Design, synthesis and analysis of novel bicyclic and bifunctional protease inhibitors

Agnès M. Jaulent and Robin J. Leatherbarrow1

Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, UK

1 To whom correspondence should be addressed. E-mail: R.Leatherbarrow{at}imperial.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Two novel synthetic inhibitors were designed to combine the advantageous properties of Bowman Birk inhibitor (BBI) and sunflower trypsin inhibitor-1 (SFTI-1). As is the case for BBI, the novel inhibitors have two active sites that give dual independent protease inhibition. However, they also possess a small bicyclic structure, reminiscent of the single-site SFTI-1. It is found that the synthetic inhibitors retain the potent inhibitory properties of the parent structures; they are also found to be relatively resistant to proteolysis. Their inhibition properties and a comparison of their stability to proteolysis relative to SFTI-1 are described. It is found that the new inhibitors do indeed allow bifunctional inhibition, although, unlike BBI, the small size of the inhibitor prevents the simultaneous inhibition of two proteases at the same time.

Keywords: Bowman Birk inhibitor/peptide synthesis/protease inhibitor/sunflower trypsin inhibitor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The Bowman Birk family of serine protease inhibitors (BBIs) was first isolated by Bowman in 1944 (Bowman, 1944Go) and characterized by Birk's group in 1967 (Birk et al., 1967Go). The BBIs distinguish themselves from other families of protease inhibitors by their small size, usually between 60 and 90 amino acids, and a strikingly high number (seven) of disulfide bridges. Some BBIs have evolved through gene duplication to present a characteristic shape; they are made of two symmetrical homology domains, each domain possessing a loop that projects out of the main protein core. This results in the BBI having a typical ‘bow tie’ shape (Chen et al., 1992Go) as shown in Figure 1A. This symmetry translates into an important biological feature as it enables the BBIs to inhibit both simultaneously and independently two serine proteases, usually trypsin and chymotrypsin (Birk et al., 1967Go), conferring upon the BBIs bivalency of inhibition. As with other protease inhibitors, the specificity of inhibition depends primarily on the nature of one unique residue within the loop, the P1 residue (flanking the scissile peptide bond on the N-terminal side) (Schechter and Berger, 1967Go); for example, arginine or lysine in this position gives rise to a trypsin inhibitor, whereas a phenylalanine or a tyrosine residue directs the specificity towards chymotrypsin (Laskowski and Kato, 1980Go).



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Fig. 1. Design of the bifunctional inhibitors. (A) The backbone structure of the BBI protein (coordinates from Werner and Wemmer, 1992Go). The two reactive site loops are shown in red, the core of the molecule in blue and the disulfide bridges in yellow. The pseudo C2 symmetry of the BBI protein is clear from this view. (B) The two reactive sites are shown in isolation. Construction of the bifunctional inhibitors involves merging these reactive site loops, joining them together as indicated by the arrows. (C) The bifunctional inhibitor structure. The two reactive loops are merged together so that they share a central disulfide. (D) Sequence of the bifunctional inhibitor BiKK, which has a symmetrical structure with two P1 residues, each comprising a lysine.

 
One of the challenges faced by researchers in the field of protein engineering is to achieve size reduction whilst retaining some or all of the biological properties present in the natural protein. Since they were first discovered, the BBIs have been extensively studied and one of the first issues addressed was to try and isolate the active part of the inhibitor. Odani and Ikenaka (1973)Go demonstrated that the BBI protein can be bisected in such a way that each portion still retains inhibitory activity. X-ray crystallographic studies of BBIs in complex with their target proteases then revealed that only part of the protein is in direct contact with the enzyme (Lin et al., 1993Go). This reactive part of the BBI was identified as the projecting loop(s) of the bow-tie-shaped protein. Nishino et al. (1977)Go chemically synthesized the first BBI mimic and demonstrated that the inhibitory activity of BBIs can be reduced to a 9-mer, disulfide-enclosed loop. Size reduction of the BBI has been extensively studied since then, with numerous reports of different peptide inhibitor mimics of the BBI (reviewed by McBride et al., 2002Go).

