Anna Jaśkiewicz

Examples of Peptide–Peptoid Hybrid Serine Protease Inhibitors Based on the Trypsin Inhibitor SFTI-1 with Complete Protease Resistance at the P1P1′ Reactive Site

Research in the field of protease inhibitors is focused on obtaining potent, specific and protease‐resistant inhibitors. To our knowledge, there are no reports in the literature that consider the application of N‐substituted glycine residues (peptoid monomers) for the design of peptidomimetic protease inhibitors. We hereby present the chemical synthesis and kinetic properties of two new analogues of the trypsin inhibitor SFTI‐1 modified at the P1 position. Substitution of Lys5 in SFTI‐1 by N‐(4‐aminobutyl)‐glycine and N‐benzylglycine, which mimic Lys and Phe, respectively, made these analogues completely protease‐resistant at their P1P1′ reactive sites. The analogues synthesised appeared to be potent inhibitors of bovine β‐trypsin and α‐chymotrypsin. These noncovalent, competitive and selective peptide–peptoid hybrid (peptomeric) inhibitors might open the way to targeting unwanted proteolysis.

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.

Inhibitory activity of double-sequence analogues of trypsin inhibitor SFTI-1 from sunflower seeds: an example of peptide splicing.

Four 28‐amino acid peptides were synthesized whose sequences comprised two molecules of trypsin inhibitor sunflower trypsin inhibitor 1 (SFTI‐1) bound through a peptide bond. The peptides in their reactive positions (5 and 19 of the peptide chain) contain two Lys ([KK]BiSFTI‐1) and two Phe ([FF]BiSFTI‐1) residues, along with a combination of the amino acid residues named thereafter [KF]BiSFTI‐1 and [FK]BiSFTI‐1. Association constants of the analogues determined with trypsin and chymotrypsin, respectively, indicated that they were potent inhibitors of cognate proteinases. An MS study of the associates revealed that incubation of the compounds with the proteinases resulted in cutting out a fragment of the peptide chain to restore the native monocyclic molecule of SFTI‐1 or its analogue [Phe5]SFTI‐1. This process, analogous to that of the DNA and protein splicing, can be referred to as ‘peptide splicing’.

Inhibition of bovine α-chymotrypsin by cyclic trypsin inhibitor SFTI-1 isolated from sunflower seeds and its two acyclic analogues

Trypsin inhibitor SFTI-1 isolated from sunflower seeds (comprising 14 amino acid residues and two cycles: head-to-tail cyclisation and disulfide bridge) is the smallest naturally occurring plant serine proteinase inhibitor. In our recent paper we have shown that the elimination head-to-tail cyclisation did not change trypsin inhibitory activity as judged by measured by association equilibrium constants K a . The removal of disulfide bridge produced 2.4-fold lower activity. In the present paper we described chymotrypsin inhibitory activity. SFTI-1 inhibits significantly lower bovine α-chymortypsin (K a =(5.20±1.56)×106 M−1). The activity of the analogue with disulfide bridge only was practically the same, whereas the K a value determined for homodetic peptide was almost 3-fold lower. Considering the results obtained and the recent literature data we postulate the lower inhibitory activity against both enzymes of the analogue with head-to-tail cyclisation only reflect its lower proteolytic stability.

Selenopeptide analogs of EETI-II retain potent trypsin inhibitory activities.

Three‐disulfide‐bridged Ecballium elaterium trypsin inhibitor II (EETI‐II) is a 28‐residue peptide that belongs to the squash family of canonical trypsin inhibitors. Herein, we report synthesis and biological activity of three EETI‐II analogs. In each of analog, a pair of cysteine residues forming a native disulfide bridge was individually replaced by a pair of selenocysteine residues. All selenopeptide analogs were chemically synthesized using the Fmoc protocol and subsequently folded in the presence of oxidized and reduced glutathione. The analogs containing a diselenide bridge displayed association constants with trypsin that ranged from 2.6 × 109 to 5.1 × 109 [M−1]. Our results suggest that the selenopeptide analogs retained low nanomolar inhibitory potencies, and only the diselenide bridge adjacent to the inhibitory binding loop weakened the interactions with trypsin by approximately fivefold. We discuss these findings in the context of a broader use of selenopeptide analogs as proxies to study cysteine‐rich peptides.

