WO2000031139A1

WO2000031139A1 - Peptide inhibitor of browman-birk type

Info

Publication number: WO2000031139A1
Application number: PCT/GB1999/003945
Authority: WO
Inventors: Robert Leo Brady; Peter Shewry; Susan Luckett; Alexander Konarev; Anthony Clarke
Original assignee: University Of Bristol
Priority date: 1998-11-25
Filing date: 1999-11-25
Publication date: 2000-06-02
Also published as: AU1287100A; GB9825854D0

Abstract

There is provided a peptide comprising the serine proteinase binding loop of a BBI or a peptide mimic thereof, in which the amino and carboxyl terminal residues of the binding loop or a peptide mimic thereof are joined by a peptide bond or a peptide to form a continuous chain. There is also provided a peptide of the formula: -G-R-C-T-K-S-I-P-P-I-C-F-P-D- in which the amino and carboxyl terminal residues are joined by a peptide bond and in which there is a disulphide bridge between the two cysteine residues, and a homologue of such a peptide.

Description

PEPTIDE INHIBITOR OF BROWMAN-BIRK TYPE

This invention relates to peptides which mimic the serine proteinase-binding loop of Bowman-Birk inhibitors and pharmaceutical compositions comprising such peptides. BACKGROUND

The Bowman-Birk inhibitors (BBIs) are a family of serine proteinase inhibitors . They are small proteins (6-9 kD) which contain seven disulphide bonds. The disulphide bridges help to form a symmetrical structure consisting of two tricyclic domains, each containing an independent serine proteinase binding site. Each binding site is enclosed within a nine residue disulphide-bridged loop. The binding sites of the BBIs have a very highly conserved amino acid sequence. Based on the notation of Schechter and Berger (Biochem. Biophys . Res . Cowm.

(1967) 27, 157-162), the binding loop spans the P3 to the P6 ' sequence from one cysteine to another. The sequence of the binding loop usually conforms to the following formula, where X is any amino acid:

The most variable residues occur at the PI and P2 ' residues, with the PI defining the specificity of these binding sites for the different serine proteinases .

Several groups have demonstrated the capacity of short peptides which retain the nine-residue disulphide-linked binding loop to inhibit serine proteinases. In these studies the peptides are sometimes termed "cyclic" due to the presence of the disulphide bond.

Domingo et al . ( Int . J. Peptide Protein Res . (1995) 46 p79-87) describes the synthesis and screening of a number of 11 amino acid peptides with variations at the PI site. All of the synthetic peptides described have a glutamine residue at the P5 ' position. According to Gariani and Leatherbarrow { Journal of

Peptide Research (1997) 49 p467-475) there are two factors determining the effectiveness of peptides which mimic the BBI binding loop: the value of the inhibition constant and the rate at which the inhibitor peptide is hydrolysed. In Gariani and Leatherbarrow ' s study, a variety of peptides were investigated which included the following sequence:

The Ki value for these peptides ranged between 0.0095 - 0.46 μM. It was found that changing the P2 ' position Asn-Ile improves the inhibitor's stability to hydrolysis .

THE INVENTION

Underlying the present invention is the isolation of a novel peptide from sunflower seeds which specifically inhibits serine proteinases, notably trypsin. This peptide has a molecular weight of 1513 and has been characterised by determining its three- dimensional structure in complex with bovine β-trypsin. The sequence and conformation identified from this structure shows homology to the reactive site loop of the Bowman-Birk inhibitors. This peptide has considerably enhanced potency relative to other previously described peptides of similar length. Whereas previously described peptides have been termed " cyclic" due to the presence of a disulphide bond, the peptide of the present invention exhibits a true cyclic structure in that the backbone of the peptide chain forms a continuous chain. It has previously been thought that the presence of a glutamine residue in the P5 ' position is essential for the conformation and the activity of the BBI binding loop. Surprisingly, the isolated peptide of this invention has an isoleucine residue in this position, and it is predicted that other peptides with structural similarity to the BBI binding loop, but with isoleucine in the P5 ' position will have a higher affinity than their equivalents in the art (which have a glutamine residue in this position) . The isolated peptide has an extremely low inhibition constant (Ki = 0.0001 μM) for trypsin (measured using bovine β-trypsin) . It is also relatively rigid and stable to hydrolysis owing to the fact that its backbone is a continuous peptide chain. SUMMARY OF THE INVENTION

The first aspect of the invention concerns peptidic serine proteinase inhibitors.

