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Current Pharmaceutical Design, 2011, 17, 4308-4317
Sunflower Trypsin Inhibitor 1 as a Molecular Scaffold for Drug Discovery Adam Lesner, Anna gowska*, MagdalenaWysocka and Krzysztof Rolka Faculty of Chemistry, University of Gdansk, Gdansk, Poland Abstract: This work is focused on SFTI-1, a member of the Bowman-Birk family of inhibitors. This 14 amino acid cyclic peptide exhibits several features i.e. compact rigidity, well-defined structure and small size that could result in a wide range of potential applications. Some examples of engineering of the specificity of this inhibitor along with structure - activity relationships will be discussed herein. Additionally, potential uses of STFI-1 and its analogs as pharmaceutical agents will be described.
Keywords: SFTI-1, inhibitor, serine proteinases, peptidomimetics, peptomers. INTRODUCTION Proteases and their cognate inhibitors are important players in living organisms. Their mutual interactions are essential during the whole life cycle of a single cell starting from fertilization, embryogenesis, hormone processing, blood clotting, digestion to apoptosis [1]. Proteolytic activity is involved in all these pathways. Uncontrolled activity of proteases yields to pathology and disease [2]. There are several mechanisms established by nature in order to regulate the unwanted proteolysis such as proenzyme synthesis, compartmentalization and pH regulation. However, naturally occurring peptide and protein inhibitors are found in all tissues of living organisms [3] and seem to constitute an essential part of the entire protease-controlling system. According to the peptidase database MEROPS created by Barret [4], there are 88 families of serine protease inhibitors that have been described to date. Plants are a rich source of various peptide and protein inhibitors, which have been divided into 12 families [5]. One of these, a well-described and studied family of plant serine protease inhibitors, are Bowman-Birk inhibitors (BBIs). This family of polypeptides was first discovered by Bowman in 1946 in the seeds of soybean [6] and 20 years later characterized by Birk [7]. Later on, more peptide inhibitors with a similar sequence, 3D structure and activity were found in other legumes. They interact with enzymes in a substrate-like fashion through an exposed fragment named the binding loop which is characterized by a unique canonical conformation. For this reason, the inhibitors were at first named standard mechanism inhibitors [8], and later on called canonical inhibitors [5]. A hyper exposed amino acid residue located in the center of this loop, named after Schechter and Berger [9] P1 residue, interacts with S1 cavity of the enzyme accounting for up to 50% of the inhibitor – enzyme contacts Fig. (1) and is responsible for inhibitor’s specificity. The canonical inhibitors that belong to the BBI family are polypeptides consisting of more than 60 amino acid residues with only one exception. In 1999 Luckett et al. [10] isolated from sunflower seeds a trypsin inhibitor SFTI-1 (Sunflower trypsin inhibitor 1), the smallest one, and, in addition, the most potent one among inhibitors of the Bowman-Birk family. Its primary structure is shown below:
SFTI-1 appeared to be a 14 amino acid circular peptide Fig. (2) and was found to be homologous to the much bigger BBI family members [11]. The P1-P1’ reactive site of this inhibitor is located *Address correspondence to this author at the Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland; Tel: ++48585235359; Fax: ++48585235472; E-mail:
[email protected] /11 $58.00+.00
Fig. (1). Schechter Berger notation.
Fig. (2). Schematic organization of SFTI-1 structure.
between Lys5-Ser6. Owing to its small size and a strong trypsin inhibitory activity determined as association equilibrium constant (Ka) or inhibition constant (Ki) (the appropriate values are 1.11010 -1 -9 M [12] and 10 M [10], respectively), SFTI-1 is considered to be a very attractive template for designing proteinase inhibitors with the potential use as pharmacological agents [13,14]. However, it is known that peptides are not ideal therapeutic agents because of their high sensitivity to protease degradation and limited cell permeability. Many strategies were developed to overcome these problems including selective and total pegylation [15], introduction of non-proteinogenic amino acid residues [16] or those with reverse configuration. Another common limitation reported for peptide drug candidates is their cytotoxicity [17]. SFTI-1 STRUCTURE As just mentioned, SFTI-1 consists of 14 amino acid residues. There are two cycles in its structure. One is formed by a disulfide bridge between Cys3 and Cys11, whereas the other by the circular backbone (formally shown as the head-to-tail cyclization between amino group of Gly1 and -carboxyl group of Asp14) [1,18]. Several intramolecular hydrogen bonds found in its 3D structure are
© 2011 Bentham Science Publishers
Current Pharmaceutical Design, 2011, Vol. 17, No. 38
Sunflower Trypsin Inhibitor 1 as a Molecular Scaffold for Drug Discovery
responsible for a rigid, well-defined structure of the compound. There are two basic elements identified within the SFTI-1 peptidic backbone - the binding loop and the secondary loop. The binding loop, Thr4-Ile10, with a P1-P1’ reactive site comprises Lys5 and Ser6. The peptide bond formed by these residues undergoes proteolysis upon binding to trypsin. However, most of the inhibitor molecules remain intact. The hydrolysis constant, defined as a ratio between the virgin and modified (with the hydrolyzed P1-P1’ peptide bond) inhibitor, has been established at 9:1 [19]. The secondary loop is a pentapeptidic fragment built up from the N-terminal dipeptide Gly1-Arg2 and the C-terminal tripeptide Phe12-Pro13-Asp14 linked via a peptide bond between the Gly and Asp residues. It does not interact directly with protease, but it stabilizes the overall structure of this small peptide. The 3D structure of the inhibitor forms two anti-parallel beta strands that are linked to each other by a turn Fig. (3) that results from cis geometry of the peptide bond between Ile7 and Pro8. This kind of the rigid structure is called a beta hairpin [20].
Fig. (3). Stick and ball projection of a SFTI-1 -hairpin motive [11].
Synthesis of SFTI-1 As a Bowman-Birk inhibitor of unusual small size (fourteen amino acid residues in SFTI-1 constitute only half of the second smallest naturally occurring canonical inhibitor CMTI-III [21]), this inhibitor is extremely prone to chemical modifications taking place in the regular peptide chemical synthesis protocols. However, several non-chemical approaches to SFTI-1 modification have been reported to date. The synthetic methods applied to SFTI-1 are summarized in Table 1. Most often SFTI-1 and its analogs were synthesized by the classical solid phase method followed by the disulfide bridge formation performed on the resin or in solution [27]. In order to obtain a cyclic backbone, an additional step, a head-to-tail cyclization (via Gly1 and Asp14) of a linear precursor, is required. Usually, it is Table 1.
