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WO1998014208A9 - Ligands reactifs et complexes covalents de ligands et proteines - Google Patents

Ligands reactifs et complexes covalents de ligands et proteines

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WO1998014208A9
WO1998014208A9 PCT/US1997/017483 US9717483W WO9814208A9 WO 1998014208 A9 WO1998014208 A9 WO 1998014208A9 US 9717483 W US9717483 W US 9717483W WO 9814208 A9 WO9814208 A9 WO 9814208A9
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alkyl
amino
bonded
amino acid
peptide
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  • Ligand protein interactions influence many factors of biological systems. Ligands bind to proteins according to particular kinetic profiles. For example, antigenic peptides of T cells first bind to class I or class II major histocompatibility complex (MHC) molecules before interacting with T cell receptors on CD 8+ T cells or CD + T cells to elicit immune responses. Stern, L.J. & Wiley, D.C. (1994), Structure 2, 245-251. In general , conservative residue substitutions in the middle portion of antigenic peptides of T cells are introduced to selectively inhibit activation of T cells without significantly altering binding affinity to the relevant MHC molecules. See, Sloan-Lancaster, J., Evavold, B.D. & Allen, P.M.
  • MHC-blocking peptides have included the replacement of several residues from the middle portion of antigenic peptides with moieties such as 4-aminobutyric acid, 6-aminohexanoic acid, and phenanthridine to act as spacers.
  • moieties such as 4-aminobutyric acid, 6-aminohexanoic acid, and phenanthridine to act as spacers.
  • the reversible kinetics of binding lead to the release of the ligand from the binding site, thereby influencing recognition of the ligand-protein complex.
  • By changing the kinetics of binding it is possible to alter the response to the cellular immune system.
  • covalent complexes of peptides with MHC molecules have been constructed previously by generating recombinant proteins with elongated N-termini that extended into the binding grooves of MHC class I (see, e.g., Mottez, E., Langlade-Demoyen, P., Gournier, H., Martinon, F., Maryanski, J. , Kourilsky, P. & Abastado, J.-P.
  • the present invention features compounds that bind as ligands to a binding region of a protein.
  • the compounds react with residues in the binding region, selectively forming a covalent bond between the protein and the ligand.
  • the formation of the covalent bond changes the reversibility of the binding kinetics between the ligand and the protein.
  • the invention relates to a compound of the formula (I)
  • An alkyl group is a branched or unbranched C x to C 6 alkyl or benzyl
  • aryl is a substituted or unsubstituted C 6 to C 14 carbocyclic or heterocyclic aromatic having one or two rings.
  • a substituted aryl is an aryl bearing one or more ⁇ to C 6 substituents including alkyl, alkoxy, or acyl groups, or halogen, hydroxy, amino, or amido groups.
  • a substituted amino is an amino group having one or two branched or unbranched C ⁇ to C 6 alkyl or benzyl groups.
  • R x is halogen, alkyl sulfonate, carboxylate, or aryl sulfonate and R 2 is hydroxy, amino, alkylamino, ammonium, or alkyl ammonium, or together R x and R 2 are O, NH 2 ⁇ or NH;
  • X is a C 2 to C 4 alkyl;
  • R 3 is H;
  • R 4 is -NH-W, where W is an amino acid residue bonded at its carbonyl group or a peptide moiety bonded at its terminal carbonyl group; and
  • W is an amino acid residue bonded at its carbonyl group and Y' is a peptide moiety bonded at its terminal amino group.
  • R x is alkyl sulfonate and R 2 is an ammonium, or together R : and R 2 are 0, or NH; X is C Pain alkyl; and the amino acid residue is a glycine residue.
  • a portion of the peptide moiety is identical to a portion of a selected peptide or an antigenic peptide.
  • the peptide moiety can preferably contain between 2 and 10 amino acid residues.
  • the compound is G(az)IDKPILK or G (az) AFVTIGK, where (az) is an unnatural amino acid residue where the side chain is a C 6 alkyl having a terminal aziridine group .
  • a further aspect of this invention relates to a method of forming a ligand-protein complex.
  • the method includes the steps of providing a protein having an arginine-specific binding region, the binding region including a nucleophilic residue, and adding a ligand to bind with the protein at the arginine-specific binding region, the ligand having a moiety which reacts with the nucleophilic residue.