According to a first embodiment of the first aspect of the invention, there is provided a peptide inhibitor comprising the serine proteinase binding loop of a BBI or a peptide mimic thereof, wherein the amino and carboxyl terminal residues of the binding loop or a peptide mimic thereof are joined by a peptide bond or a peptide to form a continuous chain. A peptide mimic of a BBI binding loop is a peptide having the same qualitative binding affinity as the serine proteinase-binding loop of a BBI but with a different amino acid sequence. It particularly includes peptides which have one or more conservative amino acid changes which peptides interact with the binding pockets of the serine proteinase active site in an analogous manner to the BBI binding-loop .

The term "continuous" is used to indicate that the peptide backbone is a closed loop, so that there are no free ends and, once the loop is closed, none of the amino acids is considered to be an "amino terminal" or "carboxyl-terminal" residue. The term "continuous" distinguishes the peptides of the present invention from the "cyclic" peptides of the prior art. Previously characterised peptides corresponding to the BBI binding loop were called "cyclic" peptides because their cysteine side-chains form a disulphide bridge. In the continuous peptide chain of the present invention, the ring is completed by the formation of a peptide bond between the two terminal residues of the binding loop, or by a peptide, so the ring is completed by a main-chain bond.

The term "peptide" is used to indicate a short stretch of amino acids, commonly between 4 and 40 amino acids. Preferably the continuous peptide of this first embodiment is between 9 and 20 amino acids, more preferably between 11 and 17 amino acids, and most preferably about 14 amino acids .

The peptides of this invention may be isolated form their natural source, cleaved from larger proteins or made synthetically (for example by using a peptide synthesizer or by recombinant techniques) . Cyclic peptides may be made by solid-phase synthesis of linear peptides followed by head-to-tail cyclisation after resin cleavage. The readiness of an open chain to cyclise depends on the size of the ring to be closed and on the presence of "turn-inducing" amino acids such as proline, glycine or a D-amino acid. One or more coupling reagents may be used, for example DPPA (diphenylphosphorylazide) , TBTU (0- (benzotriazol-1-yl) - 1, 1, 3 , 3-tetramethyuronium tetrafluoroborate) , BOP (benzotriazolylxytris- (dimethylamino) - phosphoniumhexafluorophosphate) , DCC (dicyclohexylcarbodiimide) , HOBt (1- hydroxybenzotriazole) and HOAt (l-hydroxy-7- azabenzotriazole) , or derivatives thereof. On-resin cyclisation is also possible, which procedure involves a step to free the required carboxyl group.

Synthetic peptide variations such as retroinverso D peptides, variations to improve stability to hydrolysis are also intended to be within the scope of the general term "peptides".

Preferably the peptide comprises a disulphide bridge .

Preferably the peptide comprises the following sequence :

-C-T-X-S-I-P-P-Q/I-C-

in which X is any amino acid.

Most preferably the peptide inhibitor in accordance with this first embodiment of the first aspect of the invention has the following sequence:

L-rG-R-C-T-K-S-I-P-P-I-C-F-P-D- 1 S 1

According to a second embodiment of the first aspect of the invention there is provided a peptide of the formula :

L-G-R-C-T-K-S-I-P-P-I-C-F-P-D-

I _{s s}_i

or a homologue thereof.

The term "homologue" refers to peptides which comprise one or more deviations from the described sequence, but which retain the same qualitative binding affinity as the described peptide. The deviation may be a substitution, deletion or insertion of a single amino acid. Preferably the deviation will be a substitution.

