Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-02
2004-03-23
Celsa, Bennett (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S023000, C435S320100
Reexamination Certificate
active
06709814
ABSTRACT:
FIELD OF THE INVENTION
the compositions and methods of the invention relate to the use of dimerization peptides that self-associate and their use with other proteins to effect the formation of compact structures.
BACKGROUND OF THE INVENTION
Proteins interact with each other largely through conformationally constrained domains. Although linear peptides with freely rotating amino and carboxyl termini can have potent functions as is known in the art, the conversion of such peptide structures into pharmacologic agents is frequently difficult. Therefore the presentation of peptides in conformationally constrained structures can result in the generation of pharmaceuticals with high affinity to its target protein. Constrained peptides have many valuable features compared to their linear analogs. These include: (i) enhanced stability to proteolysis [Szewczuk et al., Biochemistry 31:9132-9140 (1992)) due to the lack of unconstrained N- or C-terminal amino acid residues accessible to amino- or carboxypeptidases and a non-extended structure which diminishes endopeptidase susceptibility; (ii) a restricted conformation space that can result in a higher binding affinity for cognate binding proteins due to a reduced entropic cost of binding [Hruby, Life Sci., 31:189-199 (1982); Rizo and Gierasch, Ann. Rev. Biochem. 61:387-418 (1992)]; (iii) the geometry to mimic reverse turns, loops or other secondary structures [Rose et al., Adv. Proptein Chem., 37:1-109 (1985); Stradley et al., Biopolymers 29:263-287 (1990); Rizo et al., in
Molecular Conformation and Biological Interactions
(P. Balaram and S. Ramaseshan, eds.), Indian Academy of Sciences Publications (Bangalore, India), p 469-496 (1991)]; and (iv) a conformationally restricted scaffold which allows easier pharmaophore and drug development.
Thus constrained peptides can form the basis for the isolation of new ligands and receptors and subsequently for the rational design of small molecules which may be useful as drugs. The desirability of this approach was shown using cyclic peptide libraries which have been used to discover and refine potent ligands of a variety of receptors [O'Neil et al., Proteins: Structure Function and Genetics 14:509-515 (1992); Giebel et al., Biochem. 34:15430-35 (1995); Spatola and Crozet, J. Med. Chem. 39:3842-46 (1996); Koivunen et al., J. Biol. Chem. 268:20205-10 (1993); Koivunen et al., J. Cell. Biol. 124:373-380 (1994)], enzymes [McBride et al., J. Mol. Biol. 259:819-27 (1996); Eichler et al., Mol. Divers. 1:233-240 (1996)], and other proteins [Wang et al., J. Biol. Chem. 270:2323942 (1995)].
Several constrained protein scaffolds, capable of presenting a protein of interest as a conformationally-restricted domain are described in the literature and include minibody structures (Bianchi et al., J. Mol. Biol. 236(2):649-59 (1994), loops on beta-sheet turns, coiled-coil stem structures (Myszka and Chaiken, Biochemistry 33:2363-2372 (1994), zinc-finger domains, cysteine-linked (disulfide) structures, transglutaminase linked structures, cyclic peptides, helical barrels or bundles, leucine zipper motifs (Martin et al., EMBO J. 13(22):5303-5309 (1994); O'Shea et al., Science 243:53842 (1993), etc.
In addition, self-aggregation has been described for regulatory peptides such as the neuropeptide head activator [as further outlined below; Bodenmuller et al., EMBO J. 5(8):1825-1829 (1986)], substance P [Poujade et al., Biochem. Biophys. Res. Commun. 114:1109-1116(1983)], metenkephalin [Mastropaolo et al., Biochem. Biophys. Res. Commun. 134:698-703 (1986)], and neuropeptide Y [Minakata et al., J. Biol. Chem. 264:7907-7913 (1989)].
Pertinent to the subject of this invention is a peptide derived from the neuropeptide head activator (HA) isolated from the freshwater coelenterate
Hydra
(Bodenmuller et al., supra). Bodenmuller et at. demonstrated that under physiological conditions the HA peptide (pEPPGGSKVILF) dimerizes to form a biologically inactive molecule.
Dimerization of the monomer form yields a stable structure, which does not dissociate into its monomeric components at concentrations as low as 10
−13
M. Further analysis of HA fragments revealed that a fragment containing only the last six amino acid residues from the carboxy terminus of the HA peptide (pSKVILF) dimerized more efficiently that HA itself. However, a fragment containing only the last 4 amino acid residues (pVILF) and a fragment derived from the amino-terminal end of HA (pEPPGGSK) did not lead to dimer formation. Most importantly, their analysis showed that both the replacement of the carboxy-terminal phenylalanine and a modification thereof (e.g., introduction of an iodine in the para (4′) position of the aromatic ring) abolished dimerization completely or decreased dimerization tendency drastically.
Aldwin et al. (U.S. Pat. No. 5,491,074), referring to SKVILF as ‘association peptide’, added additional amino acid residues at either its amino terminal sequence or to its carboxy-terminus and found that some of the resulting proteins could form dimeric peptides. However, Aldwin et al. did not demonstrate or anticipate the addition of more than one ‘association peptide’ to one polypetide of interest. Accordingly, it is an object of the invention to provide dimerization peptides for use in a variety of applications.
SUMMARY OF THE INVENTION
Peptides which have a moderate or high affinity for each other, when added as extensions to both the N- and C-terminus of a protein, can be used to help fold the protein into a compact structure. Compared to cognate linear proteins and disulfide-cyclized proteins, this new compact structure is more stable to cellular and other proteases, and is significantly more conformationally constrained than the linear peptides. The compact structure can have other functional sequences embedded within its sequence, and is preferable to linear and less constrained peptides for intracellular and extracellular library screens, and for targeting to specific intracellular locations. It can be used, with appropriate flanking residues on each end of the varied residues in a random peptide sequence, to create structurally-biased peptide libraries. By virtue of its stability and constraints, this scaffold can prolong the activity of any embedded peptide sequences in the presence of proteases.
Peptides having the property of self-aggregating herein are referred to as dimerization peptides (DP). The dimerization peptides of this invention comprise the sequence FLIVK (from amino-terminal to carboxy-terminal). Examples of dimerization sequences which enhance the folding of a protein of interest include, but are not limited to, FLIVK, EFLIVKS, KFVLIKS, VSIKFEL, LIVKS, EFLIVK, KFLIVK, FESIKVL, and LKSIVEF. These dimerization peptides (DP) can be used in several combinations to yield proteins of the general structure ‘DP-protein’ or ‘DP-protein-DP’ wherein ‘DP’ is a dimerization peptide, ‘protein’ comprises at least two amino acid residues. In addition other amino acid sequences including, but not limited to, linker sequences, tag sequences, targeting sequences and stabilization sequences are generally included.
Other sequences include those with a high content of hydrophobic amino acids and 1 or 2 charged residue side chains. Generally, a sequence at each terminus of the dimerization peptide composed of 5, 6, 7 and 8 amino acids with at least 34 highly hydrophobic residues (taken from F, I, L, M, V, W, and Y) will function in this fashion.
The compositions of this invention are displayed intracellularly or extracellulary and are useful to identify binding proteins and molecules and to modulate intracellular signaling pathways. In one aspect of the invention, a library of constrained proteins is evaluated in vivo for its bioactive potential. Thus, the invention accesses molecules or targets within living cells and provides for the isolation of the constrained protein which has a phenotypic effect on this living cell. This method c
Anderson David
Gururaja Tarikere
Celsa Bennett
Diehl James J.
Francis Carol L.
Keddie James S.
Rigel Pharmaceuticals Inc.
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