  • the moiety of the ligand and the nucleophilic residue of the protein form a covalent bond between the ligand and the protein.
  • the arginine-specific binding region includes a thiol , a thiol and a carboxyl , or two carboxyl groups, and the moiety is halogen, haloalkyl, haloalkyl carbonyl, alkyl sulfonate, aryl sulfonate, carboxylate, a Michael acceptor, aziridine, or epoxide.
  • the arginine-specific binding region includes a thiol and a carboxyl and the moiety is aziridine, or epoxide.
  • the step of providing the protein includes the step of altering a binding region of the protein to introduce the arginine-specific binding region.
  • Another aspect of this invention relates to a covalently linked ligand-protein complex including a protein having an arginine-specific binding region and a ligand covalently bonded to the binding region of the protein, said ligand having the formula
  • R ⁇ and R 2 is hydroxy, alkyl, alkyl carbonyl, amino, alkyl amino, ammonium, or alkyl ammonium, and the other of R x and R 2 is the covalent bond to the protein;
  • X is C x to C 6 alkyl, substituted aryl, or deleted;
  • R 3 is H, alkyl, or aryl;
  • Y is alkyl, alkoxy, amino, substituted amino, hydroxyl, a peptide moiety bonded at its terminal amino group or an amino acid residue bonded at its amino group; and
  • the arginine-specific binding region includes a thiol, a thiol and carboxyl, or two carboxyl groups;
  • R x is the covalent bond between the ligand and the protein;
  • R 2 is hydroxy, alkyl, alkyl carbonyl, amino, alkyl amino, ammonium, or alkyl ammonium;
  • R 3 is H;
  • X is a C 2 to C 4 alkyl.
  • R 2 is the covalent bond between the ligand and the protein (i.e., a thioether or ester linkange) .
  • the invention includes one or more of the following advantages.
  • the ligand selectively reacts with groups in the binding region of the protein. Thus, undesired reactions outside of the binding region are unlikely to occur.
  • the covalent nature of the linkage between the ligand and the protein changes the kinetics of ligand binding dramatically by making the ligand binding essentially irreversible.
  • a "selected peptide” is any peptide or variant thereof that is associated with an undesired physical condition, such as, but not limited to, an immune system- selective peptide (i.e., a peptide that induces an immune response) .
  • an antigenic peptide mentioned herein can be either a naturally occurring peptide or a variant thereof (e.g., containing an acetylated alpha- amino group, an amidated alpha-carboxyl group, a D- and/or beta- or gamma-amino acid residue, or any suitable organic moiety) ; indeed, there are only two criteria, i.e., it is a peptide and it binds to a class I or class II MHC molecule in the manner described in Stern, L.J. & Don C. Wiley, Structure (1994) 2, 245-251.
  • FIG. 1 is a schematic diagram depicting the synthesis and structure of a compound according to the invention.
  • Fig. 2 is a drawing of a model of the P2 pocket of HLA-B27 binding arginine (Fig. 2A) , a model of an aziridine-containing ligand (Fig. 2B) and a covalently bonded residue (Fig. 2C) .
  • Fig. 3 is a graph of three HPLC runs depicting the stability of the aziridine ligand to free thiols in solution.
  • Fig. 4 is an image of an SDS-PAGE (15%) of HLA-B27 complexed with the aziridine-containing peptide (G(az) IDKPILK) (lane 1) and the control peptide (GRIDKPILK) (lane 2) .
  • Ligands bind to proteins according to particular kinetic profiles. Short peptides containing arginine at peptide position 2 (P2) bind to arginine-specific binding regions of class I major histocompatibility complex (MHC) glycoproteins, such as HLA-B27.
  • MHC major histocompatibility complex
  • HLA-B27/peptide complex is recognized by particular T cells. This recognition leads to both the development of the repertoire of T cells in the cellular immune system and the activation of cytotoxic T cells.
  • the reversible kinetics of binding lead to release of the ligand from the binding site, thereby affecting the recognition of the ligand-protein complex. By changing the kinetics of the reversible binding, it is possible to alter the response to the cellular immune system.
  • Reactive ligands can be used to change the kinetics of binding by reacting with the protein when bound.
  • Suitable reactive ligands for changing the kinetics of binding with protein substrates are compounds of the formula I.