Preferably the homologue will comprise fewer than 10 deviations, more preferably fewer than 7 deviations and most preferably fewer than 2 deviations from the described peptide.

Preferably the peptide according to the first aspect of the invention has an isoleucine residue in the P5 ' position.

Preferably the peptide according to the first aspect of the invention has a K_x value of less than lOnM, more preferably less that InM, most preferably less than lOOpM.

The peptide of the first aspect of the invention may be used in a number of applications. For example, transgenic expression of the peptide in plants may confer insect resistance. The peptide itself may be used generally in anti-microbial, anti-insect and food preservation applications.

A peptide having the formula:

-G-R-C-T-K-S-I- .p.p. ^■I-C-F-P ^~1 I 5— -s- inhibits trypsin, cathepsin G, elastase, chymotrypsin and thrombin. Peptides which are specific for any one or more of these enzymes or which are specific for another serine protease may be produced by protein engineering, based on this peptide.

According to a second aspect of the invention, there is provided a pharmaceutical composition comprising a peptide in accordance with the first aspect of the invention. The pharmaceutical composition may be used to inhibit blood coagulation enzymes such as Factor Xa, urokinase plasminogen activator or plasmin or mast cell proteases such as tryptase.

The potential applications of the inhibitor, together with the target serine proteases, are given in the following table:

According to the third aspect of the invention, there is provided a method for the production of a peptide in accordance with the first aspect of the invention which comprises the step of isolating a peptide from sunflower seeds.

Preferably the method comprises the following steps : i) preparing an aqueous extract of sunflower seeds ; ii) isolating the peptide; and iii) purifying the peptide.

Preferably the peptide is isolated and/or purified by serine proteinase affinity chromatography .

In order to produce peptides which are not naturally occurring in sunflower seeds, it is necessary to express the peptide in the sunflower seeds before carrying out the method of the third aspect of the invention. For example, the sunflower plant may be transfected with a gene encoding the desired peptide.

The invention shall now be further illustrated by reference to the following examples, in which:

Example 1: Purification and characterisation of the peptide;

Example 2: Competition assays to calculate the inhibitory properties of the peptide;

Example 3 : Structural studies ; and Example 4: Comparison of SFTI-1 with other similar peptides .

In the examples, reference is made to the following Figures, in which:

Figure 1: Isolation and purification of SFTI-1 (the peptide) . Figure 1(a) shows the elution profile of the peptide from a trypsin affinity column, which was then further purified by reverse-phase HPLC (Figure Kb)) .

Figure 2: Amino acid sequence of the peptide, as deduced from amino acid composition analysis, mass spectra and electron density of the peptide-trypsin complex.

Figure 3: Inhibition curve for peptide activity. Activity was assayed using BAPNA as the substrate (for details see materials and methods) and a trypsin concentration of 0.21 μM.

Figure (a) : Stereoview showing omit map electron density corresponding to SFTI-1. The map was calculated from Fo-Fc coefficients, calculated using the final refined coordinates of the complex from which the inhibitor was removed and the coordinates subjected to several rounds of further refinement. The map is contoured at 2 sigma.

Figure 4 (b) : Stereoview showing the bound conformation of the peptide in the trypsin-SFTIl complex. Residues of the inhibitor only are shown.

Figure 5 : Ramachandran plot showing peptide bond conformations for the trypsin-peptide inhibitor complex. Phi/psi relationships for inhibitor residues are depicted as crosses (x) , and for trypsin residues as dots (•) . Drawn using PROCHECK. Figure 6: Cα traces showing superimpositions of the peptide with the Mung bean inhibitor, Adzuki bean inhibitor, and soybean inhibitor.

Figure 7 : Stereoview showing the interactions of the active site loop of the inhibitor with bovine trypsin. Lys5-I projects into the SI pocket of the enzyme, delineating specificity for trypsin- like serine proteases .

