Peptidomics of food-derived biopeptides explores compounds generated during food hydrolysis, highlighting protease-mediated hydrolysis by lactic acid bacteria as a cost-effective and efficient method for producing bioactive peptides. This study describes the peptidomics of peptides from whole milk fermentation by Limosilactobacillus fermentum (LBF 433), identified using nanoLC-MS/MS and analyzed with in silico prediction tools. A total of 232 peptides were identified, averaging 13.56 residues and a molecular mass of 1544.18 Da. The analysis revealed an average isoelectric point of 7.1 and a net charge of 0.894 at pH 7.0. In terms of composition, the peptides averaged 2.60 aliphatic, 1.71 aromatic, 5.66 non-polar, 4.49 polar, and 2.77 charged residues. The average hydrophobicity percentage was 42.23%, and 56.03% of the peptides showed good water solubility. The acid-base profile indicated 61.63% were basic, 13.36% acidic, and 25% neutral. These findings suggest that the peptides are stable in acidic environments, such as the gastrointestinal tract, due to their average molecular mass and low isoelectric point. The positive net charge at neutral pH and presence of basic amino acids suggest antimicrobial potential. Hydrophobic residues favor interactions with membranes, implying immunomodulatory and anti-inflammatory effects, making these peptides suitable for nutraceutical and pharmaceutical applications.

The modulation of peptidic scaffolds through stapling reactions has been established as a powerful tool for recapitulating the bioactivity of native ⍺-helices in targeting protein-protein interactions (PPIs). However, accessing such helices have largely relied on protecting group manipulations or the use of non-natural building blocks during peptide synthesis. As such, there is a growing need for a stapling strategy involving only natural amino acids in their unprotected states. Herein we report a rapid, mild, and highly chemoselective stapling reaction using a new class of molecular linchpins called 2-ketobenzaldehydes that installs a highly fluorescent thiol-isoindole crosslink. This methodology also exhibited good positional selectivity favoring the helical i and i+4 linkage on fully unprotected peptides in the presence of competing reaction sites, offering exceptional late-stage functionality for easier access of stapled ⍺-helices. In our efforts to further validate this chemistry, we have successfully shown in vitro cytotoxicity (IC₅₀ = 5.10 µM) equipotent to an all-hydrocarbon stapled peptide candidate. Furthermore, in harnessing the innate fluorescence of thiol-isoindole, this staple can be directly used as a probe for cell imaging in the qualitative assessment of stapled-peptide cell permeability, thus bridging therapeutic potential with analytical probe development.

Amyloidoses consist of a group of diseases associated with protein misfolding leading to the formation and accumulation of insoluble fibrils in multiple organs. In fact, few therapeutic approaches exist to treat patients suffering from amyloidosis, and one of the reasons for this is the complexity of the aggregation process, involving many conformations. In this study, using the islet amyloid polypeptide (IAPP) as a model polypeptide, we evaluated a therapeutic strategy based on monomer stabilization. Although IAPP mainly adopts a random conformation in aqueous media, this peptide displays a transient helix-α secondary structure, offering the possibility of stabilizing monomeric IAPP. In this context, a library of helically constrained IAPP derivatives was designed, including fragments of different chain lengths (short, medium or long). Conformational restriction was induced by a stapled technique through the formation of an intramolecular triazole between residues at positions i and i+4 carried out on solid support. By monitoring amyloid formation with the thioflavin T, some compounds showed strong inhibition, a result confirmed by the absence of fibers by atomic force microscopy and by CD spectroscopy. The results obtained suggest that this innovative approach could make it possible to stabilize proteins displaying primarily an natively disordered conformation.

In Canada, it is estimated that 30 to 50% of all healthcare involves wounds. Specifically, chronic wounds critically affect living conditions of patients and add to the burden on healthcare systems. Matrices and hydrogels have emerged as promising treatment options, acting as scaffolds for cells and delivery systems for therapeutic agents. Self-assembled peptide-based matrices have attracted great interest owing to their biocompatibility, degradability, robustness and low immunogenicity. In this study, we aim to produce fully synthetic peptide-based matrices functionalized with bioactive sequences for wound healing. The I10 synthetic peptide, known to assemble into cross-β-sheet fibrils, was functionalized with cell-adhesion (PRa), cell migration-promoting (FGF2) or antimicrobial (IG19) sequences. All I10-based peptides individually self-assembled into β-sheet- and β-turn-rich fibrils, as observed by circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). Coassembly of the functionalized peptides with a charged version of I10 (K-I10-COOH) allowed formation of hydrogels containing both β-sheet- and β-turn-rich fibres, as confirmed by CD spectroscopy, AFM, and scanning (SEM) and transmission (TEM) electron microscopy. These peptide assemblies showed good adhesion properties for skin cells, including keratinocytes (HaCaT) and fibroblasts (1BR3, L929), encouraging further evaluation of cell migration properties, antimicrobial activity and healing potential in a mouse full-thickness wound model.

Many alternatives to human donor corneas are being developed to meet the global shortage of donated tissues. However, corneal transplantation remains the gold standard for diseases resulting in thinning corneas. In this work, transparent low-energy photoactivated extracellular matrix peptide-mimicking materials are developed for intrastromal injection to restore stromal thickness. The injectable biomaterials are comprised of short peptides and glycosaminoglycans (chondroitin, hyaluronic acid) that assemble into a hydrogel when pulsed with low-energy blue light. The dosage of pulsed-blue light needed for material activation is minimal at 8.5 mW cm^-2, thus circumventing any blue light cytotoxicity. Intrastromal injection of these light-activated biomaterials in rat corneas show that two iterations of the formulations remain stable in situ without stimulating significant inflammation or neovascularization. The use of low light intensities and the ability of the developed materials to stably rebuild and change the curvature of the cornea tissue make these formulations attractive for clinical translation.

