Spliced analogues of trypsin inhibitor SFTI‐1 and their application for tracing proteolysis and delivery of cargos inside the cells

Abstract

A series of analogues of trypsin inhibitor SFTI-1 were designed and synthesized to monitor peptide splicing. In the middle part of the SFTI-1 analogues, which is released upon incubation with proteinase, the RGD sequence or an acceptor of fluorescence for FRET was introduced. The results of studies with trypsin confirmed that the designed analogues underwent peptide splicing. Furthermore, we showed that a FRET displaying SFTI-1 analogue was internalized into the HaCaT keratinocytes, where it was degraded. Therefore, both proteolysis and the reduction of the disulfide bridge of the peptide took place. As a result, such analogues are a convenient tool to trace the proteolytic activity inside the cell. However, the cytotoxicity of SFTI-1 analogues grafted with the RGD sequence did not correlate with their susceptibility to peptide splicing. Nevertheless, these peptides were slightly more active than the reference peptide (GRGDNP). Interestingly, one of the analogues assigned as [desSer ]VI, under experimental conditions, appeared significantly more cytotoxic towards cancer cells U87-MG in contrast to the reference peptide.

Antimicrobial Activity of Chimera Peptides Composed of Human Neutrophil Peptide 1 (HNP-1) Truncated Analogues and Bovine Lactoferrampin

“…As shown by HPLC and MS analysis, the conjugate was not present in the reaction mixture after 1 h of incubation with trypsin (see Figure S2 in Supporting Information). These results significantly differ from our previous stability studies of trypsin inhibitor SFTI-1 and its analogues. , We have shown that intramolecular disulfide bonds in SFTI-1 and its analogues were not reduced in serum, and their presence in molecules significantly increased proteolytic resistance of these compounds …”

“…These results significantly differ from our previous stability studies of trypsin inhibitor SFTI-1 and its analogues. 17,18 We have shown that intramolecular disulfide bonds in SFTI-1 and its analogues were not reduced in serum, and their presence in molecules significantly increased proteolytic resistance of these compounds. 18 Conformational Analysis.…”

“…5­(6)-Carboxyfluorescein (Novabiochem, Merck Germany) was attached to the HNP peptide (HNP­[Cys5]- CF ) through the ε-amino group of Lys located at its C-terminus or to the α-amino group of lactoferrampin ( CF -LFampB-C). After deprotection (removal of MTT with 2% TFA in dichloromethane) of the ε-amino group of Lys or α - amino group of the second peptide, a mixture of 5(6)-carboxyfluorescein, HATU, HOAt, and DIPEA (molar ratio 1:1:1:2) in DMF was added. The reaction was carried out at room temperature overnight in the dark.…”

“…Formed precipitate was removed by centrifugation for 5 min at 13,000 rpm. The obtained supernatants were analyzed by RP-HPLC and MS after storing on ice for 1 h, applying conditions described above . In order to determine peptide stability at each time point, peptide peak area from recorded chromatogram was compared with the peptide peak area obtained just after mixing peptide and plasma, and expressed as its percentage.…”

Design and chemical syntheses of potent matriptase‐2 inhibitors based on trypsin inhibitor SFTI‐1 isolated from sunflower seeds

“…[53] Human serum from male plasma (Sigma-Aldrich, Germany) was prepared for the assay as described. [53] Human serum from male plasma (Sigma-Aldrich, Germany) was prepared for the assay as described.…”