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Recently, we described a process of trypsin-assisted peptide splicing of analogs of trypsin inhibitor SFTI-1, that seems to be very similar to proteasome-catalyzed peptide splicing. Here, we show, for the first time, that a peptide-peptoid hybrid (peptomer) can also be spliced by trypsin. Incubation of a double sequence SFTI-1 analog, containing two peptoid monomers, with equimolar amount of trypsin leads to formation of monocyclic peptomer as the main product. We proved that the peptide bond formed by a peptoid monomer is not only digested by trypsin but also participates in the enzyme-assisted splicing process. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 206-212, 2015. [ABSTRACT FROM AUTHOR]

Abstract: A series of trypsin inhibitor SFTI-1compounds modified in substrate-specific P1 position was synthesized by the solid-phase method. Lys5 present in the wild inhibitor was replaced by Phe derivatives substituted in para position of the phenyl ring, l-pyridylalanine and N-4-nitrobenzylgycine. Their inhibitory activities with bovine α-chymotrypsin and cathepsin G were estimated by determination of association equilibrium constants (K a). All analogues inhibited bovine α-chymotrypsin. The highest inihbitory activity displayed peptides with the fluorine, nitro and methyl substituents. They were 13–15-fold more active than [Phe5]SFTI-1 used as a reference. They are the most potent chymotrypsin inhibitors of this size. Substitution of Lys5 by Phe did not change the cathepsin G inhibitory activity. Introduction of Phe(p-F), Phe(p-NH2) and Phe(p-CH3) in this position retained the affinity towards this proteinase, whereas Phe(p-guanidine) gave an inhibitor more than twice as active, which appeared to be stable in human serum. On the other hand, a peptomeric analogue with N-4-nitrobenzylglycine failed to inhibit cathepsin G. Despite the fact the introduced amino acids were non-coded, the peptide bonds formed by them were hydrolyzed by chymotrypsin. We postulate that additional interaction of para-substitutents with the enzyme are responsible for the enhanced inhibitory activity of the analogues. [Copyright &y& Elsevier]

The conversion of primary structures of novel, highly selective and sensitive, internally quenched peptide substrates of the human 20S proteasome into peptide aldehydes is presented. Such covalent and reversible inhibitors differ in their primary structures and chemical moieties attached to their N‐terminal amino group. Inhibitory potency is primarily tested against proteolytic subunits of human constitutive 20S proteasome (β1c, β2c, and β5c subunits), but in some cases, also against 20S immunoproteasome β5i. Most of the peptide aldehydes act nonspecifically and bind equipotently to the corresponding proteolytic subunits of both forms of proteasome (β5c and β5i). Two inhibitors are 2.7‐times more specific for immunoproteasome over its constitutive counterpart. The antitumor activity of the selected inhibitors and MG132 (used here as the reference) is analyzed using MTT assay against nonmalignant human fetal osteoblastic hFOB 1.19, malignant human osteosarcoma MG‐63, human pancreatic cancer MiaPaCa‐2, and human acute leukemia Jurkat T cell lines. All inhibitors tested are able to decrease cell viability in a concentration‐dependent manner. One of the peptide aldehydes exerted stronger, as compared to the MG132, activity against human glioblastoma cell line U87‐MG. Moreover, its combination with dinaciclib, which is a novel, potent inhibitor of cyclin‐dependent kinases, reduces cellular metabolic activity more potently as compared to either proteasome inhibitor or dinaciclib alone. [ABSTRACT FROM AUTHOR]

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 6 ]VI, under experimental conditions, appeared significantly more cytotoxic towards cancer cells U87-MG in contrast to the reference peptide., (© 2016 Wiley Periodicals, Inc.)

Sunflower trypsin inhibitor (SFTI-1) is recognized as an attractive scaffold to designed potent inhibitors of various proteases. We have recently found that its analogues inhibit noncovalently both human and yeast 20S proteasomes. Here, a set of novel and more potent in vitro inhibitors is presented. The inhibitory potency of the peptides was assessed with human 20S proteasome in the presence or absence of sodium dodecyl sulfate and with human 26 proteasome. Their antiproliferative action against tumor (human melanoma cells A375) and normal cells (46 BR.1N human fibroblasts and HaCaT keratinocytes) was determined. The selected fluoresceine-labeled inhibitors were able to internalize into A375 cells and were sometimes present as foci in the cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 685-696, 2016., (© 2016 Wiley Periodicals, Inc.)

Recently, we described a process of trypsin-assisted peptide splicing of analogs of trypsin inhibitor SFTI-1, that seems to be very similar to proteasome-catalyzed peptide splicing. Here, we show, for the first time, that a peptide-peptoid hybrid (peptomer) can also be spliced by trypsin. Incubation of a double sequence SFTI-1 analog, containing two peptoid monomers, with equimolar amount of trypsin leads to formation of monocyclic peptomer as the main product. We proved that the peptide bond formed by a peptoid monomer is not only digested by trypsin but also participates in the enzyme-assisted splicing process., (© 2015 Wiley Periodicals, Inc.)

This article describes the synthesis and enzymatic study of newly synthesized analogs of trypsin inhibitors SFTI-1 that were fluorescent labeled on their N-terminal amino groups. Two fluorescent derivatives of benzoxazole (3-[2-(4-diphenylaminophenyl)benzoxazol-5-yl]-L-alanine-[(4NPh2 )Ph]Box-Ala and 3-[2-(2',4',5'-trimethoxyphenyl)benzoxazol-5-yl]-L-alanine-[2,4,5-(OMe)3Ph]Box-Ala) were used as efficient fluorescent labels. The compounds obtained preserved their inhibitory activity and were efficient inhibitors of bovine trypsin or chymotrypsin. Nevertheless, their association inhibition constants were one or two orders of magnitude lower than those determined for unlabeled monocyclic SFTI-1 or [Phe(5)]SFTI-1, respectively. The conjugates obtained were found to be proteolytically stable in the presence of cognate enzymes. Applying such fluorescent peptides, we were able to investigate enzyme-inhibitor complex formation using fluorescent techniques. We found that such compounds were rapidly internalized by the fibroblast or cancer cells with no cytotoxic effects., (Copyright © 2013 Wiley Periodicals, Inc.)

A number of monocyclic SFTI-1 analogues modified in the conserved inhibitor P1' position by Pro, its L-hydroxyproline (Hyp) derivative as well as mimetics with different ring size were synthesized by the solid-phase method. Replacement of Ser6 by Pro, Hyp, and a four-member ring, L-azetidine-2-carboxylic acid (Aze), retained trypsin or chymotrypsin inhibitory activity. The determined association equilibrium constants of these analogues with a cognate enzyme were about two orders of magnitude lower than those obtained for ones with conserved Ser6. In all analogues, with the exception of one, [Phe5,Aze6]SFTI-1, the P1-P1' reactive site remained intact. The results provide first evidence that the conserved Ser in the P1' position of Bowman-Birk inhibitors can be successfully replaced by an amino acid with a secondary amine group.