METHOD TO PREPARE C-TERMINAL MODIFIED PEPTIDES

Abstract: Non-peptide molecules of formula (I) may be used to mimic the structure of peptides and to bind with proteins which naturally bind peptide ligands. In said formula, C' is a substituted or unsubstituted carbon atom; X is a C1 to C4 chain in which each of the carbon atoms may be substituted or unsubstituted; and R<1> and R<2> are groups mimicking the side chains of a natural or unnatural amino acid. The molecules may either be used on their own, or may be incorporated into larger molecules such as polypeptides. In either case, they may be used as therapeutics to alter the activity of the protein. Additionally, they may be used to assay the proteins or to act as affinity ligands in their purification. Methods of manufacturing polypeptides incorporating the molecules of the invention are also described.

Description: METHOD TO PREPARE C-TERMINAL MODIFIED PEPTIDES This invention relates to the manufacture of peptides and peptide analogues. Many enzyme substrates and receptor ligands consist of polypeptides. In the search for novel agents which interact with such enzymes and receptors and modify their action in a therapeutically beneficial manner, it is sensible to concentrate on molecules which possess some degree of structural and conformational analogy with the natural substrate or ligand. Very often the identity of the natural substrate or ligand is not known, but in many cases it can be inferred that a polypeptide is involved. It is therefore desirable to be able to manufacture a diverse range of compounds based on naturally occurring peptide structures, which can be screened for efficacy in interacting with enzymes and receptors of interest . It is known that the interaction of proteins controls key aspects of cellular function. For example it is protein-protein interactions which control the signalling process causing a cell to divide, the malfunctioning of which may lead to cancer and other proliferative diseases. It has previously been demonstrated that an isolated peptide having the same sequence as the active portion of a natural protein may be used to bind with the natural protein-binding partner, and to elicit the same biological response. Processes of this type are described, for example, in W097/11174, 096/14339, W096/35715 and 097/42222. The applicants have now found that a range of non- peptide molecules may be use to mimic the structure of specific peptides within the natural polypeptide binding partners of various proteins which naturally bind protein ligands, and which may be used to interact with those proteins. The molecules, which either may be used on their own or may be incorporated into larger molecules such as polypeptides, have the following general formula: R 2 wherein X = (CH 2 ) n , and n = 1, 2, 3 or 4 0 or (CH 2 ) X -C- (CH 2 ) y/ where x and y each = 0, 1, 2 or 3 and x + y ≤ 3 The remaining carbon atoms in the ring structure may also be substituted if desired, and the formula represented more generally as follows : formula I where c' is a substituted or unsubstituted carbon atom; X is a C x to C 4 chain in which each of the carbon atoms may be substituted or unsubstituted; and R 1 and R 2 are groups mimicking the side chains of a natural or unnatural amino acid. In a primary aspect, therefore, the invention provides a method of forming a complex between a protein and a molecule having analogous structure to at least a portion of the natural binding partner of the protein, wherein the molecule consists of or contains a group of formula I . One important use of molecules of this type will be as pharmaceuticals capable of affecting the activity of the bound protein, whether by inhibition or potentiation, or as templates for the design of such pharmaceuticals. Other important applications, however, will include the use in assays, as affinity ligands in purification and as diagnostic molecules for the natural binding partners. In the structure of formula I, the carbonyl group and the adjacent methylene and CR 1 R 2 groups mimic the structure of an amino acid (apart from the amino group) . The identity of groups R 1 and R 2 and of any further substituents may vary widely, and may for example be chosen from