The most effective peptide mimic of the BBIs, however, is sunflower trypsin inhibitor-1 (SFTI-1), a natural peptide isolated from sunflower seeds (Luckett et al., 1999Go). SFTI-1 is not only the smallest naturally occurring peptidic protease inhibitor isolated to date, with only 14 residues, it is also the most potent of all BBI proteins, combining size reduction and retention of biological properties. This achievement arises from SFTI-1's unique structure: whilst SFTI-1 possesses the characteristic 9-mer disulfide-cyclized loop of the BBIs, its remaining five residues form a second, non-reactive backbone-cyclized ring. SFTI-1 is therefore bicyclic, which is thought to provide increased rigidity. Several groups have reported the chemical synthesis of SFTI-1 and confirmed it to be a very potent trypsin inhibitor, with reported inhibition constants in the sub-nanomolar to picomolar range (Luckett et al., 1999Go; Jaulent et al., 2001Go; Korsinczky et al., 2001Go; Long et al., 2001Go; Zablotna et al., 2002Go). SFTI-1 has also been shown to be a potent cathepsin G and matriptase inhibitor (Long et al., 2001Go), making it of potential pharmaceutical interest. Crucially, however, and in contrast to the BBIs, SFTI-1 can only inhibit one protease at a time since it possesses only one reactive loop. Indeed, all BBI mimics reported in the literature to date are monovalent inhibitors.

We wanted to design and synthesize bifunctional inhibitors that would combine features from both BBI and SFTI-1: on the one hand the ability of the BBI proteins to allow dual inhibition and on the other hand the small size and constrained nature of SFTI-1. In this paper, we describe the design and synthesis of two novel bifunctional inhibitors, one that is symmetrical and directed towards trypsin and one that includes distinct activities and separately targets trypsin and chymotrypsin. We compare the inhibitory potency of the bifunctional inhibitors with that of the BBI protein, SFTI-1 and an 11-mer peptide encompassing SFTI-1's reactive loop. The resistance of the novel bifunctional inhibitors to trypsin hydrolysis is evaluated and the stoichiometry of complex formation with their target proteases investigated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Reagents

Protected amino acids were purchased from Advanced ChemTech Europe (Cambridge, UK) and Merck Biosciences (Nottingham, UK). N-Hydroxybenzotriazole (HOBt),2-(1H-benzotriazol-1-yl)-1,1,3,tetramethylmethyluroniumhexafluorophosphate (HBTU) and all resins were obtained from Merck Biosciences. Peptide synthesis-grade dimethylformamide (DMF) was purchased from Rathburn Chemicals (Walkerburn, UK), high performance liquid chromatography (HPLC)-grade acetonitrile from Fischer (Loughborough, UK). Boc-Gln-Ala-Arg-AMC (Boc, tert-butyloxycarbonyl; AMC, aminomethylcoumarin) and Suc-Ala-Ala-Pro-Phe-AMC (Suc, 3-carboxypropionyl) were supplied by Bachem (UK) and dimethyl sulfoxide (DMSO) by VWR International (Poole, UK). All other chemicals, including bovine trypsin N{alpha}-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated and bovine chymotrypsin N{alpha}-p-tosyl-L-lysine chloromethyl ketone (TLCK)-treated were purchased from Sigma (Gillingham, UK). Ultrapure water was obtained using an Elix Water Purification System, followed by a Milli-Q Gradient Ultrapure Water System, both from Millipore (UK).

The purity of the proteases was determined immediately prior to use by active site titration (Bender et al., 1966Go; Chase and Shaw, 1967Go), which was performed using p-nitrophenyl acetate as the chymotrypsin substrate and p-nitrophenyl guanadinobenzoate for trypsin.

Inhibition assays were conducted at 25°C on a CytoFluor Series 4000 spectrofluorimeter microplate reader (Perseptive Biosystems, Framingham, MA).