EXAMPLES

In the examples, the peptide in accordance with the present invention is termed SFTI-1. Example 1

Separation of an aqueous extract of defatted sunflower seeds by trypsin affinity chromatography (Fig. la) followed by RP-HPLC (Fig. lb) gave a single homogenous component with an M_r, determined by ESMS, of

1513. Difficulties were experienced in obtaining N- terminal amino acid sequencing data, with only a small fraction of the sample (typically 15%) proving susceptible to Ν-terminal cleavage, and with 2 distinctive origins (Table 3) . The sequence of the peptide was subsequently determined directly from the electron density map (see below) . This showed a cyclic structure of 14 amino acid residues (Fig. 2) , hence explaining the difficulty encountered in N-terminal sequencing. The calculated M_r, 1513, agrees well with that from ESMS, and the sequence was further confirmed by amino acid composition analysis (Table 3) .

Sequence comparisons demonstrated that the peptide is related to a fragment present in members of the Bowman-Birk family of proteinase inhibitors, showing high sequence identity to the disulphide-stabilised loop region which contains the first active site (which is usually specific for trypsin) (Fig. 2) . However, the peptide is the smallest naturally occurring plant protein inhibitor reported to date, corresponding to the reactive loop and parts of the two adjacent strands. It contains a single disulphide bond, and has a novel cyclic structure. Example 2 The peptide inhibited bovine trypsin with a stoichiometric ratio of 1:1 (Fig. 3) and a Ki of 0.1 nM

(100 pM) was derived from the competition assays. SFTI-1 was also observed to inhibit cathepsin G (K₁~<0.15nm) , elastase (K.-IOS (±12) μM) , chymotrypsin (K_x~7.4 (±1.5) μM) , and thrombin (1^-136 (±21) μM) . No inhibition was observed with Factor Xa. Example 3 a) Structure of the peptide

The fourteen amino acids of the peptide form two anti-parallel β-strands connected at the reactive site end by an extended loop region, and by a hairpin turn at the opposite end (Fig 4) . These strands are constrained by the single disulphide bond (between Cys3 and Cysll) , dividing the peptide into a nine residue loop region, and a five residue turn. There is a sharp turn in the peptide chain at Pro8-I, and the Ile7-I - Pro8-I peptide bond is in the cis conformation. The electron density is contiguous for the main chain throughout the peptide (Fig. 4) , and all of the residues adopt reasonable conformations as determined by PROCHECK including Ramachandran plot analysis (Fig 5) . The side chain of the C-terminal residue Serl3 is not properly defined in the electron density, and hence presumably disordered within the crystals . The mean temperature factor for inhibitor main chain atoms is 25.4 A², compared with 16.7 A² for -lithe main chain atoms of trypsin. This is consistent with the surface location of the peptide within the crystal complex.

The overall conformation of the peptide is extremely similar to the reported structures for the equivalent, trypsin-inhibitory loop fragments from within the structures of reported Bowman-Birk inhibitors. Figure 6 shows superimpositions of the peptide with the Mung bean inhibitor, adzuki bean inhibitor, and soybean inhibitor. The root mean square deviations (rmsd) of equivalent main chain atoms are 0.14, 0.39 & 0.66 A² respectively. There is also similarity with the bitter gourd inhibitor, although this is restricted to the first seven residues of the peptide as this inhibitor diverges considerably from the consensus fold after this point . Note that in all cases the peptide represents only a subset of the fold observed in these other inhibitors. The exception is the Mung bean inhibitor (lsmf) , but this is a synthetic fragment of a larger inhibitor, and the construct co-crystallised is actually 22 amino acids, with only the core fragment observed in the electron density maps.