4309
done by a standard chemical protocol applying e.g. PyBOP as a coupling reagent. However this step, in the case of SFTI-1, could be also achieved by closing a peptide bond between Lys5 and Ser6 which corresponds to the P1-P1’ reactive site. This kind of cyclization is catalyzed by the proteinase and it is unique for canonical inhibitors. This phenomenon has been reported for SFTI-1 by two research teams: that of Craik [19] and ours [28]. Craik and coauthors synthesized several disulfide bridged analogs with one particular peptide bond open. In the case of the monocyclic SFTI-1 analogue open at its scissile bond (Ser6 and Lys5 located at the Nand C-termini, respectively), the enzymatically assisted re-synthesis of its P1-P1’ reactive site was observed. On the other hand, doublesequence SFTI-1 analogs cross-linked by a single disulfide bridge formed between two flanked Cys3 and Cys25 (the two remaining ones were replaced by -aminobutyric acid (Abu) residues), after proteolytical processing ended up with re-synthesized monocyclic SFTI-1. In the first step, two reactive sites were cleaved, the middle fragment (the so-called insert peptide) was removed, followed by proteolytically assisted closing of the reactive P1-P1’ bond Fig. (4). This rearrangement, by analogy to the mechanisms observed for RNA and proteins, has been suggested to be named “peptide splicing”. Such proteolytical release of the insert might potentially be utilized as a delivery system of peptide(s) embedded in the SFTI-1 scaffold. Very recently, Mylne et al. [30] published interesting results that correspond with the subject of this paper. They described an unusual biosynthetic route in which SFTI-1 and possibly other cyclic peptides emerge from albumins. Experimental evidence obtained by molecular biology methods in conjunction with MS analysis led the authors to the conclusion that asparaginyl endopeptidase (AEP) that preferentially cleaved peptide bonds after Asn or with less affinity after Asp, is responsible for SFTI-1 cyclization. AEP cleaves albumin precursor PawS1 at Asn35 and Asp49. During the final cleavage by AEP, an active thioester acyl intermediate is formed and it reacts with amino group of Gly36 to form a Gly36Asp49 peptide bond. The proposed mechanism, although it does not involve splicing, explains how small homodetic peptides are produced in vivo. It should be emphasized that, similarly to the mechanism of peptide splicing proposed by us, the presence of the disulfide bridge is crucial for the SFTI-1 release from PawS1. The same group published the excellent review [31] presenting the mechanism of biosynthesis of cyclic peptides in plants with special attention focused on SFTI-1 molecule. They postulate that original preproSFTI-1 is extensively processed by a set of aspartyl and cystein proteases. SFTI-1 was also successfully synthesized using the native chemical ligation method as it was demonstrated by the Craik’s team [25]. The fragments were synthesized using Fmoc chemistry and only recently Kang and coauthors [24] have described biosynthesis of this peptide using the ribosome drop-off mechanism in
Chemical and Non-Chemical Methods of SFTI-1 Synthesis
Synthesis Method
Inhibitory Specificity
Reference
Solid phase peptide synthesis
various set of proteinases
[18,19,22]
Native chemical ligation
trypsin
[59]
Biosynthesis
trypsin
[23,30]
Ribosome drop off
trypsin
[24]
Semi-synthesis
trypsin
[25]
Codon programming
trypsin
[26]
4310 Current Pharmaceutical Design, 2011, Vol. 17, No. 38
Lesner et al.
Fig. (4). Mechanism of the proteolytical „peptide splicing”. [28, 29].
vivo. Briefly, they completed the synthesis of the cyclic peptide by the combination of peptidyl-tRNA drop-off and the spontaneous thioester rearrangement. This process is important from the therapeutic point of view, since by the means of genetic manipulation organisms could be forced to synthesize the SFTI-1 molecule on the mRNA template. Codon programming is another biosynthetic method utilized for synthesis of SFTI-1 employing the translational apparatus of the living cell. It was described for the first time by Kawakami and coauthors in 2010 [26]. This group used peptidyl-tRNA drop-off followed by the self-rearrangement of the resulting peptide to a Cterminal diketopiperazine-thioester, which non-enzymatically generated cyclized peptide due to the S-N shift. STRUCTURE-ACTIVITY RELATIONSHIP Numerous SFTI-1 analogs have been synthesized but we will review only those with potential pharmacological applications Table 2. In general, the modification of SFTI-1 was focused on several fundamental structural features. The first described analogs were devoid of one cycle (i.e. either head-to-tail cycle or disulfide bridge) [12]. We have shown that elimination of the head-to-tail cyclization did not practically affect inhibitory activity and proteolytic resistance [12,32]. On the other hand, elimination of the disulfide bridge by the replacement of Cys3 and Cys11 by Abu residues suppressed inhibitory activity 2 – 3 times as shown by the determined association equilibrium constants (Ka). Remarkably lower, as compared with the native SFTI-1, was also the proteolytic stability. Our results were compatible with those published by the Craik’s team [33]. They also showed that the solution structure of the monocyclic head-to-tail cyclized analog was similar (but less rigid) to that of the native SFTI-1 [34]. The studies presented above proved that to simplify the structure of SFTI-1, at least one of its cyclic fragments could be eliminated without affecting inhibitory activity. As a matter of fact, monocyclic SFTI-1 with the disulfide bridge is the only equipotent compound exhibiting proteolytic resistance matching that of the wild SFTI-1. This is the reason why this analog has been selected as a lead structure for further studies. Since it has been demonstrated that the disulfide bond ensures high activity of SFTI-1 analogs, this structural feature was subjected to modification. The purpose of the modification was to establish a red-ox stable cycle, which could remain intact in the case of the cell penetration and provide the proper active conformation of SFTI-1. One approach has been proposed by Jiang et al. [35] where both Cys entities within the SFTI-1 sequence were replaced by ethylene and olefin bridges. The obtained analogs displayed inhibition constants (Ki) with matriptase similar (ethylene) or one order of magnitude lower (olefin) as compared to that of monocyclic SFTI-1. The same team introduced a methylenedithioether bridge [36] as an alternative for that just mentioned. The potency of the obtained inhibitor against matriptase was high (Ki = 0.16 FM). A carbonyl bridge was introduced by gowska et al. [37] as a
more hydrophilic, red-ox stable alternative for the disulfide bridge. A series of 10 SFTI-1 analogs with rings varying in size (from Dap (-diaminopropionic acid) to Lys) were synthesized on the solid phase followed by subsequent ring closure on a solid support. All the peptides displayed the inhibitory activity one order of magnitude lower than that of the parent inhibitor. Another approach to the modification of SFTI-1 consisted of the replacement of the disulfide bridge by a diselenide bridge (L-selenocysteine residues were introduced instead of Cys) [38]. This selenium counterpart strongly inhibited the activity of bovine -trypsin with an inhibition constant (Ki) one order of magnitude lower than that determined for the wild SFTI-1, but with a significantly higher stability in terms of oxidative potential. Recently gowska and coworkers [39] replaced the Cys3 and/or Cys11 located in inhibitor’s positions P3, P6’, respectively, by its derivatives: L-penicylamine, L-homocysteine and Nhomocysteine. All the analogs with modified position P3 displayed a dramatic decrease in the association constant (up to 6 orders of magnitude) with cognate proteinase as compared to that of the unmodified monocyclic inhibitor. Conversely alternation of position P6’ created analogs that exhibited similar inhibitory potency as that of the reference inhibitor. Since P3 position is in the immediate neighborhood of the reactive site of the inhibitor, it could disturb proper recognition of the key residue at position P1. Modifications made in the disulfide bridge of the inhibitor are summarized in Table 3. The size of a drug candidate is another very important issue to be considered. As mentioned above, SFTI-1 is an already exceptionally small naturally occurring proteinase inhibitor. Consequently, a further reduction of its size would also reduce its cost of synthesis, an important option when bulk quantities of the compound are required. Data originating from several research groups indicate that the smallest fragment of SFTI-1 able to inhibit trypsin and chymotrypsin is a peptide containing the binding loop only. Such analog, [desGly1,desArg2, des Phe12,desPro13,des Asp14]SFTI1, inhibited the cognate enzyme only 10 times less than the parent inhibitor [41]. This finding correlates well with additional data provided by the Leatherbarow’s team [42]. The aim of their study was to design a potent -tryptase inhibitor. An eleven amino acid peptide (with a core disulfide flanked binding loop) was further extended in its C- and N- termini. The inhibition constant Ki of the starting peptide with tryptase was 31 FM. A further extension of the peptide chain decreased this value significantly, and the most potent compound [desGly,Ser2,Gln10,Tyr12,Ala13,Lys14, Gly15]SFTI-1 inhibited the human tryptase with a Ki value of 48 nM. Another study, presented independently by the Leatherbarow’s [41] and Robinson’s teams [43], indicated that a minimal peptide displaying trypsin inhibitory activity was a nonapeptide. The Leatherbarrow’s team designed a peptide based on the BBI binding loop that resembled the sequence within the SFTI-1 disulfide bridge. On the other hand, the Robinson’s team identified a heptapeptide fragment
Sunflower Trypsin Inhibitor 1 as a Molecular Scaffold for Drug Discovery
Table 2.