  • Each of R x and R 2 is hydroxy, halogen, haloalkyl, haloalkyl carbonyl, alkyl sulfonate, aryl sulfonate, carboxylate, a Michael acceptor, amino, alkylamino, ammonium, or alkyl ammonium.
  • R ⁇ and R 2 are O, NH, NH 2 + , or NZ, where Z is alkyl, or aryl, thus forming an epoxide, aziridine or substituted aziridine ring.
  • X a spacer group, is C x to C 6 alkyl, substituted aryl, or deleted.
  • R 3 is H, alkyl, or aryl.
  • one of R x and R 2 is a reactive group that will react with a nucleophile (e.g., -SH, -NH 2 , -OH, or -COO") .
  • a nucleophile e.g., -SH, -NH 2 , -OH, or -COO
  • one of the groups is a leaving group or contains a leaving group.
  • the leaving group can be a halogen, haloalkyl, haloalkyl carbonyl, alkyl sulfonate, aryl sulfonate, alkyl carboxylate, ammonium, alkyl ammonium, epoxide, aziridine, or substituted aziridine, where the alkyl group is a branched or unbranched C x to C 6 alkyl or benzyl and aryl is a substituted or unsubstituted C 5 to C 14 carbocyclic or heterocyclic aromatic having one or two rings.
  • the reactions between the group at R x or R 2 and the nucleophile take place at a binding region of a protein.
  • the compounds of the invention can be used to prepare reactive ligands, or are themselves reactive ligands that can be used to make covalent ligand-protein complexes in vi tro or in vivo .
  • R 4 and R 5 is a reactive group or the peptide moiety or amino acid residue is ultimately a group that binds selectively to a protein.
  • a portion of the peptide moiety, the amino acid residue, or a combination thereof is a selected peptide or an antigenic peptide, which can be either a naturally occurring peptide or a variant thereof (e.g., containing an acetylated alpha-amino group, an amidated a-carboxyl group, a D- and/or beta- or gamma- amino acid residue, or any suitable organic moiety) .
  • a portion of the peptide moiety, the amino acid residue, or their combination is a peptide and it binds to a class I or class II MHC molecule in the manner described in Stern, L.J.
  • short peptides containing arginine at peptide position 2 bind to arginine-specific binding regions of class I major histocompatibility complex (MHC) glycoproteins, such as HLA-B27.
  • MHC major histocompatibility complex
  • HLA-B27/peptide complex is recognized by particular T cells.
  • Fig. 1 is a schematic diagram depicting the synthesis and structure of a compound according to the invention.
  • Abbreviations are Boc, butoxycarbonyl ; Ph, phenyl; DEAD, diethyl azodicarboxylate; THF, tetrahydrofuran; DMS, dimethyl sulfide; TFA, trifluoroacetic acid; Gly, glycine; BOP, benzotriazol-1- yloxy-tris (dimethylamino)phosphonium hexafluorophosphate; HOBT, 1-hydroxybenzotriazole; DIEA, diisopropyl ethylamine; MCPBA, m-chloroperoxybenzoic acid; EtOAc, ethyl acetate; Ms, mesyl ; Me, methyl; HBTU 2-(lH- benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluoro
  • the first two steps shown in Fig. 1 can be modified to prepare variants of Compound 1, where the length of the alkenyl group can be varied, or other functionalities can be added using ordinary synthetic methods.
  • Boc-amine (Compound 1) was prepared according to the methods described in Arnold, L.D., Kalantar, T.H. & Vederas, J.C. (1985) J. Am. Chem. Soc. 107, 7105-7109, and Arnold, L.D., Drover, J.C. G. & Vederas, J.C. (1987) J. Am. Chem. Soc. 109, 4649-4659 and the dipeptides were prepared according to the methods described in Arnold, L.D., Kalantar, T.H.
  • the olefinic group of Compound 1 can be transformed by ordinary methods, e.g., addition reactions.
  • the subsequent reactions of Compound 1 and its analogues can be carried out to link other natural or unnatural amino acid residues or peptide moieties.
  • a ligand-protein complex is formed by exposing a protein having an arginine-specific binding region which includes a nucleophilic residue to a ligand which binds with the protein at the arginine-specific binding region.
  • the ligand is a compound of the invention, thus having a moiety which reacts with the nucleophilic residue.