Peptide synthesis

The automated synthesis of the linear backbone of the bifunctional peptides was carried out on an Advanced ChemTech Apex 396 multiple peptide synthesizer (Advanced ChemTech Europe, Cambridge, UK), using a standard Fmoc/tBu (9-fluorenylmethoxycarbonyl/tert-butyl) peptide synthesis strategy (Carpinoand Han, 1972Go; Atherton and Sheppard, 1989Go). Amino acid derivatives were used with the following side-chain-protecting groups: Lys (Boc); Thr and Ser (tBu); Cys (Trt). Fmoc removal was achieved with 20% (v/v) piperidine in DMF. The peptide chains were elongated on a pre-loaded Fmoc-Ser(tBu)-2-chlorotrityl resin and coupling of the next amino acid (5-fold excess to the peptidyl-resin) mediated by HOBt/HBTU (1 equiv. each to the amino acid) and N-ethyldiisopropylamine (DIPEA) (2 equiv. to the amino acid). After final Fmoc deprotection, the fully protected peptides were cleaved from the resin by adding a solution of acetic acid (AcOH)–trifluoroethanol (TFE)–dichloromethane (DCM) (1:1:3) for 1.5 h (Barlos et al., 1991Go). The backbone cyclization was performed by dissolving the peptides in DMF (0.5 mg/l) containing 1% (v/v) DIPEA, adding to this solution 1-hydroxy-7-azabenzotriazole (HOAt) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (1.1 equiv. each) and stirring for 18 h. Deprotection of the side chains was effected by adding to the peptides a mixture of trifluoroacetic acid–ethanedithiol–water–triisopropylsilane (TFA–EDT–H2O–TIS) (94:2.5:2.5:1) for 1.5 h. The final oxidation step to form the disulfide was performed by stirring the crude peptides for 24 h in 20% (v/v) aqueous DMSO solution (0.5 mg/ml) containing 5% (v/v) AcOH and adjusting the pH to 6.0 (Tam et al., 1991Go; Domingo et al., 1995Go).

Purification of the crude peptides was performed on a Gilson reversed-phase (RP) HPLC system using a Delta-Pak C18 cartridge (15 µm, 25 x 100 mm) column. Analytical RP-HPLC was performed using a Beckman System Gold HPLC system equipped with a C18 Symmetry column (4 µm, 4.6 x 250 mm). RP-HPLC columns were obtained from Waters (UK). After two-fold dilution of the oxidizing solution in water, purification was effected using a 5–85% linear gradient of acetonitrile into water (both solvents containing 0.1% TFA) over 35 min. Peptides were detected by measuring the absorbance at 223 nm. Fractions were collected, loaded on to the analytical HPLC column and combined when judged homogeneous. The peptides were obtained as white powder after a final lyophilization step.

The identity of the peptides was confirmed using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Mass values of 1678.08 and 1696.94 for BiKK and BiKF, respectively, were found to be in full agreement with the theoretical calculated masses (1678.1 and 1697.1). Their purity as evaluated by analytical HPLC was found to be >97%.

The 11-mer BBI mimic was also synthesized on an Advanced ChemTech Apex 396 multiple peptide synthesizer on Fmoc-Tyr(tBu)-Wang resin, using the same methods as used for BiKK and BiKF, without the steps of cleavage from the resin and backbone cyclization. SFTI-1 was synthesized as described elsewhere (Jaulent et al., 2001Go).

Determination of inhibition constants

All solutions for trypsin assays were prepared in 50 mM Tris–HCl buffer, 20 mM CaCl2, pH 8.0. Inhibitor solutions (0–200 µl) of a given peptide ([BiKK] = 0.8 µM, [BIKF] = 1.8 µM, [BBI] = 0.8 µM, [11-mer] = 4 µM, [SFTI-1] = 0.2 µM) were made up to a total volume of 200 µl. To 50 µl of these solutions was added 50 µl of trypsin solution (2 nM). After an incubation time of 5 min, the reaction was started by addition of 100 µl of a solution of Boc-Gln-Ala-Arg-AMC substrate (6 µM). For chymotrypsin assays, solutions were prepared in 144 mM Tris–HCl, pH 7.8. The assays were otherwise conducted in the same fashion as the trypsin assays, using Suc-Ala-Ala-Pro-Phe-AMC as the substrate (20 µM) and a chymotrypsin solution of 8 nM. Inhibitor concentrations were [BBI] = 4 µM and [BiKF] = 8 µM. Initial substrate hydrolysis was monitored by measuring the fluorescence to follow AMC release, with excitation at 360 nm and emission at 460 nm. Initial velocity data were then fitted using the GraFit software package (Leatherbarrow, 2001Go) to the inhibition equation:

(1)

The assays were performed in triplicate with fresh solutions of all reagents.