Current Pharmaceutical Design, 2011, Vol. 17, No. 38
4311
Ref.
Modification of SFTI-1 – Summary
P5
P4
P3
P2
P1
P1’
P2’
P3’
P4’
P5’
P6’
P7’
P8’
P9’
Enzyme
Ki
Gly
Arg
Cys
Thr
Lys
Ser
Ile
Pro
Pro
Ile
Cys
Phe
Pro
Asp
trypsin
1.1 e-10
[11]
des
des
des
des
des
trypsin
6.8 e-8
[77]
trypsin
1.0 e-8
[76]
Gln Arg
Ka
chymotrypsin
2.0 e9
[32]
cathepsin G
3.7 e6
[44]
Tyr
chymotrypsin
1.9 e10
[44]
Trp
chymotrypsin
1.4 e-6
[43]
HLE
6.5 e-8
[51]
Phe
des
des
des
Nle
D-Pro
Ala
Phe
Gln
Tyr
des
des
Phe(4-F)
chymotrypsin
3.0 e10
[44]
Phe(4-NO2)
chymotrypsin
2.6 e10
[44]
Phe(4-CH3)
chymotrypsin
2.5 e10
[44]
Phe(4-guanidine)
cathepsin G
8.1 e7
[44]
NPhe
chymotrypsin
3.8 e8
[49]
NLys
trypsin
1.1 e8
[49]
NPhe(4-NO2)
chymotrypsin
2.4 e9
[44]
Gln
Arg
kallikrein
Ile
Ala
Abu
Nleu
HLE
[51]
1.7 e-9
[45]
Phe
chymotripsin
1.3 e-7
[57]
trypsin
7.4 e-8
[58]
trypsin
1.1 e-7
[58]
trypsin
2.5 e-7
[58]
1.1 e-8
[43]
1.0 e-7
[43]
Ala
Ala
D-Pro
des
Val
6.5 e8
chymotripsin
Ala
des
[78]
Tyr
Ala
des
3.6 e-9
D-Pro
Pro
des
des
des
trypsin
Val
HLE
1.4 e7
[52]
(R,S)HmVal
trypsin
7.0 e8
[52]
HmSer
trypsin
1.4 e9
[52]
Leu
PPE
3.0 e-7
[64]
proteinase K
7.4e-6
[64]
2-NaI
matriptase
4.5 e-7
[36]
Nphe
cathepsin G
2.1 e7
[60]
Pro
trypsin
9.3 e7
[66]
Pro
chymotrypsin
5.4 e7
[66]
Hyp
trypsin
6.4 e7
[66]
Hyp
chymotrypsin
9.5 e7
[66]
Aze
trypsin
8.4 e7
[66]
Aze
chymotrypsin
1.5 e8
[66]
Ala
Leu
Nphe
Phe
Phe
Phe
Npip
Phe
Npip
Nleu
4312 Current Pharmaceutical Design, 2011, Vol. 17, No. 38
Table 3.
Lesner et al.
SFTI-1 Disulfide Bridge Modification
Disulfide Bridge Modification
Target/Activity
Ref.
Olefin bridge
matriptase
[35]
Ethylene bridge
matriptase
[35]
Peptoid residue bridge
trypsin, chymotrypsin
[39]
Carbonyl bridge
trypsin
[37]
Selenocystine bridge
trypsin
[38]
Methylene dithioether bridge
matriptase
[36]
Homoallyloglycine bridge (RCM)
chymotrypsin
[40]
(TKSIPPI) flanked by a D-Pro-Pro cycle able to inhibit trypsin at a level of 100 nM. A substitution of a single amino acid residue located in the substrate specificity P1 position (originally occupied by Lys5) has been studied most extensively. As shown in Table 2, modifications of the SFTI-1 sequence led to potent inhibitors of such physiologically important enzymes as matriptase (Ki = 0.92 nM), tryptase (Ki = 4.8 nM), kallikrein related peptidase (Ki =3.6 nM), cathepsin G (Ki = 0.15–5 nM), proteinase K (Ki ~10 JM), etc. No SFTI-1 analogs have been found to inhibit activity of human thrombin and urokinase, at micromolar levels or below. Hydrophobic residues and their structural derivatives introduced into the P1 position of SFTI-1 analogs significantly enhanced the chymotrypsin inhibitory activity. Thus, monosubstitution of Lys5 by Phe increased the Ka value determined with bovine chymotrypsin by three orders of magnitude [44]. This is in line with the work of the Leatherbarrow’s team [45] focused on the selection of chymotrypsin inhibitors from the peptide library modified in three positions by a series of 20 proteinogenic amino acid residues. A starting structure was an undecapeptide containing a binding loop of BBI. Among the peptides present in the library, the one with Phe or Tyr in position P1 displayed the most potent chymotrypsin inhibitory activity. Taking these results into consideration, our team decided to synthesize and determine inhibitory activity of a series of SFTI-1 monocyclic analogs (with disulfide bridge only) modified in this position by Phe derivatives substituted in the phenyl ring [44]. Our intention was to determine the influence of the parasubstituent of Phe5 on enzyme – inhibitor interaction. Among the synthesized analogs, the highest chymotrypsin inhibitory activity, 15 times as high as that of the reference compound, ([Phe5]SFTI-1), was determined for the one with Phe(p-F). Almost equipotent activity was displayed by peptides with uncharged substituents introduced in this position of the phenyl ring, [Phe(p-NO2)5]SFTI-1 and [Phe(p-CH3)5] SFTI-1. Beside the modification of P1 position of SFTI-1, another approach was described by Swedberg et. al. [46]. They re-engineered the inhibitor binding loop to induce the increase in the number of hydrogen bonds that are formed in contact with protease (KLK4, kalikrein-related proteinase) associated with prostate cancer. In silico study followed by the chemical synthesis yield to the selective KLK4 inhibitor called SFTI-1-FCQR Asp14. This inhibitor displayed impressive inhibitory constant at subnanomolar level (Ki = 0.0386 nM). Much effort of our teams has been made to investigate the possibility of SFTI-1 modification by N-substituted glycine derivatives (peptoid monomers) as mimics of proteinogenic amino acids. This kind of amino acid mimetics were introduced by the Zuckermann team in 1995 [47]. Polymers composed of these building blocks are
completely protease resistant. The hybrid combination of peptides and peptoids (see Fig. (5)), named after Ostergaard and Holm [48] “peptomers” (peptide-peptoid hybrid polymers) appeared to be a promising class of inhibitors. The introduction of N-4-aminobutylglycine (Nlys), N-phenyl-glycine (Nphe) [49] and Nphe(p-NO2) [44] (first one mimicking Lys and two remaining Phe, respectively) into the monocyclic SFTI-1 not only preserved high inhibitory activity but also conferred a considerable proteolytic resistance of the modified analogs. In the case of some peptomeric SFTI-1 analogues, the P1-P1’ reactive site remained intact. It is worth mentioning that in the canonical inhibitor, upon interaction with the cognate enzyme, this peptide bond is hydrolyzed, and the hydrolysis constant between virgin and modified inhibitor is usually close to unity. Another interesting finding is a potent chymotrypsin inhibitory activity (K a = 6.2107 M-1) [50] of a linear analog of the inhibitor with Nphe in the discussed position and with Cys residues isosterically replaced by (Abu). This fact is very intriguing since this has been the first report on a linear peptide capable of inhibiting the protease in a non-covalent manner. Inhibitors with elastase inhibitory activity were selected from the peptide library, designed and synthesized by the Leatherbarrow’s team [51]. The minimal peptide sequence (a 7-residue binding loop of BBI, flanked by the disulfide bridge) was modified by a set of proteinogenic amino acid residues in three positions corresponding to P2, P1 and P4’. The lowest inhibition constant for human neutrophil elastase (Ki = 65 nM) was determined for analogs with Ala followed by Val in position P1, in contrast to the porcine pancreatic elastase, that displayed preference towards Ala followed by Thr. The sequence of the peptide inhibitor was highly homologous to the monocyclic SFTI-1, but instead of Ile in the position P5’ the Gln residue was present. Unusual accommodation in the substrate pocket of bovine chymotrypsin of the side chain of -hydroxymethylamino acid located in the position P1 of SFTI-1 has been reported by Zabotna et al., [52]. The SFTI-1 analogs with HmVal Fig. (6) in this position, in contrast to the effect caused by its parent proteinogenic Val, retain trypsin inhibitory activity (Ka = 108 M-1). Accommodation and interaction with the substrate pocket of trypsin to give rise to uncharged aliphatic amino acid side chain located in the P1 position of the inhibitor was unexpected since trypsin did not exhibit elastolytic activity. However, this could be explained in terms of inter action of the hydroxyl group of the inhibitor with the catalytic Ser195 of the enzyme. This finding is compatible with the results provided by Hovhannisyan and coworkers [53], who investigated inhibitory activity of trypsin and proteinase K of a series of simple hydroxymethyl derivates of proteinogenic amino acids Ala, Ser, Phe, Gly, The and Leu. The majority of the derivates appeared to be
