Dawid Dębowski

Introduction of non-natural amino acid residues into the substrate-specific P1 position of trypsin inhibitor SFTI-1 yields potent chymotrypsin and cathepsin G inhibitors

A series of trypsin inhibitor SFTI-1compounds modified in substrate-specific P(1) 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 alpha-chymotrypsin and cathepsin G were estimated by determination of association equilibrium constants (K(a)). All analogues inhibited bovine alpha-chymotrypsin. The highest inihbitory activity displayed peptides with the fluorine, nitro and methyl substituents. They were 13-15-fold more active than [Phe(5)]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-NH(2)) and Phe(p-CH(3)) 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.

Inhibitors of Matriptase-2 Based on the Trypsin Inhibitor SFTI-1

A series of 17 new analogues of trypsin inhibitor SFTI‐1 were designed and synthesized to obtain matriptase‐2 inhibitors. A number of the modified bicyclic peptides displayed much higher affinity towards matriptase‐2 than towards the highly homologous matriptase‐1. Replacement of Lys5 by Arg in the wild‐type SFTI‐1 led to an 11‐fold increase in the matriptase‐2 inhibitory activity. Replacement of Arg2 by its enantiomer (D‐arginine) slightly lowered the inhibition of matriptase‐2, but almost completely abolished the affinity towards matriptase‐1, thus yielding the most selective matriptase‐2 inhibitor. This is the first report describing inhibitors of the recently discovered matriptase‐2 based on the SFTI‐1 structure. The results showed that SFTI‐1 is a promising scaffold for the design of potent and selective inhibitors of this enzyme.

Peptomeric analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds

A series of linear and monocyclic analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds, modified by N-(4-aminobutyl)glycine (Nlys) and N-benzylglycine (Nphe), were obtained by the solid-phase method. Some of these peptomers displayed trypsin or chymotrypsin inhibitory activity. In contradiction to the literature data, in most analogues peptide bonds formed by these peptoid monomers were at least partially hydrolyzed by the experimental enzymes at two different pH (3.5 and 8.3). Nevertheless, the replacement of Phe present in the P(1) substrate specificity of linear inactive SFTI-1 analogue with Nphe, yielded a potent chymotrypsin inhibitor. The introduction of one cyclic element (a disulfide bridge or head-to-tail cyclization) to the analogues synthesized significantly increased their proteinase resistance.

Selection of peptomeric inhibitors of bovine α-chymotrypsin and cathepsin G based on trypsin inhibitor SFTI-1 using a combinatorial chemistry approach

A peptomeric library consisting of 360 monocyclic analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds was designed and synthesized by a solid-phase approach in order to select chymotrypsin and cathepsin G inhibitors. All peptomers contained a proteinogenic-Phe-mimicking N-benzylglycine (Nphe) at positions 5 and 12. Into the synthesized library, different peptoid monomers were introduced in the 7–10 segment. Deconvolution of the library against both proteinases through an iterative method in solution revealed that the strongest chymotrypsin inhibitory activity was displayed by two analogues, [Nphe5,12]SFTI-1 (1) and [Nphe5,12, Naem8]SFTI-1 (2), where Naem stands for N-(2-morpholinoethyl)glycine. After deconvolution against a cathepsin G analogue, [Nphe5,12, Npip8,9, Nnle10] SFTI-1 (3) (Npip = N-(3,4-methylenedioxybenzyl)glycine) appeared to be the most potent inhibitor with a high serum stability. It is worth noting that the analogues obtained by a combinatorial approach display high specificity towards one of the experimental enzymes. Another interesting feature is the lack of Pro8 in analogues 2 and 3, the amino acid residue absolutely conserved in the family of Bownan–Birk inhibitors.

Implication of the disulfide bridge in trypsin inhibitor SFTI-1 in its interaction with serine proteinases

Fourteen monocyclic analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds were synthesized by the solid-phase method. The purpose of this work was to establish the role of a disulfide bridge present in inhibitors side chains of Cys3 and Cys11 in association with serine proteinases. This cyclic fragment was replaced by the disulfide bridges formed by l-pencillamine (Pen), homo-l-cysteine (Hcy), N-sulfanylethylglycine (Nhcy) or combination of the three with Cys. As in the substrate specificity the P(1) position of the synthesized analogues Lys, Nlys [N-(4-aminobutyl)glycine], Phe or Nphe (N-benzylglycine) were present, and they were checked for trypsin and chymotrypsin inhibitory activity. The results clearly indicated that Pen and Nhcy were not acceptable at the position 3, yielding inactive analogues, whereas another residue (Cys11) could be substituted without any significant impact on the affinity towards proteinase. On the other hand, elongation of the Cys3 side chain by introduction of Hcy did not affect inhibitory activity, and an analogue with the Hcy-Hcy disulfide bridge was more than twice as effective as the reference compound ([Phe⁵] SFTI-1) in inhibition of bovine α-chymotrypsin.

Inhibition of human and yeast 20S proteasome by analogues of trypsin inhibitor SFTI-1.

Starting from the primary structure of sunflower trypsin inhibitor SFTI-1, we designed novel non-covalent inhibitors of human and yeast 20S proteasomes. Peptides with Arg residue in P1 position and two basic amino acid residues (Lys or/and Arg) in P2′ and P3′ positions strongly inhibited chymotrypsin-like and caspase-like activities, while trypsin-like activity was poorly modified. We found that some SFTI-1 analogues up-regulated exclusively the chymotrypsin-like activity of latent yeast 20S proteasome.

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin.

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine β-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3-18) and its analog [Phe(8)]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.

Bladder cancer detection using a peptide substrate of the 20S proteasome.

The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2‐aminobenzoic acid (ABZ)‐Val‐Val‐Ser‐Tyr‐Ala‐Met‐Gly‐Tyr(3‐NO2)‐NH2, is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 105 m−1·s−1) and a high kcat (8 s−1). Using this peptide, we identified chymotrypsin‐like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods.

Noncovalent inhibitors of human 20S and 26S proteasome based on trypsin inhibitor SFTI-1.

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.

Investigation of Serine-Proteinase-Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor 1 (SFTI-1)

Serine‐proteinase‐catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI‐1: both single peptides and two‐peptide chains (C‐ and N‐terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl–enzyme intermediate was preceded by hydrolysis of the substrate Lys–Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two‐peptide‐chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl–enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl–enzyme were not observed. The peptide splicing was sequence‐ not structure‐specific.