Trypsin hydrolysis of the synthetic peptides

The experiments were conducted at 25°C in 50 mM sodium acetate buffer containing 20 mM CaCl2, pH 3.5. Solutions of peptide and trypsin were mixed together so that the final concentration of the peptide was 200 µM and that of trypsin 2 µM (1 mole%). At discrete intervals, a 195 µl aliquot was taken and the reaction stopped by addition of 5 µl of TFA. The samples were then analysed directly by RP-HPLC.

For all inhibitors except BiKK, the hydrolysis followed an irreversible reaction scheme whereby the intact inhibitor (I) is transformed irreversibly into the P1–P'1 hydrolysed inhibitor (I*), with the process characterized by a first-order rate constant k (Fersht, 1985Go):

(2)

For BiKF, the 11-mer and SFTI-1, there was a clear separation between hydrolysed and intact species on the HPLC traces, which allowed the proportion of each inhibitor form to be determined by integration of their respective peaks. Data were then fitted to Equation 2 using the GraFit software (Leatherbarrow, 2001Go) to determine k. The half-life of the reaction, t1/2, was calculated from the equation:

(3)

For BiKK there were two hydrolysis products, one which has cleavage at a single site (BiKK*) and one with both sites hydrolysed (BiKK**). As each form was again resolved by HPLC, peak integration gave the proportion of all species. A full discussion of the BiKK hydrolysis kinetics is given in the Results section.

Samples from BiKK hydrolysis and BiKF hydrolysis were analysed by MALDI-MS and were found to correspond to the expected hydrolysates ([BiKK*]+ 1696.10, [BiKK**]+ 1714.15, [BiKF*]+ 1714.35).

Stoichiometry of complex formation

The stoichiometry of the complex between BBI or BiKF with trypsin and chymotrypsin was determined using chymotrypsin competitive binding assay studies at 25°C in the presence of trypsin. All solutions were prepared in 50 mM Tris–HCl buffer containing 20 mM CaCl2, pH 7.8.

Preliminary experiments (not shown) established that trypsin does not hydrolyse Suc-Ala-Ala-Pro-Phe-AMC, the chymotrypsin substrate, and that chymotrypsin is able to hydrolyse this substrate at essentially the same rate with or without trypsin present.

The experiments were conducted in the following manner. Increasing amounts (0–200 µl) of a given peptide concentration ([BBI] = 5 µM, [BiKF] = 10 µM) were added to buffer in a total volume of 200 µl over 24 wells. To 50 µl of these solutions, 50 µl of trypsin solution were added so that the protease was in a two-fold excess over the inhibitor in each well. After incubation for 5 min, 50 µl of a chymotrypsin solution (10 nM) were added. After a further 5 min, the reaction was started by the addition of 100 µl of substrate ([Suc-Ala-Ala-Pro-Phe-AMC] = 25 µM). Treatment of the data was as described above for trypsin inhibition kinetics.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Design of the bifunctional inhibitors

The aim of this study was to combine the bifunctionality of inhibition that is found in the BBIs with the small size and constrained structure of SFTI-1. In this way, it was hoped to retain favourable features of each inhibitor. The BBI is arranged such that its two reactive loops are symmetrically spaced and project out of the main core of the protein. Each of these nine-residue, disulfide-delimited projecting loops retain their function in isolation, allowing the generation of synthetic BBI mimics. We therefore elected to dispense with the core of the protein and splice the reactive site loops together, retaining just a single disulfide bridge, as depicted in Figure 1. Because of the pseudo C2-symmetry inherent in this construction, the two P1 positions in the newly designed bifunctional inhibitors are on opposite sides of the cyclic structure and are as far away from each other as the small cyclic structure allows. The symmetry of the structure also allows the disulfide to be ‘shared’ by each loop, giving a total of 16 residues.