Lectins are a varied group of proteins that play key roles in biological processes exhibiting unique structural and functional properties. Lectins are proteins that can specifically bind to carbohydrates, and play a role in processes such as cell adhesion, immune responses, and intracellular signaling. Lectins have potential to regulating blood sugar levels, defend against pathogens, and prevent cancer progression, making them promising candidates for therapeutic applications. Terminalia catappa , a large tropical tree, is known for its medicinal properties. This study aimed to purify and characterize lectins from TC seeds. The research involved extracting and partially purifying the lectin, followed by tests such as hemagglutination assays, stability under varying temperature and pH conditions, EDTA dependence, metal ion effects, sugar specificity, and antibacterial activity. Hemagglutination was observed in human blood group B+. The findings suggest that TC seed lectin is stable within a moderate temperature and pH range. Its EDTA dependence indicates it may be a metalloprotein, interacting with metal ions, except Hg²⁺. Although initial antibacterial tests showed limited activity, further research is needed to fully explore its therapeutic potentials.

The bicyclic octapeptide α-amanitin is one of the deadliest toxins found in nature and is of interest as a therapeutic agent. However, the high hepatotoxicity of amanitin must be reduced, such as by increasing drug selectivity using prodrug strategies. Hypoxia is a key feature of cancerous tumours as their rapid proliferation outstrips the oxygen provided by surrounding vasculature. Nitroreductase enzymes catalyze the reduction of aromatic nitro moieties to their respective amines and are upregulated in hypoxic cells. We hypothesize that a nitrated amanitin analogue will be selectively activated by nitroreductases in hypoxic cancer cells.

We describe a late-stage nitration of the amanitin heptapeptide monocycle precursor towards the synthesis of nitrated amanitin prodrugs. Using nitrosaccharin as an alternative nitrating reagent, the nitration proceeded selectively, under mild conditions, and with tolerance to solid-phase peptide synthesis protecting groups. The bioreduction susceptibility of the heptapeptide was assessed through incubation with nitroreductase and easily detected via the distinctive absorbance profile of the nitrated tryptathionine. Cell viability assays will determine the toxicity of the nitrated amanitins under normoxic and hypoxic conditions.

α-Amanitin, a highly toxic bicyclic octapeptide extracted from death-cap mushroom (Amanita phalloides), is one of the deadliest natural compounds known, with an LD50 of 50–100 μg/kg. It is a potent inhibitor of RNA polymerase II (Ki 1−10 nM), an enzyme critical for cellular function and survival, thereby affecting both dividing and quiescent cells. Due to its stability, potency, and unique mechanism, it shows promise as a payload for antibody-drug conjugates (ADCs) in cancer therapy. However, current extraction methods yield low amounts, underscoring the need for a synthetic production route.

The synthesis of di-hydroxy isoleucine (DHIle), a vital component, is particularly challenging. We successfully achieved a gram-scale synthesis of DHIle, streamlining the overall process and generating intermediates that were synthetically modified to produce 12 novel amanitin analogs. These analogs, compatible with solid-phase peptide synthesis, were utilized in SAR studies, revealing compounds with similar potency and selective toxicity toward HEK-293 cells. This work also introduced new bioconjugation handles for enhancing ADC applications with increased cytotoxicity and selectivity over liver-derived HepG2 cells, improving amanitin’s therapeutic index for cancer treatment. This work led to the development of new ADCs with efficacy comparable to those currently available on the market.

Cyclic azapeptide cluster of differentiation-36 receptor (CD36) modulator 298 has exhibited biomedical potential for treating diseases implicating macrophage-driven inflammation.^1,2 Previously, azapeptide 298 was prepared in mg amounts using a linear solid-phase peptide synthesis approach featuring an A³-macrocyclization on resin prior to cleavage and deprotection.^3,4 Towards scale-up, a solution-phase synthesis of azapeptide 298 has now been achieved using a fragment coupling strategy. Employing phosgene-free semicarbazide synthesis, orthogonal protection, and A³ macrocyclization in solution, the convergent approach minimizes chromatographic purification to effectively afford the cyclic azapeptide. Our presentation discloses the features of this promising means for delivering the potent CD36 modulator for preclinical investigations

Self-assembling antimicrobial peptides with aromatic amino acid residues, such as phenylalanine, often display intrinsic fluorescence due to delocalization of electron densities in dense hydrogen bonding networks and dipolar coupling of aromatic residues. These self-assembling peptides are a promising platform to develop fluorescent nanomaterials for various biomedical and optical engineering applications. Our research explores the effect of lipophilicity and net charge on the self-assembly behavior and intrinsic fluorescence properties of mini chicken Angiogenin 4 (mCA-4). mCA-4 is a synthetic, ten-residue antimicrobial peptide derived from chicken Angiogenin-4, an avian antimicrobial protein, and forms β-sheets which further self-assemble to form spherical nanostructures. Changing lipophilicity by substituting valine with isoleucine residues has no notable effects on the self-assembly and intrinsic fluorescence properties of mCA4 nanostructures. However, the replacement of a single arginine residue with citrulline was observed to impart intrinsic fluorescence. Reducing cationic charge from substituting arginine with citrulline also results in morphological changes in self-assembled structures from nanospheres to larger nanorods and fibers. In this study, simple and systematic substitutions of short peptide sequences are shown to be a potential avenue to develop new classes of intrinsically fluorescent self-assembled nanomaterials.