  • the reactive moiety of the ligand and the nucleophilic residue of the protein form a covalent bond between the ligand and the protein, resulting in a covalently linked ligand-protein complex where the protein and ligand become linked at R x or R 2 , changing the kinetics of release of the ligand from the binding site.
  • Cytotoxic T-lymphocytes recognize antigens as short peptides bound to MHC class I molecules that consist of a heavy chain (H) and 3 2 -microglobulin ( ⁇ 2 -m) .
  • H heavy chain
  • ⁇ 2 -m 3 2 -microglobulin
  • HLA-B27 has been implicated in, for example, the autoimmune disorder ankylosing spondylitis. See, Brewerton, D.A., Caffrey, M., Hart, F.D., James, D.C.O., Nicholls, A. & Sturrock, R.D. (1973) Lancet 1, 904-907, Schlosstein, L. , Terasaki, P.I., Bluestone, R. & Pearson, CM. (1973) N. Engl . J. Med. 288, 704-706, and Hammer, R.E., Maika, S.D., Richardson, J.A., Tang, J.-P. & Taurog, J.D.
  • the autoimmune response involves binding of an autoantigen peptide by HLA-B27.
  • Endogenous and viral peptides that bind to HLA-B27 have an arginine in their P2 position, as described by Jardetzky, T.S., Lane, W.S., Robinson, R.A. , Madden, D.R. & Wiley, D.C. (1991) Nature 353, 326-329.
  • the d-guanidinium group of the arginine is bound within a deep pocket where four polymorphic residues project toward it. See, Madden, D.R., Gorga, J.C, Strominger, J.L. & Wiley, D.C. (1991) Nature 353, 321-325, and Madden, D.R. , Gorga, J.C, Strominger, J.L. & Wiley, D.C. (1992) Cell 70, 1035-1048.
  • T cell receptors on CD8 + and CD4 + T cells requires the recognition of antigenic peptides bound in the groove of MHC molecules on the surface of antigen-presenting cells.
  • Antigenic peptides bind in an extended conformation to class I and class II MHC molecules.
  • X-ray crystallography studies have revealed that for all class I MHC complexes, the peptide N- and C- termini form hydrogen bonds with conserved MHC amino acid residues located at both ends of the binding site, some peptide side chains are buried in deep pockets along the binding site, and the central region of the peptide arch away from the floor of the binding site with side chains generally pointing toward T cell receptors.
  • Antigenic peptides of 8- to 10-amino acids long have been shown to adopt this mode of binding.
  • a decamer has been shown to extend out of the binding groove at the C-terminus and an octamer has been shown to preferentially leave the N- terminal end of the binding site unoccupied.
  • other modes of binding can be described: extension of a decamer out of the N- terminal end of the binding site and binding of an octamer preferentially at the N-terminal end of the groove leaving the C-terminal end of the groove unoccupied.
  • class II MHC molecules bind to longer antigenic peptides.
  • This difference in length can be ascribed to structural differences between the two binding sites and the position of key MHC amino acid residues that interact with the peptide backbone.
  • the few X-ray structures of class II MHC complexes that have been determined so far indicate that the antigenic peptide is stretched out along most of its length and adopts a bound conformation characteristic of a type II polyproline helix.
  • the peptide' s N- and C-termini extend out of the binding groove.
  • Side chains of some amino acid residues bind specifically in pockets made of polymorphic MHC amino acid residues while other side chains, spaced approximately three amino acid residues apart, point away from the binding site.
  • Interaction of T cell receptors with both types of MHC complexes involves amino acid residues on both the peptide and MHC molecules.
  • amino acid residues from the central portion of the peptide, where side chains are most exposed play a more crucial role in this recognition event
  • amino acid residues having side chains pointing up are found along the entire sequence.
  • references for the structures of class I MHC molecules include: Madden, D.R., Garboczi, D.N. & Wiley, D.C. (1993) Cell 75, 693-708; Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L. & Wiley, D.C. (1987) Nature 329, 506-512; Madden, D.R., Gorga, J.C, Strominger, J.L. & Wiley, D.C. (1991) Nature 353, 321-325; Silver, M.J., Guo, H.-C, Strominger, J.L. & Wiley, D.C. (1992) Nature 360, 367-369; Guo, H.-C, Jadertzky, T.S., Garrett, T.P.J., Lane, W.S., Strominger,
  • a ligand was synthesized with a reactive side chain designed to mimic arginine. Although other reactive groups at this position can be used, an aziridine is most preferred.