The newly designed sequence is a 16-residue, disulfide-bridged and backbone-cyclized peptide. This putative inhibitor thus comprises two reactive loops, as does the BBI protein and is small and bicyclic, as is SFTI-1. The sequence for the reactive site loop was chosen to match that of SFTI-1. Repeating this sequence throughout the second reactive loop gives rise to a bifunctional inhibitor that possesses two lysine residues at the two P1 positions. It is therefore expected that each loop should have a common specificity, which should be directed towards trypsin in the same way as SFTI-1. We have termed this inhibitor BiKK to denote the bifunctional nature of the molecule and the presence of lysine residues at each P1 site. In BBI proteins and inhibitory peptide loops, replacement of the P1 lysine residue by a phenylalanine residue is sufficient to redirect specificity from trypsin to chymotrypsin (McBride et al., 2002Go). Accordingly, substitution of one of the lysine residues from the BiKK inhibitor by phenylalanine is predicted to give a bifunctional trypsin/chymotrypsin inhibitor; this inhibitor is called BiKF.

This simple design scheme gives the advantage of yielding small peptides that are easily amenable to solid-phase synthesis. The bifunctional peptides do not incorporate any extraneous spacer region. Indeed, every residue within the peptide serves as part of the reactive loop in addition to taking part in the head-to-tail cyclization. The bifunctional inhibitors only have the inhibitory loop and can be thought of as a condensed version of the original BBI where all amino acids that do not participate directly in inhibition through contact with the protease target are eliminated.

For reference and comparison purposes, the following inhibitors were studied alongside the bifunctional inhibitors BiKK and BiKF: commercially available BBI protein and synthetic SFTI-1 were chosen as they represent the parents of the bifunctional inhibitors. A synthetic 11-mer BBI mimic was also included in this study to represent a monocyclic inhibitory loop.

Inhibition assays

Inhibition of trypsin The inhibition data are presented in Table I. In this study, trypsin was used as a representative serine protease to probe the importance that the rest of the inhibitors' scaffold exercises on the inhibitory potency of their reactive loops. All inhibitors tested were found to give potent trypsin inhibition with inhibition constants in the nanomolar range. It should be noted that the reactive loop for all these inhibitors is the same in length and nature of cyclization. The sequences within this region are the same, except for an Ile/Gln substitution at P5' and an Ile/Asn substitution at P2' in the BBI protein; the residue at P5' is not involved in the enzyme–inhibitor interface and substitutions here have only small functional effects (Brauer and Leatherbarrow, 2003Go) whereas the P2' position varies widely in BBIs. It is therefore in the nature, length and shape of the scaffold onto which the reactive loop is built that the main variation between the inhibitors is introduced.


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Table I. Inhibition of trypsin by the BBI-related inhibitors

 
SFTI-1 and BBI. The two natural inhibitors, SFTI-1 and the BBI, are the best inhibitors in the series analysed; SFTI-1 is the most potent inhibitor of all and the Ki value found closely matches that reported by other groups (Korsinczky et al., 2001Go; Long et al., 2001Go; Zablotna et al., 2002Go). These two inhibitors are naturally occurring protease inhibitors and as such present structural elements extraneous from their reactive sites that stabilize the latter. In the case of the BBI, long-range hydrophobic interactions are expected to exercise positive stabilizing interactions on the 9-mer loop. As for SFTI-1, structural studies have shown that the non-reactive cycle further extends the network of hydrogen bonds present within the reactive loop (Korsinczky et al., 2001Go). As a result of these interactions, both the BBI protein and SFTI-1 are highly potent.

11-mer. The 11-mer BBI mimic is the smallest and the least potent of all tested inhibitors. It is also the only one of the synthetic inhibitors not to incorporate a head-to-tail cyclization. Its structure includes the essential 9-mer loop, flanked on each side by one residue only. For this reason, none of the additional stabilization factors mentioned above for the BBI or SFTI-1 are available to the 11-mer; this results in reduced inhibitory potency.