Sunflower Trypsin Inhibitor 1 as a Molecular Scaffold for Drug Discovery
Current Pharmaceutical Design, 2011, Vol. 17, No. 38
4313
Fig. (5). Schematic representation of peptomer structure.
Fig. (6). Chemical formula of (R,S)-2-amino-2-(hydroxymethyl)-3-methylbutanoic acid (HmVal).
effective inhibitors of the enzyme (inhibition constants at a nanomolar level). In the case of canonical inhibitors, the inhibitor’s P1 position is responsible for up to 50% of contacts with the target enzyme [54,55] and therefore it has often been referred to as the primary specificity residue. This is the reason that the most effective way of modulation of inhibitory properties is a substitution in the P1 position. Consequently, a modification of the binding loop from positions P2 to P4’ also has an impact on the inhibitory potency of SFTI1. The Leatherowbarrow’s team [56] focused its attention to inhibitor’s P2’ position which was occupied in native inhibitor by Ile7. The inhibition constant and the rate of hydrolysis were determined for analogs modified in this position by 19 proteinogenic amino acids and additionally by Nle. It turned out that the highest inhibitory activity was observed for the peptide with Ile (originally present in SFTI-1) followed by Leu and Nle in the modified position. Little or no inhibitory activity was detected for analogs with Gly or Pro in the P2’ position. The authors suggested that the large aliphatic side chains could easily be accommodated in apolar S2’ subsite of the enzyme. In addition, they found a strong positive correlation between inhibition constant and hydrolysis rate of those inhibitors. A similar study was performed to investigate the role of the conserved P2 position in BBI family, which is predominantly occupied by Thr [57]. Nineteen peptides, whose sequences were based on the BBI binding loop (with Phe in the P1 position) were modified in the position by the set of the proteinogenic amino acids. The potency of chymotrypsin inhibition was tested and again demonstrated that Nature made a best selection. The peptide with Thr in the position, whose sequence displayed a high homology (8 out of 9
residues) with truncated SFTI-1, displayed the highest inhibition constant. The preference towards neutral hydrophilic residues such as Ser or Gln in this position was observed. Also the inhibitor with Thr was the most proteolytically resistant one. Positions P3’ and P4’ are both occupied in SFTI-1 by Pro residues, with the former creating the cis and the other the trans geometry of the peptide bond with preceded amino acid, respectively. The Leatherbarrow’s team [58] analyzed the effect of substitution of each Pro residue by Ala in monocyclic SFTI-1. Position P3’ was several times more sensitive to such manipulation than position P4’. Replacement of Pro8 (P3’) resulted in a significant decrease in inhibitory potency (the Ki value dropped from 0.03 to 1.49 nM). In case of Pro9 (P4’), no change in the inhibitory activity was observed (Ki matched that of the native inhibitor). These results indicate that cis-Pro in the inhibitor’s P3’ position is the key factor that influences the whole SFTI-1 structure and thus the recognition by proteinase. This suggestion was confirmed by the Craik’s team [59]. They synthesized 12 L-alanine-modified SFTI-1 analogues where in each analogue a different position of the inhibitor was substituted. The highest decrease in trypsin’s inhibitory potency was noticed when Ala was in the position P1 and in position P3’. The outcome of the first substitution could be expected since the inhibitor lost its key substrate specificity residue Lys5. The other mutation emphasizes the crucial role of Pro8 in the formation of the 3D structure of SFTI-1. The effect of Pro8 and Pro9 substitutions was also investigated in our laboratory [60] with a series of N-substituted glycine derivatives with a bulky aromatic group in the side chain. A small peptomeric library based on the [Nphe5]SFTI-1 template modified in the P2’ – P5’ fragment was synthesized using a split and mix method. The deconvolution applying iterative approach in solution [61] against chymotrypsin and cathepsin was carried out. For the first proteinase, the presence of Pro8-Pro9 (P3’-P4’) gave analogs with a highest chymotrypsin inhibitory activity. With cathepsin G, we were able to select a potent inhibitor displaying the Ka value one order of magnitude higher than that determined for [Phe5]SFTI-1 used as a reference. In this inhibitor, Pro8-Pro9 was exchanged by a Npip-Npip peptoid fragment, where Npip is a peptoid monomer obtained by nucleophilic substitution by piperonylamine. The deconvolution of synthesized library has shown that in fact other peptoid monomers introduced in the discussed positions had equipotent influence on cathepsin G’s inhibitory activity as did Pro residues. The role of position P1’ was elucidated by several research groups. Among the BBI family of inhibitors, this position is occu-
4314 Current Pharmaceutical Design, 2011, Vol. 17, No. 38
pied by Ser with the highest frequency but it seems to be inessential for the inhibitory activity. For instance, Odani and Ikenaka [62] have shown that the substitution of Ser located in the P1’ position of soybean BBI by other uncharged, proteinogenic amino acids preserved chymotrypsin’s inhibitory activity. These results are compatible with more recent reports. Thus, Brauer and Leatherbarrow [63] reported that substitution of this amino acid residue by Ala in the BBI binding loop suppressed trypsin inhibitory activity (as expressed by dissociation constant, Kd) four-fold, but this modification did not destroy the structural integrity of the inhibitor. The same substitution introduced into the SFTI-1 heptapeptide transplanted onto a hairpin-induced template yielded a seven-fold lower trypsin inhibition as compared with one containing Ser [63]. Substitution of SerAla in the wild SFTI-1 preserved trypsin’s inhibitory activity, but the Kd value was eleven times lower [63]. The affinity of this analog towards trypsin was also supported by screening the biosynthesized SFTI-1 library [24]. In addition, a complete substitutional analysis of monocyclic SFTI-1 using SPOT synthesis showed the affinity towards trypsin of the inhibitor analogs modified in the P1’ position [64]. Recently, our team [65] obtained several SFTI-1 analogs with Ala, L-homoserine (Hse), and its peptoid counterparts (Sar and Nhse) in this position. Since all the peptides were designed to be chymotrypsin inhibitors, the P1 site was occupied by either Phe or Nphe. Except Sar, all the remaining residues were well tolerated by chymotrypsin when introduced in P1’ position of the monocyclic SFTI-1. In the other series of monocyclic