  • the reactive ligand contains an aziridine and binds covalently by reaction of the aziridine upon binding in the arginine-specific P2 pocket of HLA-B27.
  • a peptide was designed such that a covalent bond with HLA-B27 will form between the ligand and the protein spontaneously and selectively upon binding.
  • An aziridine-containing amino acid mimic of arginine was selected (Fig. 2B) . According to Fig. 2B) . According to Fig.
  • the aziridine-containing ligand can bind in place of the guanidinium group with the aziridine amine, forming a hydrogen bonded salt bridge to Glu-45.
  • the free thiol of Cys-67 is poised for in-line nucleophilic attack of the aziridine group.
  • Fig. 2C shows the expected geometry of the covalent complex between Cys-45 and the aziridine- containing peptide that results from the nucleophilic attack of the thiol which opens the aziridine group.
  • the positively charged secondary amine of the aziridine ring is expected to form a hydrogen-bonded charged-pair with Glu-45 in the arginine-specific pocket, positioning the eta-carbon of the aziridine ring for an in-line, ring- opening attack by the thiolate of Cys-67 (Fig. 2C) .
  • Selectivity is expected from the polarizability of the aziridine by Glu-45 and the propinquity of the Cys-67 thiol to the bound aziridine ring.
  • the aziridine- containing ligand is shown to alkylate specifically cysteine 67 of HLA-B27.
  • free cysteine in solution nor an exposed cysteine on a class II MHC molecule can be alkylated, showing that specific recognition between the anchor side-chain pocket of an MHC class I protein and the designed ligand (propinquity) is necessary to induce the selective covalent reaction with the MHC class I molecule.
  • the aziridine within the ligand was cyclized from the mesylate in solution, before addition to the folding buffer containing recombinantly expressed, purified HLA- B27 heavy chain and ⁇ 2 -m.
  • Activation and regulation of immune responses can be influenced by the compounds of the invention, since the compounds bind to specific protein binding pockets irreversibly. It is possible to activate T cell responses by treatment with a preformed ligand-protein complex or by treatment with the reactive ligand or a precursor thereof.
  • Nonamer peptides containing the aziridine were synthesized by coupling the dipeptide to synthetic heptamers corresponding to the seven C-terminal amino acids of two peptides known to bind to HLA-B27, GRIDKPILK and GRAFVTIGK. See, Jardetzky, T.S., Lane, W.S., Robinson, R.A. , Madden, D.R. & Wiley, D.C. (1991) Nature 353, 326-329.
  • the peptide sequence of the ligand shown in Fig. 1 and described here is based upon a ribosomal peptide GRIDKPILK. Other peptide sequences and analogues can be substituted in the scheme shown in Fig.
  • Boc-amine (Compound 1) .
  • Boc-amine Compound 1 was prepared according to the methods described in Arnold, L.D., Kalantar, T.H. & Vederas, J.C (1985) J. Am. Chem. Soc. 107, 7105-7109, and Arnold, L.D., Drover, J.C.G. & Vederas, J.C. (1987) J. Am. Chem. Soc. 109, 4649-4659.
  • Boc-Glycine- (2-amino-hept-6-enoic acid) dipeptide methyl ester (Compound 2) .
  • Compound 1 211 mg, 0.911 mmol
  • CH 2 C1 2 5.0 mL
  • trifluoroacetic acid 5.0 mL
  • the mixture was swirled, allowed to stand for 20 minutes, diluted with 4 mL of toluene, and concentrated in vacuo to afford the crude deprotected amine.
  • the deprotected amine was dissolved in 5.0 mL of CH 2 C1 2 .
  • Diisopropyl ethylamine (0.476 mL, 0.273 mmol) was added to the solution containing the deprotected amine, followed by Boc-glycine (175 mg, 1.00 mmol) and benzotriazol-1-yloxy- tris (dimethylamino)phosphonium hexafluorophosphate (442 mg, 1.00 mmol) .
  • Boc-glycine (175 mg, 1.00 mmol
  • benzotriazol-1-yloxy- tris (dimethylamino)phosphonium hexafluorophosphate (442 mg, 1.00 mmol)
  • the mixture was stirred for 6 hours and then quenched by adding 5 drops isopropyl amine.