BiKK and BiKF. The novel bifunctional inhibitors BiKK and BiKF are both potent trypsin inhibitors, with inhibitory constants of 9.4 and 18.0 nM, respectively. The BiKK and BiKF inhibitors are entirely homologous in sequence, length, cyclization nature and extent, but differ in the nature of one of their P1 residues. BiKK possesses a lysine residue at each of its P1 positions and therefore has two potential sites for inhibition by trypsin, whereas BiKF possesses only one. BiKK is twice as good an inhibitor as BiKF; this is as expected, simply on a concentration basis, if both loops on BiKK can each act as an independent inhibitory site. Although the design of these molecules anticipates that they will have two functional sites, owing to the small size and very constrained nature of the molecule it is conceivable that steric hindrance or unfavourable geometry introduced by the unnatural cyclization could prevent the loops from adopting the necessary conformation for inhibition. This result, therefore, is consistent with the overall structure of both 9-mer reactive loops being undisturbed by their sharing a disulfide bridge, with each loop providing a separate inhibition site. Both BiKK and BiKF can therefore be said to be bifunctional.

Inhibition of chymotrypsin BBI proteins typically are able to inhibit trypsin and chymotrypsin via their two reactive site loops. The BiKF sequence was designed to incorporate chymotrypsin specificity in the second reactive site and was found to be active as a chymotrypsin inhibitor, with a Ki value in the nanomolar range (Table II). In contrast, BiKK is not able to inhibit chymotrypsin to any significant extent. This confirms that that the selectivity of one of the reactive loops of the bifunctional inhibitor can be redirected without disturbing the interaction of the first loop with its target.


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Table II. Inhibition of chymotrypsin by the BBI-related inhibitors

 
Stoichiometry of the inhibitor–protease complex: can the bifunctional inhibitors inhibit two proteases simultaneously?

We have demonstrated that the BiKK and the BiKF inhibitors are truly bifunctional as each of their loops interacts independently with its cognate protease. This is also the case for the full BBI protein, which is not only capable of inhibiting two proteases independently, but can also do so simultaneously (Birk et al., 1967Go). However, the bifunctional inhibitor peptides are far smaller than the BBI protein, which means that it is possible that they do not allow simultaneous inhibition of two proteases.

The stoichiometry of the complexes formed between inhibitor, trypsin and chymotrypsin was tested in the following manner. Chymotrypsin inhibition assays were performed following incubation with an excess of trypsin, in order to ensure that all inhibitor present was complexed with trypsin. These experiments were undertaken using BiKF and BBI and the results are summarized in Table II.

It was found that with the BBI protein, pre-incubation with trypsin did not result in any significant loss of chymotrypsin inhibition. In marked contrast, for BiKF, pre-incubation with trypsin resulted in a total loss of chymotrypsin inhibition. In the reverse experiment (data not shown), pre-incubation with chymotrypsin did not prevent trypsin inhibition for the BBI, but did entirely stop it for the BiKF. Under the conditions in these assays, there is no significant degradation of either inhibitor. Therefore, we conclude that these results suggest that the size of the BiKF is incompatible with simultaneous binding of trypsin and chymotrypsin; in contrast, the BBI protein is large enough to allow this. Therefore, although the bifunctional inhibitors inhibit two proteases independently through their two binding loops, they are unable to bind simultaneously to their two target proteases, unlike the bivalent BBI.

Hydrolysis by trypsin

Small peptidic inhibitors of proteases often show poor resistance to hydrolysis as they are effectively substrates that are hydrolysed very slowly. The situation is similar for natural proteinaceous inhibitors, such as the BBI, although the hydrolysis of the complex formed on binding to the enzyme is not favoured (Laskowski and Kato, 1980Go). In addition to conferring better biological activity, cyclization is a well-known means of reducing a peptide's sensitivity to proteolytic enzymes such as trypsin (Hruby et al., 1990Go; Li and Roller, 2002Go). It was therefore interesting to compare the cyclic synthetic peptides, BiKK, BiKF, SFTI-1 and the 11-mer, for their resistance towards turnover by trypsin.