SFTI-1 analogs, Pro and its derivatives were introduced in the P1’ position [66]. Interestingly, analogs with Pro, Hyp and a fourmember ring, L -azetidine-2-carboxylic acid (Aze), retained the trypsin or chymotrypsin inhibitory activities. Based on these results, it is worth emphasizing that the absolutely conservative in the BBI family Ser residue located in the inhibitor’s P1’ position can be replaced not only by Ala, but also by Pro, synthetic amino acids and their peptoid mimetic, retaining inhibitory activity of the modified SFTI-1 analogs. Biological Studies Since SFTI-1 is easily accessible using both chemical and biological methods, and also in addition to its extreme sensitivity to chemical modifications, several research teams [67,36,68] decided to utilize these readily available compounds in biological systems i.e. in tissue culture, animal models, etc. Matriptase is implicated in tissue remodeling, activation of proteinase-activated receptor 2 (PAR-2) and other crucial biological proteins (hepatocyte growth factor (HGF) or urokinase). This enzyme promotes metastasis and migration of cancer cells when not complexed with inhibitor. Strong inhibition of this protease could potentially reduce the tumor growth. To verify this hypothesis, fourteen SFTI-1 analogs were synthesized and their potency to inhibit matriptase, as well as thrombin, was evaluated [36]. The modifications of the wild-type inhibitor disulfide bridge that was replaced by the methylene dithioether bridge, yielding an inhibitor with a similar inhibition constant as the parent one. Additionally, positions P4, P1, P3’ and P5’ were altered to obtain a selective inhibitor of matriptase. The thrombin inhibitory activity was also evaluated. The most selective analog was [Gln10] SFTI-1. This peptide inhibited matriptase with a Ki value of 2.33 BM. At the same time, no inhibition was observed for thrombin, even when a millimole level of the inhibitor was applied. One of the first studies confined to the influence of SFTI-1 on type II transmembrane serine protease matriptase that is overexpressed on epithelial cells, was published by Lee et al. [67]. The influence of the native SFTI-1 on cancer cell proliferation and the level of hepatocyte growth and hormone activation was also investigated [67]. The growth rate of the mouse mammary epithelial EpH4/K6 cells was unchanged over a 5-day incubation period with SFTI-1. However, the inhibitor blocked completely the matriptasedepended activation of HGFA, resulting in the reduced morphogenesis and cell development [36].
Lesner et al.
Another potential application of the BBI family of inhibitors is modulating the inflammation by inhibition of the lipopolysaccharide (LPS)-induced macrophage activation. Macrophages incubated with the BBIs isolated from soybean released reduced amounts of cytokine and were less toxic in comparison with the control ones. This phenomenon could be explained in terms of inhibition of enzymes responsible for proteolytic activation of cytokines. Although the study of Li and coworkers describes these phenomena [69] for the BBI isolated from soybean only, it could pave the way for further studies of SFTI-1. Burster and coauthors [68] showed another application of SFTI-1 analogs, while demonstrating their ability to inhibit in vitro cathepsin G, one of the main proteases that participate in antigen autoprocessing. [Phe(4-guanidine)5]SFTI-1 and [Nphe5,12,Npip8,9,Nleu10]STFI-1 were incubated with the human peripheral blood mononuclear cells (PBMC) and the quantity of inhibited/blocked cathepsin G was followed over time. Both compounds slowly crossed the cell membrane within one day of incubation and were able to diminish the activity of this protease. So far, only one report has been published that investigated the possibility of application of SFTI-1 as a scaffold structure for the development of peptidic radiopharmaceuticals. Boy et al. [70] synthesized three SFTI-1 analogs modified by replacing the Phe12 by the Tyr residue in order to allow radiolabeling with 125I or 133I. The obtained peptides (monocyclic SFTI-1, bicyclic SFTI-1 and monocyclic SFTI-1 with attached at the N-terminus a chelating agent DOTA (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid that served as an 111 In chelator) strongly inhibited trypsin at subnanomolar level and were fairly stable in human serum (half life above 30 h). The cell permeability and cellular intake along with tissue distribution was investigated in the prostate-carcinoma-bearing mice model. Two human prostate cancer cell lines were also used for this study. All the SFTI-1 analogs showed a low but constant binding to the human cancer cells, with a preference to carcinoma cell lines. In the case of tumor-attacked organs, a high accumulation of radioactive SFTI-1 analogues was noticed in the heart, brain and muscle. The highest levels were observed with the bicyclic SFTI-1, probably owing to its extreme stability in the serum. The described examples of utilization of SFTI-1 and its derivatives indicate that this inhibitor interacts with designated proteinase and displays its inhibitory activity in biological systems. Moreover, SFTI-1 molecules, due to their compact 3D structures, are able to cross the cell membrane and act as inhibitors inside the cell [70]. However, the dynamics of cell permeability is not efficient enough for effectivity inside the cell. In our recent research [71] we report on the SFTI-1 conjugate (named as [(4-NPh2)Ph)]Bx-O2Oc[desArg2]SFTI-1) that has a polyethylene glycol (PEG) moiety attached along with a benzoxyazole moiety on their N-termini used as fluorogenic marker. General formula of the conjugate is presented in Fig. (7A). The incubation of the PEG-ylated SFTI-1 analogs at a micromolar level with various cancer cell lines (HEK, MDA MB 231) resulted in a fast (in 5-10 minutes) and efficient internalization of the compounds. Fig. (7B) shows an example of this experiment. The SFTI conjugate was able to penetrate the cell membrane, however, it did not travel across the nuclear membrane to the nucleus of the alive and healthy cell. When the cell becomes apoptotic, the SFTI-1 conjugate strongly associates with the nuclear fraction. These preliminary results indicate that SFTI-1 conjugates such as [(4-NPh2)Ph)]Bx-O2Oc-[desArg2]SFTI-1 display increased cell permeability and enhanced proteolytic stability. The ability of native SFTI-1 to penetrate the cell membrane was described in mid 2011 by the Australian group [72]. They investigated the migration of the set of fluorescent labeled cyclic inhibitors (including SFTI-1) across the cell membrane. Interestingly, the migration of SFTI-1 into the cells was not associated with any interactions with phospholipids, the main cell wall components. Another issue to be discussed in terms of potential pharmaceutical properties of SFTI-1 is its antimicrobial activity. The ability of
Current Pharmaceutical Design, 2011, Vol. 17, No. 38
Sunflower Trypsin Inhibitor 1 as a Molecular Scaffold for Drug Discovery
4315
Fig. (7). (A) Chemical formula of [(4-NPh2)Ph)]Bx-O2Oc-[desArg2]SFTI-1; (B) Effect of [(4-NPh2)Ph)]Bx-O2Oc-[desArg2]SFTI-1 incubation with the MDA MB 231 cell line, where O2Oc is 2-(2-(2-aminoethoxy)ethoxy)acetic acid.