  • the mixture was concentrated in vacuo and chromatographed (40% ethyl acetate :hexanes) to give 178 mg (0.542 mmol, 59%) of Compound 2.
  • Compound 3 was characterized by: IR (Film) : n 2934, 1744, 1719, 1678, 1524, 1456, 1437, 1412, 1391, 1368, 1250, 1211, 1171, 1051, 1030, 939, 864, 835, 781, 615 cm “1 ; *H NMR (CDC1 3 ) : d 1.41 (s) , 1.48 (m) , 1.52 (m) , 1.62 (m) , 1.82 (m) , 2.40 (q) , 2.69 (t) , 2.84 (t), 3.69 (s) , 3.78 (m) , 4.57 (q) , 5.26 (d) , 6.71; and 13 C NMR (CDC1 3 ) : d 24.82, 25.32, 25.38, 28.16, 31.94, 44.10, 46.77, 51.87, 51.96, 52.20, 79.92, 156.02, 169.41, 172.59
  • Azido Dipeptide (Compound 4) .
  • NH 4 C1 80 mg, 1.49 mmol
  • NaN 3 300 mg, 4.61 mmol
  • the mixture was heated to reflux and stirred for 2 hours.
  • the mixture was then cooled to 25°C, diluted with 40 mL of ethyl acetate, washed with water (2 x 20 mL) , washed with brine (10 mL) dried over MgS0 4 , and concentrated in vacuo to give 71.0 mg (0.191 mmol, 100%) of azido dipeptide Compound 4.
  • Compound 4 was characterized by: IR (Film) : n 3328, 2976, 2936, 2865, 2101, 1742, 1671, 1524, 1439, 1393, 1368, 1279, 1252, 1217, 1167, 1051, 1030, 941, 864, 632 cm “1 ; L H NMR (CDCl 3 ) : d 1.28 (m) , 1.38 (s) , 1.61 (m) , 1.79 (m) , 1.82 (d) , 3.17 (m) , 3.24 (m) , 3.66 (s) , 3.74 (m) , 4.52 (m) , 5.49 (t) , 6.90 (d) ; and 13 C NMR (CDC1 3 ) : d 24.60, 24.72, 24.82, 28.19, 32.01, 33.80, 44.09, 51.75, 51.89, 52.34, 56.86, 64.84, 70.12, 70.38, 80.
  • Boc-amine Dipeptide (Compound 5) .
  • a 5% solution of Pd on carbon in ethyl acetate (6.0 mL) was stirred at room temperature, under atmospheric pressure of hydrogen gas, for one hour.
  • Addition of Compound 4 (82.3 mg, 0.24 mmol) and Boc-anhydride (104.3 mg, 0.48 mmol) required 1.0 mL ethyl acetate, followed by two ethyl acetate washes of 1.0 mL each. After stirring 12 hours, and Celite was added. The solution was filtered and concentrated in vacuo . The residue was purified by flash chromatography, running neat ethyl acetate, to yield 86.6 mg (0.188 mmol, 78%) of Boc-amine dipeptide Compound 5.
  • Compound 5 was characterized by: IR (Film) : n 3328, 2978, 2934, 2865, 1744, 1686, 1524, 1456, 1439, 1412, 1393, 1368, 1275, 1250, 1169, 1051, 1030, 864, 781, 735 cm “1 ; ⁇ NMR (CDC1 3 ) : d 1.39 (s) , 1.41 (s) , 1.48 (t) , 1.64 (m) , 1.81 (m) , 2.92 (m) , 3.22 (br s) , 3.60 (d) , 3.69 (s) , 3.77 (d) , 4.55 (m) , 5.13 (br s) , 5.43 (d) , 6.86 (br s) ; and 13 C NMR (CDC1 3 ) : d 24.56, 24.73, 24.95, 28.08, 31.71, 33.90, 43.82, 46.35, 51.75
  • the polypeptide was cleaved from the resin using 95% trifluoroacetic acid in water to afford the mesylate polypeptide Compound 8.
  • Compound 8 was purified by reverse phase HPLC, running a gradient from 85:15 to 50:50 (water with 0.1% 0 trifluoroacetic acid:methanol) . Mass spectrometry confirmed the identity of Compound 8 (MH + 1133) .