alkyl, aryl, heterocyclic, alkanol, carboxyl, amino, imino, imido, ether, ester, thiol and thioether groups. To mimic natural amino acids, for instance, R 1 and R 2 may be chosen as follows: Ei Ei amino acid H H alanine H (CH 2 ) 2 NHC(NH)NH 2 arginine H C0NH 2 asparagine H COOH aspartic acid H SH cysteine H CH 2 C0NH 2 glutamine H CH 2 COOH glutamic acid H C 3 N 2 H 3 histidine CH 3 CH 2 CH 3 isoleucine H CH(CH 3 ) 2 leucine H (CH 2 ) 3 NH 2 lysine H CH 2 SCH 3 methionine H C 6 H 5 phenylalanine H OH serine CH 3 OH threonine H C 8 NH 6 tryptophan H C 6 H 4 OH tyrosine CH 3 CH 3 valine Other species may also be chosen as groups R 1 and R 2 , in order to produce a wide range of different amino acid mimicking molecules . One important feature of the ring structure of Formula I is that it imparts a high degree of conformational restriction, which limits the degrees of freedom available to the resultant molecules. This leads to tighter ligand binding and thus increases the chances of identifying active compounds. The preferred number of atoms in the ring lies in the range 4 to 7 : 3-membered rings exhibit excessive strain, and rings with more than 7 members are generally too flexible. Preferably, a 5- or 6-membered ring is employed. One particularly preferred group of ring structures is provided by the cyclic 1,3-diones, especially those having 6-membered rings of the following formula: Formula II The ability of such groups to mimic amino acid residues may be appreciated from the following comparison of dimedone with valine: dimedone valine Compounds of this formula (which will be referred to herein as "Cyclodiones" , a name coined by the applicants), may easily be prepared by known techniques, and are capable of undergoing a many different reactions to produce a large range of derivatives. For example, electrophilic addition may be effected at the C-2 position, and nucleophilic addition at the C-3 position. The addition of bi-functional reagents, containing both electrophiles and nucleophiles leads to bicyclic products in which further beneficial restraints are placed upon conformational flexibility. The two carbonyl groups also provide ideal sites for hydrogen bonding . In order to produce polypeptide molecules incorporating the cyclic groups of the invention, the applicants have developed a novel method of solid-phase "reverse peptide" synthesis (i.e. synthesis that proceeds in the opposite sense to conventional peptide synthesis) . The method involves the use a linker such as the well known "Wang linker" , to which an unprotected amino acid may be attached via its amino group, thus leaving its carboxyl group free for further reaction. A key advantage of this method is that it is readily possible to make modifications to the C- terminus of the peptide, in order to produce a wide range of different peptide molecules . Another advantage is that unprotected amino acids may be used, which leads to significant simplification compared to traditional synthesis methods in which protected amino acids are essential. The invention therefore provides, in another aspect, a method of manufacturing a peptide or peptide analogue comprising the steps of reacting a molecule having a free carboxyl group with a compound of formula I (as defined above) . Conveniently, the molecule having a free carboxyl group may be produced by reacting the terminal hydroxyl group of a resin-bound linker with a substituted chloroformate to produce a resin-bound molecule having an activating group attached via a carbonate linkage. An amino acid is then added, which displaces the activating group to form a carbamate linkage, resulting in a resin-bound molecule terminating in an amino acid residue with a terminal carboxyl group. These steps are illustrated below in an example in which the chloroformate is p-nitrophenyl chloroformate, and the amino acid is valine. Also illustrated are suitable reaction steps for producing the initial resin-bound linker, which are known in the art. (i) HOCH,C b H 4 OCH 2 COOH / DIC / HOBt / DCM / DMF (ii) N 2 H, .H 2 0 / DMF (iii) p- nitrophenyl chloroformate / pyridine / DCM (iv) L-valine / BSA / DMAP / DMF The product of this process (which may conveniently be termed a "polypeptide precursor") is a