Time course of hydrolysis by trypsin

When hydrolysis of the peptides was followed at pH 8 using catalytic quantities of trypsin (1 mol%), none of the peptides showed significant levels of hydrolysis products until at least 30 min of incubation, as judged by HPLC analysis from the still intact peak of the original inhibitors. This shows that all of these synthetic peptides are significantly resistant to hydrolysis by trypsin. In order to quantify their relative stabilities and elucidate the nature and number of the hydrolysis product(s), we used conditions that are known to favour hydrolysis, i.e. catalytic amounts of trypsin and acidic pH (Ozawa and Laskowski, 1966Go; Niekamp et al., 1969Go; Jensen et al., 1996Go). Under these conditions, for all peptides except SFTI-1, the hydrolysis reaction went to completion with the original peptide peak eventually becoming entirely depleted. SFTI-1 showed greater resistance than the other species and even after more than 24 h of incubation with trypsin, some SFTI-1 (~10%) was found to be still intact. This lack of susceptibility to proteolysis is correlated with SFTI-1's extremely rigid frame and particularly extensive hydrogen bond network and has been noted by other workers (Korsinczky et al., 2001Go; Zablotna et al., 2002Go).

The half-lives for hydrolysis of the inhibitor by trypsin are given in Table III. As previously noted (Korsinczky et al., 2001Go; Zablotna et al., 2002Go), SFTI-1 shows considerably improved stability compared with the 11-mer sequence that lacks the head-to-tail cyclization. There is clearly more to this than just the entropic advantage of the additional cyclization, as although BiKK and BiKF show increased half-lives relative to the 11-mer, the improvement is not as marked as that for SFTI-1. As SFTI-1 is a naturally occurring peptide, it is reasonable to speculate that evolution has played a part in producing the enhanced stability.


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Table III. Half-life of trypsin hydrolysis of the BBI-related inhibitors

 
Analysis of the hydrolysis profiles

SFTI-1, BiKF and the 11-mer each displayed similar hydrolysis traces, where a single new peak was observed. This appeared at the same rate that the original peptide disappeared. The appearance of only one peak was expected because although the hydrolysis generates new amino and carboxy fragments, these fragments are held together by the covalent disulfide bridge. All peaks corresponding to cleaved peptides were characterized by a shorter elution time, consistent with the increase in hydrophilicity that results from the two new charged ends. In all cases, the new peak was confirmed by mass spectrometry (results not shown) to correspond to the expected product of hydrolysis (M + 18).

In marked contrast, the BiKK peptide displayed more complex hydrolysis traces, with two new peaks being detected as the hydrolysis progressed. The HPLC traces of the reaction after various incubation times are shown in Figure 2. Since BiKK possesses two active sites for interaction with trypsin and since these are each able to interact independently with a trypsin molecule, this suggests that the new peaks represent products that have a single (we designate this BiKK*) and double (BiKK**) cleavage points. In both cases, the peptide is still held together through the disulfide bridge. Both BiKK* and BiKK** showed a shorter retention time than the intact BiKK peptide and the retention time of BiKK** was shorter than that of BiKK*; again, this is consistent with their expected polarities. The first peak to appear, BiKK*, can be assigned to the inhibitor hydrolysed solely in one reactive loop. BiKK** then corresponds to the product of dual hydrolysis. Mass spectrometry (not shown) confirmed that the BiKK* peak was the mono-hydrolysis product (M + 18) and BiKK** the dual-hydrolysis product (M + 36). During the progress of the hydrolysis reaction, BiKK* was observed to build up initially; the reaction then proceeded until only BiKK** was found. This behaviour is consistent with a ‘consecutive reaction’ scheme (Fersht, 1985Go), whereby the starting material, BiKK, is hydrolysed completely and irreversibly into a first intermediate, BiKK* and consecutively into the final product (BiKK**). The underlying kinetics are described by the following equations:

(4)

(5)

(6)

(7)



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Fig. 2. Time course hydrolysis of the BiKK inhibitor by trypsin. This figure shows the HPLC profiles of the trypsin hydrolysis for the BiKK inhibitor in the course of time. Prior to incubation, the BiKK shows a single peak (blue). Hydrolysis proceeds with the formation of a singly hydrolysed product, BiKK* (red) followed by, at later times, the doubly hydrolysed product, BiKK** (green).