The influence of substrate peptide length on human β‐tryptase specificity

Combinatorial chemistry approach was applied to design chromogenic substrates of human β‐tryptase. The most active substrate, Ala‐Ala‐Pro‐Ile‐Arg‐Asn‐Lys‐ANB‐NH2, was selected from among over 9 million heptapeptides. The amide of 5‐amino‐2‐nitrobenzoic acid (ANB‐NH2) attached at the C‐terminus served as a chromophore. In order to determine the optimal length of the tryptase substrate, a series of N‐terminally truncated fragments of this substrate was synthesized. Pro‐Ile‐Arg‐Asn‐Lys‐ANB‐NH2, with the determined value of the specificity constant (kcat/KM) above 9 × 106 M−1 s−1, appeared to be the most specific substrate of tryptase. This substrate was twice as active as the parent heptapeptide substrate. We postulate that the optimal size of the pentapeptide substrate for the interaction with human β‐tryptase is associated with the unique structure of this proteinase, comprising four almost identical monomer subunits arranged in a square flat ring with its substrate pockets faced inside, forming a tetramer with a central pore that can be penetrated by this short peptide. Copyright

Modifications outside the proteinase binding loop in Cucurbita maxima trypsin inhibitor III (CMTI-III) analogues change the binding energy with bovine β-trypsin

Five 26‐peptide analogues of the trypsin inhibitor [Pro18]CMTI‐III containing Leu or Tyr in position 7 and Val or Tyr in position 27: 1 (Leu7, Tyr27), 2 (Tyr7, Val27), 3 (Tyr7, Tyr27), 4 (Leu7, Val27) and 5 (Leu7, Ala18, Tyr27) were synthesized by the solid‐phase method. Analogues 1–4 displayed K a with bovine β‐trypsin of the same order of magnitude as the wild CMTI‐III inhibitor, whereas for analogue 5, this value was lower by about 3 orders of magnitude. This indicated that for the analogues with Pro (but not with Ala) in position 18, the side‐chain interactions between positions 7 and 27 did not play a critical role for the stabilization of the active structure. In addition, these results also suggest that Tyr7 is involved in an additional aromatic interaction with position 41 of the enzyme.

Conformational studies of [Abu3, 11]-SFTI-1, an analogue of the trypsin inhibitor isolated from sunflower seeds

With only 14 amino acid residues, the trypsin inhibitor SFTI‐1 is the smallest naturally occurring serine proteinase inhibitor. It consists of two cyclic fragments (with head‐to‐tail cyclization and a disulfide bridge). In our previous paper, we showed that the removal of the disulfide bridge produced 2.4‐fold lower activity. Here, we present the total conformational analysis of the [Abu3, 11]‐SFTI‐1 analog by means of 2D NMR spectroscopy in conjunction with theoretical methods. The peptide was synthesized by Fmoc SPPS. It was cyclized with PyBop and DIPEA in DMF. The NMR studies were performed in DMSO‐d6 at 303 K. Conformations of the peptide studied were calculated by the following three approaches: distance geometry (DG), molecular dynamics (MD) and determination of the statistical weights of conformations. The first two algorithms use a CHARMM force field, whereas the last uses an ECEPP/3 force field. Our calculations resulted in three sets of conformers with 7, 9 and 6 representatives, respectively. All our results were compared with published ones. It was found that the peptide has an ill‐defined structure. Despite its conformational flexibility, the binding loop (3–11 fragment) displayed geometry similar to the corresponding fragments of the other SFTI‐1 analogs and to the inhibitor itself. Furthermore, the peptide bond between the Ile7 and Pro8 residues adopts cis geometry, which is essential for inhibitory activity. Copyright