key intermediate in the peptide formation process of the invention. It may be used in a number of ways to produce polypeptide molecules in the "reverse" manner, the incorporation of Cyclodiones being only one example. In order to produce a polypeptide, the precursor may be reacted with a further substituted chloroformate to produce a mixed carbonic anhydride (i.e. a molecule having a -CO-O-CO-O- linkage) . This too is an extremely important intermediate, as it directly permits the stepwise addition of further amino acids at will . Addition of an amino acid results in cleavage of the anhydride bond, the evolution of C0 2 , and the formation of a peptide linkage. Because the terminal amino acid residue is left with a free carboxyl group, the process may then be repeated with further amino acids as many times as desired. This reaction is illustrated by the following example, in which phenylalanine is added to the mixed carbonic anhydride: (v) p-nitrophenyl chloroformate / pyridine / DCM (vi ) L-phenylalanine The resulting polypeptide may then be directly reacted with a group of Formula I or Formula II, in the presence of a suitable coupling reagent (such as DCC/DMAP) , as illustrated in the following example: - τ : UNK; ■ DCC/DMAP aq. TFA It will be appreciated that the essential feature of the polypeptide molecule, from the point of view of reactivity with the group of Formula I or Formula II, is the terminal carboxyl group. It follows that the group of Formula I or Formula II could equally well be reacted directly with the polypeptide precursor. In a variant of the general method, the terminal amino acid residue may be cyclised by treatment with a coupling reagent to form an oxazolone, which is itself a significant intermediate in the synthesis of peptides and peptide- mimicking molecules. For example, the oxazolone may readily be alkylated at the C-4 position to produce an ot, a- dialkylated amino acid, which is a group known to induce certain elements of secondary structure. Alternatively, the oxazolone may be reacted with a further (free) oxazolone to produce a "Rϋgheimer dimer" . Both reactions are illustrated in the following example: Thus, in an alternative aspect, the invention provides a method of manufacturing a peptide or polypeptide comprising the initial steps of: (a) forming a resin-bound peptide molecule with a terminal amino acid residue having a free carboxyl group; and (b) performing a cyclising condensation reaction between the carbonyl of the peptide linkage and the free carboxyl group of the terminal amino acid residue to form a terminal oxazolone group. As will be seen, the invention permits the creation of a very substantial range of different peptides and peptide- mimicking molecules . These in turn will provide a large "library" of compounds for potential therapeutic use. The benefit of the polypeptide-mimicking molecules provided by the invention may be seen from the following illustrative example relating to the interaction of Cyclodiones with cyclophilin. Cyclophilins are potential drug targets in a number of different disease areas, including immunosuppressive disorders, parasite infection 1 , rheumatoid arthritis 2 and AIDS 3 ' 4 . Currently, the only known cyclophilin-binding drug is cyclosporin A, an immunosuppressant undecapeptide drug used to prevent organ rejection after transplant operations 5,6,7 . We have proved that Cyclodiones can mimic peptides by forming a crystal complex of the parent dimedone with the protein human cyclophilin A. A protein X-ray structure resolved to a resolution of 0.2 nm clearly shows the presence of dimedone in the cyclophilin A active site. The position of the dimethyl group of the dimedone group mimics the position of a valine side chain adopted by the cyclosporin A inhibitor. The following experimental details are included by way of illustration. Recombinant human cyclophilin (hCypA) was provided by Novartis AG and concentrated to 14 mg/ml in Hepes 20mM, NaCl 100 M and NaN 3 0.02% (w/v) . Crystals of the hCypA were grown by vapour diffusion at 17°C by the hanging drop method. The precipitating solution in the well consisted of lOOmM Tris.HCl (pH 8.0), 22% (w/v) PEG 8000, 5% (v/v) DMSO, 0.02% NaN 3 . The initial 8μl drop