A limited number of hydrogen bonds, in concert with the disulphide, appear to stabilise the fold of the inhibitor (Fig 4b) . There are only two intramolecular main chain hydrogen bonds formed between main chain atoms: these are from residues Lys2-I to Phel2-I, and Thr4-I to Ilel0-I. Within the loop region the side chain hydroxyl group of Thr4 forms a bifurcated bond with the main chain nitrogen of Ilel0-I and side chain hydroxyl group of Ser6-I. The latter is also bonded to the main chain carbonyl group of Pro8-I. This pattern of intramolecular bonding appears to replicate the stabilisation previously described for other similar inhibitors. Further, extensive interactions with trypsin undoubtedly additionally stabilise the observed inhibitor conformation . b) Trypsin-inhibitor association

The peptide binds in the active site of trypsin in a very similar manner to the equivalent fragment from the BBI inhibitors. Central to this interaction is the extended conformation of the lysine side chain at position 5 (PI) . This residue, which is invariably Lys or Arg in either trypsin peptide inhibitors or substrates, projects into the SI pocket of the enzyme. Both direct and water-mediated contacts are made with the specificity-determining aspartate (Aspl89) at the base of this pocket, to the hydroxyl group of Serl90 and to the main chain carbonyl of Gly219. Again in common with other BBI's, the carbonyl and amine groups of the "scissile" bond between Lys5-I and Ser6-I are within hydrogen- bonding distance of the Ser (195) and His (57) of the trypsin catalytic triad. This orientation is the result of the intramolecular interactions of the Ser6 side chain in the inhibitor (see above) . There is an extensive network of hydrogen bonds and ion pairs formed between the inhibitor and enzyme, and these are summarised in Table 4.

As can be seen in Table 4, virtually all enzyme/inhibitor contacts observed in similar structures are conserved. In the most closely related structure, that of the synthetic peptide based on the Mung bean inhibitor (1SMF) , the only difference within the "reactive loop" is at position 10 (lie in the peptide (STFI-1) , Glu in SMF) . In the structure of the SFTI- 1/trypsin complex, the isoleucine side chain is close to the surface and makes no contacts with the enzyme, and hence this change does not affect the interaction of the inhibitor and the enzyme .

The majority of the hydrogen bonds between the inhibitor and trypsin are formed within the primary binding segment of the inhibitor, Cys3-Thr4-Lys5-Ser6- Ile7, which occupies the P3-P2' positions in the enzyme active site. This binding loop is extremely well defined in the electron density map, reflecting its firm immobilisation through these interactions with the highly complementary enzyme surface. The spatial arrangement of the distant side of the active loop of the inhibitor

(Pro9-I - Cysll-I) is very similar to the relationship between the "secondary contact region" of the other inhibitors and their reactive sites. The antiparallel β- sheet region Ilel8-Val34 in BPTI (bovine pancreatic trypsin inhibitor) , is replaced by a cis-Proline at the

P4 ' position in the Bowman-Birk inhibitors . This proline (equivalent to Pro8-I in SFTI-1) produces a sharp turn in the inhibitor chain. This distant side of the inhibitor, which through its cyclic nature is especially rigid in this peptide inhibitor compared to similar regions in the BBI inhibitors, is likely to play an important role in the inhibitory activity. By stabilising the overall inhibitor conformation, this region prevents the necessary structural change that would normally accompany proteolysis . Despite the relatively small size of the inhibitor, extensive solvent-accessible surface area

(2038 A², calculated with QUANTA (MSI) ) is occluded at the inhibitor/enzyme interface. c) Conformation of the enzyme active site.

Although the electron density confirms the scissile bond is uncleaved within the "average" structure observed in the crystal, the lability of a small fraction of the sample to N-terminal cleavage at this site (Table 3) suggests up to 15% of the sample may be cleaved. This implies slow hydrolysis of the peptide by trypsin or related enzymes, although the equilibrium suggests binding of the substrate is preferred to its conversion to product . The crystal structure hence represents an enzyme-substrate inhibitory complex. In common with many other peptide inhibitors, the carbonyl group from the PI residue (Lys5-I) is in close contact with the active site Serl95 OG (2.9 A, Fig. 7). This leaves His57 NE2 and Serl95 OG of the trypsin catalytic triad in close contact (2.7 A), implying the conformation of the inhibited enzyme is close to the active conformation. This differs from the arrangements observed in crystal structures of bovine trypsin in the absence of an inhibitor and in the presence of benzamidine or related inhibitors where the active site His and Ser are separated by 3 A or greater and hence unlikely to represent a catalytically productive conformation.

