Bowman-Birk inhibitors (BBIs) are found primarily in seeds of legumes and in cereal grains. These canonical inhibitors share a highly conserved nine-amino acids binding loop motif CTP1SXPPXC (where P1 is the inhibitory active site, while X stands for various amino acids). They are natural controllers of plants’ endogenous proteases, but they are also inhibitors of exogenous proteases present in microbials and insects. They are considered as plants’ protective agents, as their elevated levels are observed during injury, presence of pathogens, or abiotic stress, i.a. Similar properties are observed for peptides isolated from amphibians’ skin containing 11-amino acids disulfide-bridged loop CWTP1SXPPXPC. They are classified as Bowman-Birk like trypsin inhibitors (BBLTIs). These inhibitors are resistant to proteolysis and not toxic, and they are reported to be beneficial in the treatment of various pathological states. In this review, we summarize up-to-date research results regarding BBIs’ and BBLTIs’ inhibitory activity, immunomodulatory and anti-inflammatory activity, antimicrobial and insecticidal strength, as well as chemopreventive properties.

The brittle nature of the polymer beads used in SPPS dictates mild mixing techniques with low mass transfer. We demonstrate that vigorous overhead mechanical stirring with superior mass transfer properties kept the beads intact and significantly accelerates reaction kinetics and efficiency.

Hydrophilic peptides constitute most of the active peptides. They mostly permeate via tight junctions (paracellular pathway) in the intestine. This permeability mechanism restricts the magnitude of their oral absorption and bioavailability. We hypothesized that concealing the hydrophilic residues of the peptide using the lipophilic prodrug charge masking approach (LPCM) can improve the bioavailability of hydrophilic peptides. To test this hypothesis, a cyclic N-methylated hexapeptide containing Arg-Gly-Asp (RGD) and its prodrug derivatives, masking the Arg and Asp charged side chains, were synthesized. The library was evaluated for intestinal permeability in vitro using the Caco-2 model. Further investigation of metabolic stability ex vivo models in rat plasma, brush border membrane vesicles (BBMVs), and isolated CYP3A4 microsomes and pharmacokinetic studies was performed on a selected peptide and its prodrug (peptide 12). The parent drug analogues were found to have a low permeability rate in vitro, corresponding to atenolol, a marker for paracellular permeability. Moreover, palmitoyl carnitine increased the P of peptide 12 by 4-fold, indicating paracellular permeability. The P of the prodrug derivatives was much higher than that of their parent peptides. For instance, the P of the prodrug 12P was 20-fold higher than the P of peptide 12 in the apical to basolateral (AB) direction. Whereas the permeability in the opposite direction (BA of the Caco-2 model) was significantly faster than the P AB, indicating the involvement of an efflux system. These results were corroborated when verapamil, a P-gp inhibitor, was added to the Caco-2 model and increased the P AB of prodrug 12P by 3-fold. The prodrug 12P was stable in the BBMVs environment, yet degraded quickly (less than 5 min) in the plasma into the parent peptide 12. Pharmacokinetic studies in rats showed an increase in the bioavailability of peptide 12 > 70-fold (from 0.58 ± 0.11% to 43.8 ± 14.9%) after applying the LPCM method to peptide 12 and converting it to the prodrug 12P. To conclude, the LPCM approach converted the absorption mechanism of the polar peptides from a paracellular to transcellular pathway that tremendously affects their oral bioavailability. The LPCM method provides a solution for the poor bioavailability of RGD cyclohexapeptides and paves the way for other active hydrophilic and charged peptides with poor oral bioavailability.

Seven conjugates composed of well-known fluoroquinolone antibacterial agents, ciprofloxacin (CIP) or levofloxacin (LVX), and a cell-penetrating peptide transportan 10 (TP10-NH2) were synthesised. The drugs were covalently bound to the peptide via an amide bond, methylenecarbonyl moiety, or a disulfide bridge. Conjugation of fluoroquinolones to TP10-NH2 resulted in congeners demonstrating antifungal in vitro activity against human pathogenic yeasts of the Candida genus (MICs in the 6.25–100 µM range), whereas the components were poorly active. The antibacterial in vitro activity of most of the conjugates was lower than the activity of CIP or LVX, but the antibacterial effect of CIP-S-S-TP10-NH2 was similar to the mother fluoroquinolone. Additionally, for two representative CIP and LVX conjugates, a rapid bactericidal effect was shown. Compared to fluoroquinolones, TP10-NH2 and the majority of its conjugates generated a relatively low level of reactive oxygen species (ROS) in human embryonic kidney cells (HEK293) and human myeloid leukemia cells (HL-60). The conjugates exhibited cytotoxicity against three cell lines, HEK293, HepG2 (human liver cancer cell line), and LLC-PK1 (old male pig kidney cells), with IC50 values in the 10–100 µM range and hemolytic activity. The mammalian toxicity was due to the intrinsic cytoplasmic membrane disruption activity of TP10-NH2 since fluoroquinolones themselves were not cytotoxic. Nevertheless, the selectivity index values of the conjugates, both for the bacteria and human pathogenic yeasts, remained favourable.