  • Aziridine Polypeptide (Compound 9) .
  • Compound 8 was treated with sodium hydroxide (10 equivalents at room temperature for half an hour) , or lithium hydroxide in methanol, to afford aziridine polypeptide Compound 9.
  • Mass spectrometry confirmed the identity of Compound 9 (MH + 1055) .
  • Compound 9 was subjected to Edman degradation and microsequencing, which confirmed its composition to be G (az) IDKPILK, where (az) is the unnatural aziridine amino acid residue.
  • the polypeptide G(az)AFVTIGK was prepared using a different resin-bound peptide.
  • the aziridine within a peptide was cyclized from the mesylate in solution before addition to the folding buffer containing recombinantly expressed, purified HLA-B27 heavy chain and jS 2 -m.
  • the solution contained approximately equimolar distributions of mesylate precursor Compound 8 and aziridine-containing ligand Compound 9 (verified by reverse phase HPLC) and the solution was added to the folding buffer (both Compound 8 and Compound 9 have a final concentration of 0.1 mM in the folding buffer), which was incubated at 10°C After half an hour and approximately every 12 hours later, 100 mL of the folding buffer was removed and subjected to reverse phase HPLC, running a gradient of 100:0 to 40:60 (0.1% trifluoroacetic acid in water : acetonitrile) over 30 minutes and monitoring at 214 nm.
  • HLA-DR1 containing the only free cysteine, Cys-30, does not bind arginine and lacks the glutamate and threonine of the arginine binding pocket in HLA-B27 (see, Stern, L.J., Brown, J.H. , Jardetzky, T.S., Gorga, J.C, Urban, R.G. , Strominger, J.L. & Wiley, D.C.
  • the aziridine ligand alkylates Cys-67 during the folding of recombinant HLA-B27 despite a 5000 molar equivalent excess of cysteine in the glutathione of the refolding buffer.
  • the ligand was described in Garboczi, D.
  • the lower yields might result from less efficient hydrogen bonding to the aziridine moiety compared to the planar guanidinium group of the control peptide, or decreased reactivity of one diastereomer of the aziridine ligand Compound 9 (kinetic resolution) .
  • the folding of HLA-B27 was dependent upon a peptide ligand, in this case containing aziridine (Compound 9) .
  • HLA-A2 an MHC molecule lacking specificity for arginine at P2 , failed to fold in the presence of the aziridine-containing peptide, as expected.
  • Characterization of Covalent Ligand-Protein Bond The aziridine-containing ligands were shown to form covalent bonds with HLA-B27 by a shift in mobility of the heavy chain on SDS-PAGE; reaction with Cys-67 was shown by tryptic digestion, mass spectrometry (MS) , and Edman microsequencing.
  • Fig. 4. depicts the results of an SDS-PAGE (15 %) of HLA-B27 complexed with the aziridine-containing peptide (G(az) IDKPILK (lane 1) and the control peptide (GRIDKPILK) (lane 2) .
  • the SDS-PAGE of HLA-B27 complexed with aziridine-peptides revealed a mobility shift of the heavy chains of the complex due to addition of the aziridine-containing ligand (Fig.4, lane 1), when compared to heavy chain from complexes with the same peptides with arginine in the P2 position (Fig. 4, lane 2) .
  • the mobility of /3 2 -m remained unchanged.
  • the aziridine-modified complex was digested with trypsin and the fragments separated by reverse phase HPLC.
  • the carboxyamidomethylated tryptic peptides were separated by narrow-bore HPLC using a Vydac C18 2.1 x 150 mm reverse phase column on a Hewlett-Packard 1090 HPLC/1040 diode array detector. Putative difference peaks from the chromatogram were chosen based on differential UV absorbance at 210, 277, and 292 nm.
  • Microcapillary HPLC/electrospray ionization/tandem mass spectrometry on a Finnigan TSQ7000 triple quadrupole mass spectrometer (San Jose, CA) as described in Hunt, D. F., Henderson, R.A. , Shabanowitz, J., Sakaguchi, K. , Michel, H., Sevilir, N. , Cox, A.L., Appella, E. & Engelhard, V.H. (1992) Science 255, 1261- 1263.