consisted of 50mM Tris.HCl (pH 8.0), 11% (w/v) PEG 8000, 2.5% (v/v) DMSO, 0.02% NaN 3 , 0.4mM hCypA . The dimedone ligand was introduced into the crystal (0.2mm x 0. lmm x 0.025mm) using a stepwise soaking procedure in which the DMSO concentration was gradually reduced and the ligand concentration gradually increased. This procedure was required to prevent crystal damage and also to prevent competition of DMSO binding at the active site. In the first step a single crystal of hCypA was soaked in a precipitating solution containing 30mM ethyl-1-piperidine- glyoxylate and a reduced (4%) concentration of DMSO. After one hour the crystal was transferred to a fresh soaking solution containing 30 mM ligand and 3% DMSO. The crystal was transferred a total of 6 times over a period of 6 hours. The final soak was for 2.5 hours in a solution containing no DMSO and 30mM dimedone. Flash freezing of the crystal in liquid nitrogen was carried out after soaking in a cryoprotectant solution consisting of lOOmM Tris.HCl (pH 8.0), 22% (w/v), PEG 8000, 0.02% NaN 3 , and 30mM ligand 30% glycerol . X-ray data were collected on a MAR image plate (30cm diameter) detector with CuKa (λ=l.5412A) radiation from a rotating anode X-ray generator. Data sets were processed with the computer programs DENZO and scaled with SCALEPACK. The refined structure of hCypA from the X-ray structure of the hCypA/Cyclosporine A complex was used as a starting model. A rotation and translation search using AMORE was used to position the molecule and gave a good starting model. Subsequent rigid body refinement and refinement of individual atom isotropic temperature factors gave an R factor of 21% for data between 15 and 1.7A. A difference Fourier map calculated at this stage showed clear electron density for the bound ligand. The structure was built using the WITNOTP software. Full refinement including all water molecules were added giving an R= 18.7%. The dimedone ligand was found to occupy the proline binding pocket . TABLE 1 Crystallographic data for the hCypA/Di edone complex 1 - 1 - * Rmerge" (∑| I-<I> I /∑| 11 ) where I is the observed intensity and <I> is the average intensity from observations of symmetry related reflections, respectively. This X-ray structure reveals that dimedone is able to mimic a valinyl residue, present in the natural ligand cyclosporine, as shown below: Novel cyclophilin binding ligands are highly likely to be medically useful as inhibitory drugs . The discovery that inhibition of cyclophilin prevents its incorporation into the HIV protein coat indicates that families of inhibitors unrelated to the immunosuppressant cyclosporins may provide potential anti-HIV drugs. The development of species- specific cyclophilin inhibitors may also provide a route to novel anti-parasitic drugs. References : 1. Page, A. P.; Kumar, S . ; and Carlow, C.K.S. "Parasite cyclophilins and antiparasite activity of cyclosporin A" Paras i tolQQV Today 11 (1995) 385-388. 2. Billich, A.; Winkler, G.; Aschauer, H. ,- Rot, A.; and Peichl, P. "Presence of cyclophilin A in synovial fluids of patients with rheumatoid arthritis" J. Experimental Medicine 185 (1997) 975-980. 3. Luban, J. ; Bossolt, K. L . ; Franke, E.K.; Kalpana, G.V. ; and Goff, S.P. "Human-immunodeficiency-virus type 1 gag protein binds to cyclophilin A and cyclophilin B" Cell 73 1067-1078. 4. Sherry, B. ; Zybarth, G.; Alfano, M. ,- Dubrovsky, L.; Mitchell, R. ; Rich, D; Ulrich, P.; Bucala, R. ; Cerami, A; and Bukrinsky, M. "Role of cyclophilin A in the uptake of HIV-1 by macrophages and t lymphocytes" Proceedings of the National Academy of Sciences of the Uni ted States of America 95 (1998) 1758-1763. 5. Beveridge, T. ; and Calne, R.Y. "Cyclosporine (sandimmun (r) ) in cadaveric renal-transplantation - 10- year follow-up of a multicenter trial" Transplantation 59 (1995) 1568-1570. 6. Pflugl, G.; Kallen, J.; Schirmer, T.; Jansonius, J.N. ; Zurini, M.G.M.; and Walkinshaw, M.D. "X-ray structure of a decameric cyclophilin cyclosporin crystal complex" Nature 361 91-94. 7. Mikol, V.; Kallen, J. ; Pflugel, G.; and Walkinshaw, M.D. "X-ray structure of a monomeric cyclophilin-A- cyclosporin-A crystal complex at 2-center-dot-l- angstrom resolution" J. Molecular Biology 234 1119- 1130.