Table 4. HV-BBI SFTI-1
Comparison of the Amino Acid Sequence of SFTI-1 and a Peptide Isolated from Skin Secretion of Huia Versabilis S
V
I
G
C
W
T
K
S
I
P
P
R
P
C
F
V
K
G
R
C
-
T
K
S
I
P
P
-
-
C
F
P
D
SFTI-1 or any of its analogs to kill bacteria or other life threatening pathogens has not been described so far. However, Song et al. [73] isolated from the frog skin (Huia versabilis) an octadecapeptide trypsin inhibitor named HV-BBI which displays a high (reaching 70%) sequential homology with SFTI-1 (see Table 4). It is well known [74] that skin extracts from frogs are a rich source of a wide range of peptides, including antimicrobial ones. It is believed that both classes of peptides (inhibitors and antimicrobials) are part of the amphibian defense system. Our preliminary results have shown that HV-BBI is not only a potent trypsin inhibitor (Ka = 5.5108 M1 ) but also displays a high antimicrobial activity (below 50 Cg/ml) against various Gram-negative bacteria such as Staphylococcus aureus and E. coli. This is a convenient starting point to design low-molecular compounds with combined inhibitory and antimicrobial activities, both being of interest to medicinal practice. An interesting application of SFTI-1 is its utilization as a recognition tool for selection and identification of proteinases. For
instance, a wild type SFTI-1 was immobilized via thiol-disulfide exchange reaction onto a gold surface and used for quantitative analysis of inhibitor – trypsin interactions [75]. Pereira et al. [76] immobilized monocyclic SFTI-1 analogs modified in the P1 position on agarose gel. The covalent linkage was achieved by the reaction of an aldehyde moiety of an oxidized Ser residue, additionally attached at the peptide N-terminus and a hydrazine group of agarose. It was demonstrated that these immobilized SFTI-1 analogs could serve as affinity probes for isolation of serine proteinases with different specificities. In summary, we would like to emphasize that SFTI-1 is an extremely interesting template for drug discovery and pharmaceutics. The simple synthesis that could be performed using several approaches, including ones used as a routine practice in chemical and biological laboratories, is one of the many advantages of SFTI-1. The SFTI-1 inhibitory specificity is exceptionally prone to modifications, but in the meantime its structure remains unchanged and
4316 Current Pharmaceutical Design, 2011, Vol. 17, No. 38
rigid enabling proper interaction with its target protease. It is also worth underlining that it is possible to introduce a wide range of synthetic entities into the SFTI-1 polypeptide chain to produce active peptidomimetics. A single exchanged residue in the substrate specificity P1 position could bring a total shift in its specificity. More attention should be focused in the future on the SFTI-1 and its analogs studied in biological systems. There are several areas in which SFTI-1 analogs could be useful, including antimicrobials and cell growth stimulators, both inside and outside of the cell. In order to make these investigations successful, the SFTI-1 analogs should be both red-ox and proteolytically stable with a well-defined specificity and 3D structure.
Lesner et al.
[22]
[23]
[24]
[25]
ACKNOWLEDGMENTS This work supported by Ministry of Science and Higher Education under grant number ID 147819.
[26]
REFERENCES
[27]
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
[12]
[13]
[14] [15] [16]
[17] [18]
[19]
[20]
[21]
Rawlings ND, Barrett AJ. Families of serine peptidases. Method Enzymol 1994; 244: 19-61 Puente XS, Sanchez LM, Overall CM, Lopez-Otin C. Human and mouse proteases: a comparative genomic approach. Nat Rev Genet 2003; 4: 544-58. Krowarsch D, Cierpicki T, Jelen F, Otlewski J. Canonical protein inhibitors of serine proteases. Cell Mol Life Sci 2003; 60: 2427-44. Rawlings ND, Barrett AJ, Bateman A. MEROPS: the peptidase database. Nucleic Acids Res 2010; 38: D227-33. Bode W, Huber R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 1992; 204: 433-51. Bowman DE. Differentiation of soy Bean antitryptic factors. Proc Soc Exp Biol Med 1946; 63: 547-50. Birk Y, Gertler A, Khalef S. A pure trypsin inhibitor from soya beans. Biochem J 1963; 87: 281-4. Laskowski M Jr, Kato I. Protein inhibitors of proteinases. Annu Rev Biochem 1980; 49: 593-626. Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 1967; 27: 157-62. Luckett RS, Garcia JJ, Barker AV, et al. High-resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds. J Mol Biol 1999; 290: 525-33. Zabotna E, Ka mierczak K, Ja kiewicz A, Stawikowski M, Kupryszewski G, Rolka K. Chemical synthesis and kinetic study of the smallest naturally occurring trypsin inhibitor SFTI-1 isolated from sunflower seeds and its analogues. Biochem Bioph Res Comm 2002; 292: 855-7. Qi RF, Song ZW, Chi CW. Structural features and molecular evolution of Bowman-Birk protease inhibitors and their potential application. Acta Biochim Biophys Sin (Shanghai) 2005; 37: 283-92. Korsinczky ML, Schirra HJ, Craik DJ, Sunflower trypsin inhibitor1. Curr Protein Pept Sci 2004; 5: 351-64. Fuertges, F.; Abuchowski, A. The clinical efficacy of poly(ethylene glycol)-modified proteins J. Control. Release 