  • the chromatogram of the proteolyzed fragments was compared with a tryptic digest of the MHC class I protein formed with the arginine-containing parent-peptide
  • GIDKPILK Matrix-assisted laser desorption time-of- flight MS was used to screen rapidly putative difference peaks, which were deficient in UV absorbance at 277 and 292 nm (lacking tyrosine and tryptophan) .
  • Edman microsequencing revealed this fraction to contain the peptide ligand in equimolar amounts with the HLA-B27 heavy chain fragment containing Cys-67.
  • thermodynamic stability of Covalent MHC-peptide Complexes The thermodynamic stability of the class I MHC molecules from mouse and human have been shown to depend on the sequence of the bound peptide. See, Bouvier, M. & Wiley, D.C. (1994) Science 265, 398-402, Fahnestock, M.L., Tamir, I., Narhi, L.O. & Bjorkman, P.J. (1992) Science 258, 1658- 1662, and Fahnestock, M.L., Johnson, J.L., Renny Feldman, R.M. , Tsomides, T.J., Mayer, J. , Narhi, L.O. & Bjorkman, P.J. (1994) Biochemistry 33, 8149-8158.
  • the thermal transition of the HLA-B27 complexes was measured using circular dichroism (CD) .
  • thermoelectric temperature controller was used to obtain thermal denaturation curves in triplicate, from 25-95 'C, as described in Bouvier, M. & Wiley, D.C. (1994) Science 265, 398-402.
  • the addition of 50 mM NaCl to the typical 10 mM MOPS (pH 8.0) buffer was found to prevent aggregation of HLA-B27.
  • the additional salt had no effect on the measured melting temperature (T m ) of the HLA-A2 complex formed with a hepatitis B-derived peptide.
  • the covalent complexes of HLA-B27 with two aziridine-containing-peptides, G(az) IDKPILK (Compound 9) and G (az) AFVTIGK, both denature at approximately the same temperature, 62 °C G(az) AFVTIGK was prepared by coupling a different peptide to Compound 8
  • the non-covalent complexes of HLA-B27 with the cognate peptides (GRIDKPILK and GRAFVTIGK) melted at 72°C and 60°C, respectively.
  • the data indicate that covalently bound peptides with an aziridine replacing arginine stabilize HLA molecules approximately like non-covalently bound peptides, in one case stabilizing slightly more and in one less.
  • Covalent MHC-peptide complexes like those reported here may find use generating anergy if administered alone (see, Kozono, H. , White, J., Clements, J. , Marrack, P. & Kappler, J. (1994) Nature 369, 151-154) or activation if administered with co-ligands for inducing costimulatory signals as suggested by Schwartz, R.H. (1992) Cell 71, 1065-1068.
  • a non-peptide ligand capable of crossing cellular membranes to block selectively HLA-B27, based on the same aziridine specificity can also be prepared.
  • the strategy can be used to form covalent complexes of peptides or other ligands with HLA- B27 does not add any new amino acids to the surface of the MHC molecule (that might be immunogenic) , but instead forms the covalent attachment deep in a pocket buried by the bound peptide.
  • the aziridine strategy can be generalized to other proteins (i.e., recombinant class I MHC molecules) by mutating residues in the binding region (i.e., the P2 pocket residues) to those in HLA-B27.
  • the reverse experiment, engineering an HLA-A2 P2 specificity pocket into HLA-B27 by mutating residues 9, 24, 45, 66, 67, and 70 has been reported by Colbert, R.
  • peptide analogs obtained by back modification of the above-described MHC-blocking peptides, e.g., replacement of at least one of the peptide bonds with -CH 2 -NH-, CH 2 -S-, or the like.
  • alkyl groups can be readily substituted by alkenyl, alkynyl or alkyl ether groups.

Abstract

Des ligands se lient à des protéines conformément à certains profils cinétiques particuliers. La cinétique de liaison réversible entraîne la libération du ligand du site de liaison, ce qui modifie la capacité de reconnaissance du complexe ligand-protéine. En modifiant la cinétique de la liaison réversible, il est possible de modifier la réaction face au système immunitaire cellulaire. On peut utiliser, pour modifier la cinétique de liaison, des ligands réactifs qui réagissent avec la protéine une fois la liaison formée.
PCT/US1997/017483 1996-09-30 1997-09-30 Ligands reactifs et complexes covalents de ligands et proteines Ceased WO1998014208A1 (fr)

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