are expressed in mucosal tissues outside the gut, including in the respiratory tract, urinary tract, uterus, eyes, and salivary glands, where they have similar mucosal repair and protective functions. TFF3 is highly expressed in the gastrointestinal mucosa, in the small intestine and colon, where it protects, maintains, and repairs the gastrointestinal tract.[4,10−17] In the central nervous system (CNS), TFF3 is secreted by neurons and regulates physiological effects such as neuroinflammation[18] and behavioral processes including learning and memory[19] and depression.[20,21] The anti-inflammatory effects of TFF3 on microglia cells (reduced expression and secretion of proinflammatory cytokines) and its capacity to mitigate ischemic cerebral injuries by reducing cell death via suppression of caspase-3 activity further support TFF3’s neuroprotective role in the CNS.[18,22]. The mature secreted and folded TFF3 peptide (TFF31‐59) contains a highly conserved trefoil domain (TFF310‐50) that defines the other members of the TFF.[3,24] The trefoil domain contains six conserved cysteine residues (CX9‐10CX9CX4CCX10C motif) three intrachain disulfide bonds in the configuration forming CysI−V, CysII‐IV, and CysIII−VI.[3,24] This disulfide bond arrangement creates a compact three-loop structure resembling a trefoil shape, which is considered to be metabolically stable based on TFF3’s functional role in the gastrointestinal tract.[3,24,25] Its gastrointestinal stability has, not been systematically investigated, and some reports indicate that TFF3 might not be that stable.[26−28]
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