1990; 11: 139-48. Molhoek EM, van Dijk A, Veldhuizen EJ, Haagsman HP, Bikker FJ.Improved proteolytic stability of chicken cathelicidin-2 derived peptides by D-amino acid substitutions and cyclization. Peptides 2011; 32: 875-80. Cardozo AK, Buchillier V, Mathieu M, et al. Cell-permeable peptides induce dose- and length-dependent cytotoxic effects. Biochim Biophys Acta 2007; 1768: 2222-34. Korsinczky ML, Schirra HJ, Rosengren KJ, et al. Solution structures by 1H NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant. J Mol Biol. 2001; 311: 579-91. Marx UC, Korsinczky ML, Schirra HJ, et al. Enzymatic cyclization of a potent Bowman-Birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide. J Biol Chem 2003; 278: 21782-9. Jaulent AM, Brauer AB, Matthews SJ, Leatherbarrow RJ. Solution structure of a novel C2-symmetrical bifunctional bicyclic inhibitor based on SFTI-1. J Biomol NMR. 2005; 33: 57-62. Leluk J, Otlewski J, Wieczorek M, Polanowski A, Wilusz T. Preparation and characteristics of trypsin inhibitors from the seeds of
[28]
[29] [30]
[31] [32]
[33] [34] [35]
[36]
[37] [38]
[39] [40]
[41] [42]
[43]
[44]
squash (Cucurbita maxima) and zucchini (Cucurbita pepo var. Giromontia). Acta Biochimica Polonica 1983; 30: 127-38. Zabotna E, Ja kiewicz A, gowska A, Miecznikowska H, Lesner A, Rolka K. Design of serine proteinase inhibitors by combinatorial chemistry using trypsin inhibitor SFTI-1 as a starting structure. J Pept Sci 2007; 13: 749-55. Austin J, Kimura RH, Woo Y-H, Camarero JA. In vivo biosynthesis of an Ala-scan library based on the cyclic peptide SFTI-1. Amino Acid 2010; 38: 1313-22. Kang TJ, Hayashi Y, Suga H. Synthesis of the bacbone cyclic peptide sunflower trypsin inhibitor-1 promoted by the inducted peptidyl-tRNA drop-off. Angew Chem Int Ed Engl 2011; 50: 215961. Clark RJ, Craik DJ. Native chemical ligation applied to the synthesis and bioengineering of circular peptides and proteins. Biopolymers 2010; 94: 414-22. Kawakami T, Ohta A, Ohuchi M, Ashigai H, Murakami H, Suga H. Diverse backbone-cyclized peptides via codon reprogramming. Nat Chem Biol 2009: 5: 888-90. Jaulent AM, Leatherbarrow RJ. Design, synthesis and analysis of novel bicyclic and bifunctional protease inhibitors. Protein Eng Des Sel 2004; 17: 681-7. gowska A, Lesner A, Bulak E, et al. Inhibitory activity of double-sequence analogues of trypsin inhibitor SFTI-1 from sunflower seeds: an example of peptide splicing. FEBS J. 2010: 277: 2351-9. Colgrave ML, Korsinczky MJ, Clark RJ, Foley F, Craik DJ. Sunflower trypsin inhibitor-1, proteolytic studies on a trypsin inhibitor peptide and its analogs. Biopolymers 2010; 94: 665-72. Mylne JS, Colgrave ML, Daly NL, et al. Albumins and their processing machinery are hijacked for cyclic peptides in sunflower. Nat Chem Biol 2011; 7: 257-9. Conlan BF, Gillon AD, Craik DJ, Anderson MA. Circular proteins and mechanisms of cyclization. Biopolymers 2010; 94: 573-83. Zabotna E, Ka mierczak K, Ja kiewicz A, Kupryszewski G, Rolka K. Inhibition of Bovine -Chymotrypsin by Cyclic Trypsin Inhibitor SFTI-1 Isolated from Sunflower eeds and Its Two Acyclic Analogues. Lett Pept Sci 2002; 9: 131-4. Trabi M, Craik DJ. Circular proteins--no end in sight. Trends Biochem Sci. 2002; 27: 132-8. Korsinczky ML, Clark RJ, Craik DJ. Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1. Biochemistry 2005; 44: 1145-53. Jiang S, Li P, Lee S-L, et al. Design and synthesis of redox stable analogues of sunflower trypsin inhibitors (SFTI-1) on solid support, potent inhibitors of matriptase. Org Lett 2007; 9: 9-11. Li P, Jiang S, Lee SL, et al. Design and synthesis of novel and potent inhibitors of the type II transmembrane serine protease, matriptase, based upon the sunflower trypsin inhibitor-1. J Med Chem 2007; 50: 5976-83. gowska A, Bulak E, Ja kiewicz A, et al. Analogues of trypsin inhibitor SFTI-1 with disulfide bridge substituted by various length of carbonyl bridges. Protein Pept Lett 2010; 17: 1223-7. Guo X, Shi J, Tang Z, Cui D. Synthesis and Biological Activity of Seleno Sunflower Trypsin Inhibitor Analog. Chem Biol Drug Des 2006; 68: 341-4. gowska A, Dbowski D, ukajtis R, et al. Implication of the disulfide in trypsin inhibitor SFTI-1 in its interaction with serine proteinases. Bioorg Med Chem 2010; 18: 8188-93. Miles SM, Leatherbarrow RJ, Marsden SP, Coates WJ. Synthesis and bio-assay of RCM-derived Bowman-Birk inhibitor analogues. Org Biomol Chem 2004; 7: 281-3. McBride JD, Watson EM, Brauer ABE, Jaulent AM, Leatherbarrow RJ. Peptide mimics of the Bowman-Birk inhibitor reactive site loop. Biopolymers 2002; 66: 79-92. Scarpi D, McBride JD, Leatherbarrow RJ. Inhibition of human beta-tryptase by Bowman-Birk inhibitor derived peptides: creation of a new tri-functional inhibitor. Bioorg Med Chem 2004; 12: 6045-52. Descours A, Moehle K, Renard A, Robinson JA. A new family of beta-hairpin mimetics based on a trypsin inhibitor from sunflower seeds. Chembiochem 2002; 3: 318-23. gowska A, Dbowski D, Lesner A, Wysocka M, Rolka K. Introduction of non-natural amino acid residues into the substratespecific P1 position of trypsin inhibitor SFTI-1 yields potent chymotrypsin and cathepsin G inhibitors. Bioorg Med Chem 2009; 17: 3302-7.