Mixing of polystyrene resins in solid-phase synthesis is performed by shaking or gentle agitation of the reaction vessel to avoid breaking the brittle beads. These mixing strategies result in poor diffusion to and into the beads. Using a large excess of reagents is the common way to compensate for these deficiencies. We use fast overhead stirring for performing coupling reactions on a solid support. We show that fast overhead stirring enhances the efficiency of amide bond formation on the solid support compared to the state-of-the-art mixing method, while preserving the integrity of the beads. We find that fast overhead stirring minimizes the effect of decomposition of the activated species by increasing the diffusion-dependent coupling reaction. This allows decreasing the excess of reagents used for the multistep synthesis of peptides, thus providing a greener and more sustainable alternative for peptide synthesis on solid supports.

Recent studies have shown that modified human lactoferrin 20–31 fragment, named HLopt2, possesses antibacterial and antifungal activity. Thus, we decided to synthesize and evaluate the biological activity of a series of conjugates based on this peptide and one of the antimicrobials with proven antibacterial (ciprofloxacin, CIP, and levofloxacin, LVX) or antifungal (fluconazole, FLC) activity. The drugs were covalently connected to the peptide via amide, methylenecarbonyl moieties, or a disulfide bridge. The antibacterial and antifungal activities were evaluated under Clinical and Laboratory Standard Institute (CLSI) recommended conditions or in a low-salt brain–heart infusion diluted medium (BHI1/100). Results showed that conjugation of the peptide with the drug increased its antimicrobial activity up to 4-fold. Under CLSI-recommended conditions, all the compounds revealed rather low efficiency. Among conjugates, the highest antibacterial activity was recorded for the CIP-Cys-S-S-HLopt2-NH2 (III). In BHI1/100, which had lower differentiating properties, all of the conjugates revealed low MIC and MMC (minimum inhibitory and microbicidal concentrations) values. The disulfide bridge used as a linker in the most active conjugate (III) upon incubation with S. aureus cells is reduced, releasing constituent peptide and CIP-Cys. In addition, we showed that its fluorescently labeled analogue and constituent peptide are able to be internalized into both C. albicans and S. aureus cells. Moreover, the invaluable advantage of the presented conjugates was their low toxicity to mammalian cells and very low hemolytic activity. The current research can form a solid basis for further in vivo studies and drug development.

Human neurohormone vasopressin (AVP) is synthesized in overlapping regions in the hypothalamus. It is mainly known for its vasoconstricting abilities, and it is responsible for the regulation of plasma osmolality by maintaining fluid homeostasis. Over years, many attempts have been made to modify this hormone and find AVP analogues with different pharmacological profiles that could overcome its limitations. Non-peptide AVP analogues with low molecular weight presented good affinity to AVP receptors. Natural peptide counterparts, found in animals, are successfully applied as therapeutics; for instance, lypressin used in treatment of diabetes insipidus. Synthetic peptide analogues compensate for the shortcomings of AVP. Desmopressin is more resistant to proteolysis and presents mainly antidiuretic effects, while terlipressin is a long-acting AVP analogue and a drug recommended in the treatment of varicose bleeding in patients with liver cirrhosis. Recently published results on diverse applications of AVP analogues in medicinal practice, including potential lypressin, terlipressin and ornipressin in the treatment of SARS-CoV-2, are discussed.

Matriptase-2 plays a pivotal role in keeping iron concentrations within a narrow physiological range in humans. The opportunity to reduce matriptase-2 proteolytic activity may open a novel possibility to treat iron overload diseases, such as hereditary hemochromatosis and thalassemia. Here, we present 23 new analogues of trypsin inhibitor SFTI-1 designed to inhibit human matriptase-2. Influence of the modifications Gly1Lys, Ile10Arg, and Phe12His, as well as the introduction of Narg in P1 or P1 and P4 positions were examined. Selected peptides were further analyzed, together with previously reported peptides, for their inhibitory activity against related human proteases, that are, matriptase-1, plasmin, thrombin and trypsin. A highly potent inhibitor of matriptase-2, the bicycylic [Arg , Arg , His ]SFTI-1, with a K value of 15 nm was obtained.