Oral peptide delivery remains challenging because peptides are rapidly degraded by gastrointestinal proteases, and most peptide therapeutics require systemic absorption to be effective. However, for diseases confined to the gastrointestinal tract, local drug action within the intestinal lumen eliminates the need for systemic exposure, making oral, gut-restricted peptide therapeutics an increasingly attractive strategy. For gut-localised therapeutics, proteolytic degradation rather than absorption constitutes the principal barrier, requiring peptides to remain intact within the intestinal lumen long enough to reach their target site of action. Venom peptides, shaped by intense predator-prey selection into thousands of structurally and functionally distinct scaffolds, represent a compelling yet underexploited source of candidates for this purpose.
We developed a mass spectrometry-guided, stability-first discovery pipeline that prioritised resistance to gastrointestinal proteolysis rather than sequence homology or peptide family classification. Peptide mass fingerprints were generated by ESI-MS from the venoms of three phylogenetically diverse species: the scorpion Leiurus quinquestriatus, the pit viper Bothrops jararaca, and the spider Agelena labyrinthica. Each venom was incubated in simulated gastric and intestinal fluid for 12 h. Individual peptide mass signals were monitored to quantify the proportion of signal remaining after digestion, with peptides retaining more than 50% of their initial intensity prioritised ahead of sequence-level characterisation. These stable peptide masses were then interrogated by tandem mass spectrometry, with sequence tags matched to putative precursors using BLAST searches against species-specific NCBI transcriptomes, linking digestion-resistant peptides to their identities and structural families across all three venoms.
This workflow identified gut-stable sequences spanning diverse structural classes, including cysteine-stabilised αβ peptides from scorpion venom, inhibitor cystine knot peptides from spider venom, and short bradykinin-potentiating peptides from pit viper venom. Selected leads were chemically synthesised and re-evaluated in simulated gastric and intestinal fluids, confirming that digestion resistance was retained in isolated, synthetic form rather than being an artefact of the crude venom matrix.
Our study demonstrates that venoms represent a promising source of gut-stable peptides. Closer examination revealed that gastrointestinal stability is distributed across structurally diverse venom peptide scaffolds and is governed by subtle structural features rather than scaffold class, disulfide-bond content, or biological function. Beyond their intrinsic bioactivities, these gut-stable scaffolds constitute a versatile platform for epitope grafting, offering a route to oral peptide therapeutics targeting the gastrointestinal tract for the treatment of gastrointestinal disorders.