 
Fitting the hydrolysis data to this scheme allows each of the rate constants to be determined (Figure 3). At pH 3.5 these were found to be k1 = (3.08 ± 0.15) x 10–4 s–1 and k2 = (6.83 ± 0.65) x 10–4 s–1.



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Fig. 3. Trypsin hydrolysis of the BiKK inhibitor. The variation of the three main species, BiKK (uncleaved, circles), BiKK* (single cleavage, triangles) and BiKK** (double cleavage, squares) is shown. The curves show the best fit to the data using a global fit to Equations 4GoGo–7.

 
In this scheme, k1 measures the rate constant for hydrolysis of a reactive loop of the starting inhibitor that contains two reactive site loops, whereas k2 measures the rate constant for hydrolysis of the inhibitor once the first loop has been hydrolysed. The ratio of these constants therefore shows the stabilization factor that the second loop exerts over the first one. However, as the virgin BiKK possesses two hydrolysis sites per molecule as opposed to one only for BiKK*, this means that we would expect, all other things being equal, that k1 should be twice k2. On the contrary, we observe that k1 is smaller than k2. Consequently, the stabilization factor that is provided by the cyclization can be calculated to be 4.4.

This small increase in stability is also seen when comparing the hydrolysis half-lives of BiKK (or BiKF) and the 11-mer (Table III). However, it is less than that found for the naturally occurring inhibitor SFTI-1; this latter inhibitor, though, does not have the bifunctionality of the BiKK/F species.

Trypsin hydrolysis of the BBI could not be conducted as the HPLC technique used did not allow for good separation of the intact from the cleaved BBI species. Reactive-site loops of natural protein inhibitors of proteases are typically mounted on a large proteinaceous supporting scaffold, which is effectively the source of protection from rapid hydrolysis for the inhibitors; this is expected to give the BBI protein enhanced resistance to proteolysis compared with the isolated 11-mer loop. We find that the bicyclic SFTI-1 is exceptionally stable to trypsin hydrolysis. This result is corroborated by Korsinczky et al. (Korsinczky et al., 2001Go), who showed that SFTI-1 behaves more like a larger inhibitor than a small synthetic inhibitor. The bifunctional BiKK and BiKF inhibitors, on the other hand, despite being bicyclic like SFTI-1, showed a stability much more similar to that of the 11-mer. It is therefore anticipated that the bivalent inhibitors are less resistant to trypsin hydrolysis than is the full-length BBI protein.

Conclusions

We have engineered two novel bifunctional BBI mimics, BiKK and BiKF, based on SFTI-1 bicyclic structure. Although there are examples in the literature of bicyclic BBI mimics (Nishino and Izumiya, 1982Go; Ando et al., 1987Go), these are, to the best of our knowledge, the first described bicyclic, bifunctional BBI mimics.

The bifunctional inhibitors are small and easily amenable to chemical synthesis. They both encompass a macrocycle formed by the uninterrupted backbone of their 16 residues; on top of this, further constraint is introduced through a disulfide bridge. Cyclization of peptides is a well-known approach for designing therapeutically viable peptides and peptidomimetics that present both increased stability towards proteolytic enzymes and enhanced binding affinity (Hruby, 1982Go; Degrado, 1988Go; Hruby et al., 1990Go; Li and Roller, 2002Go).

By designing bicyclic inhibitors that combine two different types of cyclizations, we have obtained potent, nanomolar inhibitors of their cognate proteases that also show very good specificity and high resistance to proteolysis. Compared with the 11-mer BBI sequence, they have improved proteolytic resistance and a second functional site. In these regards they therefore combine two desirable features of the naturally occurring BBI and SFTI-1 inhibitors, albeit with some caveats: although bifunctional, they cannot inhibit two proteases simultaneously; although quite stable to proteolysis, they are not as resistant as SFTI-1.


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received July 5, 2004; revised September 11, 2004; accepted September 15, 2004.

Edited by Anthony Wilkinson





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