Claims: CLAIMS 1. A method of forming a complex between a protein and a molecule having analogous structure to at least a portion of the natural binding partner of the protein, wherein the molecule consists of or contains a group of the following formula: where C is a substituted or unsubstituted carbon atom; X is a C x to C 4 chain in which each of the carbon atoms may be substituted or unsubstituted; and R 1 and R 2 are groups mimicking the side chains of a natural or unnatural amino acid. A method according to claim 1, wherein X is a C 2 to C 3 chain. 3. A method according to claim 1 or claim 2, wherein the compound of formula I has the following formula (II) : wherein the carbon atoms in the ring structure may be substituted or unsubstituted. 4. A method according to any of claims 1 to 3 , wherein the substituents on the carbon atoms in the ring structure are chosen from hydrogen, alkyl, aryl, heterocyclic, alkanol, carboxyl, amino, imino, imido, ether, ester, thiol and thioether groups. 5. A method according to claim 3 , wherein the molecule is dimedone and the protein is cyclophilin. .6. A method of manufacturing a peptide or peptide analogue comprising the step of reacting a molecule having a free carboxyl group with a compound of Formula I : where C is a substituted or unsubstituted carbon atom; X is a C x to C 4 chain in which each of the carbon atoms may be substituted or unsubstituted; and R 1 and R 2 are groups mimicking the side chains of a natural or unnatural amino acid. 7. A method according to claim 6, wherein X is a C 2 to C 3 chain. 8. A method according to claim 6 or claim 7, wherein the compound of formula I has the following formula (II) : wherein the carbon atoms in the ring structure may be substituted or unsubstituted. 9. A method according to any of claims 6 to 8 , wherein the substituents on the carbon atoms in the ring structure are chosen from hydrogen, alkyl, aryl, heterocyclic, alkanol, carboxyl, amino, imino, imido, ether, ester, thiol and thioether groups. 10. A method according to any preceding claim, wherein the molecule having a free carboxyl group is a resin-bound peptide. 11. A method according to claim 10, wherein the resin- bound peptide is produced by the steps of: (a) reacting the terminal hydroxyl group of a resin-bound linker with a substituted chloroformate to produce a resin-bound molecule having an activating group attached via a carbonate linkage,- and (b) displacing said activating group with an amino acid and forming a carbonate linkage between the resin-bound linker and the amino group of said amino acid. 12. A method according to claim 11, wherein one or more further amino acids are added to the resin-bound peptide by: (a) reacting the free carboxyl group of the terminal amino acid residue with a substituted chloroformate to form a mixed carbonic anhydride,- (b) cleaving the anhydride bond by addition of a further amino acid, accompanied by evolution of carbon dioxide, to form a peptide linkage; and (c) repeating steps (a) and (b) as often as required. 13. A method according to claim 11 or claim 12, wherein as a final step, the resin-bound linker is detached to release a free peptide or polypeptide molecule. 14. A method of manufacturing a peptide or polypeptide comprising the initial steps of: (a) forming a resin-bound peptide molecule with a terminal amino acid residue having a free carboxyl group,- and . (b) performing a cyclising condensation reaction between the peptide linkage and the free carboxyl group of the terminal amino acid residue to form a terminal oxazolone group . 15. A method according to claim 14, comprising the further step of alkylating the oxazolone group at the C-4 position. 16. A method according to claim 15, wherein the terminal amino acid residue has an alkyl side-chain, whereby the product of the step of claim 7 is an a , α-dialkylated amino acid residue. 17. A method according to claim 14, comprising the further step of reacting the terminal oxazolone group with a further oxazolone molecule to form a Rϋgheimer dimer. 18. A method according to any of claims 14 to 17, wherein the resin-bound peptide having a free carboxyl group is produced by the steps of : (a) reacting the terminal hydroxyl group of a resin-bound linker with a substituted chloroformate to produce a resin-bound molecule having an activating group attached via a carbonate linkage,- and (b) displacing said activating group with an amino acid and forming a carbonate linkage between the resin-bound linker and the amino group of said amino acid. 19. A method according to claim 18, wherein one or more further amino acids are added to the resin-bound peptide, prior to the cyclisation step, by: (a) reacting the free carboxyl group of the terminal amino acid residue with a substituted chloroformate to form a mixed carbonic anhydride; (b) cleaving the anhydride bond by addition of a further amino acid, accompanied by evolution of carbon dioxide, to form a peptide linkage,- and (c) repeating steps (a) and (b) as often as required.

Inventors: FLITSCH SABINE LAHJA, TAYLOR PAUL, TURNER NICHOLAS JOHN, WALKINSHAW MALCOLM DOUGLAS, FLITSCH, SABINE, LAHJA, TAYLOR, PAUL, TURNER, NICHOLAS, JOHN, WALKINSHAW, MALCOLM, DOUGLAS
Applicants: FLITSCH SABINE LAHJA, TAYLOR PAUL, TURNER NICHOLAS JOHN, UNIV EDINBURGH, WALKINSHAW MALCOLM DOUGLAS, FLITSCH, SABINE, LAHJA, TAYLOR, PAUL, THE UNIVERSITY COURT OF THE UNIVERSITY OF EDINBURGH, TURNER, NICHOLAS, JOHN, WALKINSHAW, MALCOLM, DOUGLAS
Application year: 1999
Main classification: C12N9
IPC codes: A61K47, A61P43, C07K1, C07K19, C12N9