Current Pharmaceutical Design, 2011, Vol. 17, No. 38
Sunflower Trypsin Inhibitor 1 as a Molecular Scaffold for Drug Discovery [45]
[46]
[47]
[48]
[49]
[50] [51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
McBride JD, Freeman N, Domingo GJ, Leatherbarrow RJ. Selection of chymotrypsin inhibitors from a conformationallyconstrained combinatorial peptide library. J Mol Biol 1996; 259: 819-27. Swedberg JE, de Veer SJ, Sit KC, Reboul CF, Buckle AM, Harris JM. Mastering the canonical loop of serine protease inhibitors: enhancing potency by optimizing the internal hydrogen bond network. PLoS One 2011; 6: e19302. Zuckermann RN, Kerr JM, Kent SBH, Moos WH. Efficient method for the preparation of peptoids [oligo(N-substituted glycines)] by submonomer solid-phase synthesis. J Am Chem Soc 1992; 114: 10646-7. Ostergaard S, Holm A. Peptomers: a versatile approach for the preparation of diverse combinatorial peptidomimetic bead libraries. Mol Divers 1997; 3: 17-27. Stawikowski M, Stawikowska R, Ja kiewicz A, Zabotna E, Rolka K. Examples of peptide-peptoid hybrid serine protease inhibitors based on the trypsin inhibitor SFTI-1 with complete protease resistance at the P1-P1' reactive site. ChemBioChem 2005; 6: 1057-61. gowska A, Bulak E, Wysocka M, et al. Peptomeric analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds. Bioorg Med Chem 2008; 16: 5644-52. McBride JD, Freeman HN, Leatherbarrow RJ. Selection of human elastase inhibitors from a conformationally constrained combinatorial peptide library. Eur J Biochem 1999; 266: 403-12. Zabotna E, Kret A, Ja kiewicz A, Olma A, Leplawy MT, Rolka K. Introduction of alpha-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity. Biochem Biophys Res Commun 2006; 340: 823-8. Hovhannisyan N, Harutyunyan Sh, Hovhannisyan A, Hambardzumyan A, Chitchyan M, Melkumyan M, Oganezova G, Avetisyan N, The novel inhibitors of serine proteases. Amino Acids 2009; 37: 531-6. Lu W, Apostol I, Qasim MA, et al. Binding of amino acid sidechains to S1 cavities of serine proteinases. J Mol Biol 1997; 266: 441–61. Krowarsch D, Dadlez M, Buczek O, Krokoszynska I, Smalas AO, Otlewski J. Interscaffolding additivity: binding of P1 variants ofbovine pancreatic trypsin inhibitor to four serine proteases. J Mol Biol 1999; 289: 175–86. Gariani T, McBride JD, Leatherbarrow RJ. The role of the P2’ position of Bowman-Birk proteinase inhibitor in the inhibition of trypsin. Studies on P2’ variation in cyclic peptides encompassing the reactive site loop. Biochim Biophys Acta 1999; 1431: 232-7. McBride JD, Brauer ABE, Nievo M, Leatherbarrow RJ. The role of threonine in the P2 position of Bowman-Birk proteinase inhibitors: studies on P2 variation in cyclic peptides encompassing the reactive site loop. J Mol Biol 1998; 282: 447-58. Brauer ABE, Domingo GJ, Cooke RM, Matthews SJ, Leatherbarrow RJ. A conserved cis peptide bond is necessary for the activity of Bowman-Birk inhibitor protein. Biochemistry 2002; 41: 1060815. Daly NL, Chen YK, Foley FM, et al The absolute structural requirement for a proline in the P3'-position of Bowman-Birk protease inhibitors is surmounted in the minimized SFTI-1 scaffold. J Biol Chem 2006; 281: 23668-75. gowska A, Dbowski D, Lesner A, Wysocka M, Rolka K. Selection of peptomeric inhibitors of bovine -chymotrypsin and cathepsin G based on trypsin inhibitor SFTI-1 using combinatorial chemistry approach. Mol Divers 2010; 14: 51-8.
Received: October 1, 2011
Accepted: October 6, 2011
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69] [70]
[71] [72]
[73]
[74] [75]
[76]
[77]
[78]
4317
Lam KS, Hruby VJ, Lebl M, et al. The chemical synthesis of large random peptide libraries and their use for the discovery of ligands for macromolecular acceptors, Bioorg Med Chem Lett 1993; 3: 419-24. Odani S, Ikenaka T. Studies on soybean trypsin inhibitors. IV. Change of the inhibitory activity of Bowman-Birk inhibitor upon replacements of the alpha-chymotrypsin reactive site serine residue by other amino acids. J Biochem 1978; 84: 1-9. Brauer ABE, Leatherbarrow RJ. The conserved P1' Ser of Bowman-Birk-type proteinase inhibitors is not essential for the integrity of the reactive site loop. Biochem Biophys Res Commun 2003; 308: 300-5. Hilpert K, Hansen G, Wessner H, Volkmer-Engert R, Hhne W. Complete substitutional analysis of a sunflower trypsin inhibitor with different serine proteases. J Biochem 2005; 138: 383-90. ukajtis R, gowska A, Wysocka M, Dbowski D, Lesner A, Rolka K. Analogues of trypsin inhibitor SFTI-1 modified in the conserved P(1)' position by synthetic or non-proteinogenic amino acids retain their inhibitory activity. J Pept Sci 2011; 17: 281-7. gowska A, Dbowski D, ukajtis R, et al. Introduction of Pro and its analogues in the conserved P1’ position of trypsin inhibitor SFTI-1 retains its inhibitory activity. Prot Pept Lett. 2011; 11, 1158-67. Lee SL, Huang PY, Roller P, Cho EG, Park D, Dickson RB. Matriptase/epithin participates in mammary epithelial cell growth and morphogenesis through HGF activation. Mech Dev 2010; 127: 8295. Reich M, Lesner A, gowska A, Sie czyk et al. Application of specific cell permeable cathepsin G inhibitors resulted in reduced antigen processing in primary dendritic cells. Mol Immunol 2009; 46: 2994-9. Li J, Ye L, Cook DR, et al. Soybean-derived Bowman-Birk inhibitor inhibits neurotoxicity of LPS-activated macrophages. J Neuroinflammation 2011; 8: 15-22. Boy RG, Mier W, Nothelfer EM, et al. Sunflower trypsin inhibitor 1 derivatives as molecular scaffolds for the development of novel peptidic radiopharmaceuticals. Mol Imaging Biol 2010; 12: 377-85. gowska A, Wysocka M, Lesner A, et al. Unpublished data Cascales L, Henriques ST, Kerr MC, et al. Identification and characterization of a new family of cell penetrating peptides: Cyclic cell penetrating peptides. J Biol Chem. 2011 Aug 26 in press. doi/10.1074/jbc.M111.264424. Song G, Zhou M, Chen W, Chen T, Walker B, Shaw C. HV-BBI--a novel amphibian skin Bowman-Birk-like trypsin inhibitor. Biochem Biophys Res Commun 2008; 372: 191-6. Basir YJ, Colon JM. Peptidomic analysis of the skin secretions on the pickerel frog Rana palustris identifies six novel families of structurally-related peptides. Peptides 2003; 24: 379-83. Ohtsuka K, Kajiki R, Waki M, Nojima T, Takenaka S. Immobilization of sunflower trypsin inhibitor (SFTI-1) peptide onto a gold surface and analysis of its interaction with trypsin. Analyst 2004; 129: 888-9. Pereira HJ, Salgado MC, Oliveira EB. Immobilized analogues of sunflower trypsin inhibitor-1 constitute a versatile group of affinity sorbents for selective isolation of serine proteases. J Chromatogr B Analyt Technol Biomed Life Sci 2009; 877: 2039-44. Gariani T, Leatherbarrow RJ. Stability of protease inhibitors based on the Bowman-Birk reactive site loop to hydrolysis by proteases. J Pept Res 1997; 49: 467-75. Swedberg JE, Nigon LV, Reid JC, et al. Substrate-guided design of a potent and selective kallikrein-related peptidase inhibitor for kallikrein 4. Chemistry & Biology 2009; 16: 633-43.