Similarly, the geometry of the Serl95CB-OG-His57 NE2 angle is again related to the proximity of the inhibitor to the active serine residue. In SFTI-1 and the Mung bean inhibitor, this angle is very similar (approximately 96°) . In the equivalent non inhibited and benzamidine inhibited structures, this angle is very different (88° in both cases) . These observations further confirm SFTI-1 inhibits catalysis in trypsin by an identical mechanism to other peptidic inhibitors, in particular those of the BBI family. Example 4

Although SFTI-1 is the smallest BBI-related inhibitor to be purified from plants, smaller inhibitory synthetic peptides based on BBI structures have been reported. Nishino et al . (J. Biochem. 1977, 82: 901-909) and Tereda et al (Int. J. Peptide Protein Res. 15, 441- 454) reported that a range of heterodetic nonapeptides based on the inhibitory loops of BBI were active against trypsin or chymotrypsin. Table 6 shows tha amino acid composition and reported Ki values for a series of peptides similar to SFTI-1. It was found that naturally ocurring SFTI-1 exhibits the greatest potency as an inhibitor of trypsin, with a Ki value in the sub-nanomolar range. Despite the small size of this peptide, its inhibitory activity exceeds that of the 82 amino acid residue soybean BBI, to date the most potent inhibitor reported from the BBI family.

Table 1 - Summary of X-ray diffraction data statistics

Table 2. Final Model Parameters of the SFTI-Bovine-b-Trypsin structure at 1.65A resolution.

Table 3. Comparison of the directly determined amino acid composition of SFΗ-1 with that determined by X-ray crystallography. * Cysteine residues were determined by mass spectrometry after reduction and alkylation using vinylpyridine, to form S-pyridylethylcysteine (Pecys).

Table 4. SFTI-1 trypsin contacts. ISMF and ITAB are included as comparison.

Table 5. Comparison of hydrogen bonding distances (A) and angles ( ° ) at the active site.

HTBl- Benzamidine inhibited trypsin (Bartuknik et al)

Table 6. Comparison of the amino acid sequences and reported Ki values against trypsin for a series of peptides similar to SFΗ-1.

SMF is an artificial trypsin inhibitor based on Mung bean trypsin inhibitor. BBI is soybean Bowman-Birk inhibitor. CtA (cyclotheonamide A) is a cyclic pentapeptide containing unusual amino acid residues. V-Tyr is vinologous tyrosine. Dpr is β-linked-diaminopropanoic acid. K-Arg is α-ketohomoarginine.

Claims

1. A peptide comprising the serine proteinase binding loop of a BBI or a peptide mimic thereof, wherein the amino and carboxyl terminal residues of the binding loop or a peptide mimic thereof are joined by a peptide bond or a peptide to form a continuous chain.

2. A peptide according to claim 1, which includes a disulphide bridge.

3. A peptide according to claim 1 or 2, which comprises the following sequence:

-C-T-X-S-I-P-P-Q/I-C-

in which X is any amino acid.

4. A peptide according to claim 3 , which has the following sequence:

^G-R-C-T-K-S-I-P-P-I-C-F-P-D^ I 5 — s — ¹

5. A peptide of the formula:

[LG-R-C-T-K-S-I-P-P-I-C-F-P-D ]-J

or a homologue thereof .

6. A peptide according to claim 1, 2 or 3, which has an isoleucine residue in the P5 ' position.

7. A peptide according to any any previous claim which has a K value of less than lOOpM.

8. A pharmaceutical composition comprising a peptide in accordance with any previous claim.

9. A method for the production of a peptide in accordance with any one or more of claims 1 to 7, which comprises the step of isolating a peptide from sunflower seeds .