Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1996-07-24
2002-12-31
Kaufman, Claire M. (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C530S300000, C530S312000, C530S350000, C530S306000, C514S002600, C514S012200, C435S007200, C435S007210
Reexamination Certificate
active
06500934
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to bivalent agonists having affinity for GPCRs, peptide dimers useful as bivalent agonists, and methods for their preparation and use.
2. BACKGROUND OF THE INVENTION
G-Protein coupled receptors (“GPCRs”) are plasma membrane proteins capable of transducing signals across a cell membrane so as to initiate a second messenger response. To this end, GPCRs bind a variety of ligands ranging from small biogenic amines to peptides, small proteins and large glycoproteins (C. D. Strader et al.,
Annu. Rev. Biochem.
63, 101-132 (1994)). All GPCRs contain seven hydrophobic domains, which have been postulated to span the plasma membrane, connected by hydrophilic extracellular and intracellular loops. Some examples of GPCR subfamilies include the rhodopsin/&bgr;-androgenic (“&bgr;AR”) family, which includes receptors for dopamine, serotonin, substance P, bradykinin, angiotensin, somatostatin and lutropin; the secretin/vasointestinal peptide (“VIP”) family, which includes receptors for secretin, glucagon, glucagon-like peptide 1, gastric inhibitory peptide, parathyroid hormone, secretin/vasointestinal peptide, pituitary adenylate cyclase activating peptide, calcitonin and growth releasing hormone; and the metabotropic glutamate (“mGlu”) family, which includes receptors for glutamate. Over 250 GPCRs have been identified to date (M. A. Cascieri et al.,
JPM
33(4), 179-185 (1995); C. D. Strader et al.,
FASEB J.
9, 745-754 (1995)), including Ca
2+
, olefactory, prostaglandin and sweet-taste receptors.
Multivalent ligands such as immunoglobulins have significantly enhanced affinity for their binding site. Based on the entropic effects of avidity, multivalency theoretically can increase apparent binding affinity by several orders of magnitude (D. M. Crothers et al.,
Immunochemistry
9, 341-357 (1972)). Although this affinity gain is usually more modest, multivalent molecules have potentially powerful applications in clinical pharmacology. For example, synthetic bivalent ligands can be used to target toxins, drugs and, potentially, plasmid DNA to specific cell subtypes, or across the blood-brain barrier.
Thermodynamically, the binding of a multivalent antibody to adjacent epitopes on the cell surface is similar to the chelate effect (C. G. Spike et al.,
J. Amer. Che. Soc.
75, 2726-2729 (1953); D. Neri et al.,
J. Mol. Biol.
246, 367-373 (1995)). Although this effect was described originally to explain the enhanced stability of chelate rings, it is also relevant in rate accelerations of enzymic reactions and in base pair formation of polynucleotides (M. I. Page et al.,
Proc. Natl. Acad. Sci. USA
68, 1678-1683 (1971); C. Delisi et al.,
Biopolymers
10, 1809-1827 (1971)). The common feature of these reactions is that following the initial reaction (e.g., binding of one antibody “arm” to its antigen), each succeeding reaction (e.g., binding of the second antibody “arm” to an adjacent epitope) is more favorable because the entropy loss is decreased.
Specific examples of bivalent molecules capable of binding to adjacent epitopes include small bivalent antibodies composed of either antibody fragments (F
ab
) or single chain antibodies (F
v
) (P. Pack et al.,
Biochemistry
31, 1579-1584 (1992); P. Holliger et al.,
Proc. Natl. Acad. Sci. USA
90, 6444-6448 (1993); W. D. Mallender et al.,
J. Biol. Chem.
269, 199-206 (1994)). In addition, other multivalent molecules have been designed to bind to adjacent epitopes, including bivalent carbohydrates and a variety of synthetic drug delivery systems (R. T. Lee et al.,
Biochemistry
23, 4255-4261 (1984); R. Duncan et al.,
Clin. Pharmacokinet.
27, 290-306 (1994)).
Bivalent peptides, such as receptor-adhesive modular proteins (“RAMPs”), have been used in an alternative approach to cell targeting (M. Engel et al.,
Biochemistry
30, 3161-3169 (1991); C. A. Slate et al.,
Int. J. Peptide Protein Res.
45, 290-298 (1995)). These large synthetic peptides, which contain two ligand sites separated by a spacer region and a dimerization domain, were designed with the hope of binding to two membrane receptors simultaneously. In its original design, the dimerization domain consisted of a two stranded parallel alpha helical coiled coil, and the ligand region was composed of two identical integrin receptor binding peptides. However, although the two ligand domains were separated by at least 50 angstroms, no increased affinity of their dimeric constructs was demonstrated, suggesting that such dimeric peptides could not bind to two receptors at the same time. Previous studies suggested that the minimal distance between two GPCRs is 40 angstroms (Å) (G. F. X. Schertler et al.,
Nature
362, 770-772 (1993)), while structural studies of immunoglobins have demonstrated that the distance between antigen binding sites is 100-250 Å (D. M. Crothers et al.,
Immunochemistry
9, 341-357 (1972)).
Previous studies indicated that short, crosslinked gonadotropin releasing hormone (“GnRH”) peptide dimers that were incubated with anti-GnRH antibodies to form larger dimers having a bridge length of 150 Å, increased agonist activity, relative to their corresponding short dimers, in a functional luteinizing hormone (“LH”) release assay (P. M. Conn et al.,
Endocrinology
111, 335-337 (1982)).
In addition, it has been shown that a reversible association of an antibody with two GnRH antagonists resulted in the association having agonistic activity (P. M. Conn et al.,
Nature
296, 653-654 (1982)). However, in that case, because there was no covalent or ionic bonding between the two GnRH antagonists and the antibody, such an reversible association is easily disrupted, leading to cessation of agonist activity.
It has been speculated that a dimer of any small molecule that binds to a single transmembrane receptor that is known to work through dimerization, with an appropriate spacer portion to allow mutual contact of the active moieties with their receptors, could possibly possess agonist activity (B. Seed,
Chemistry
&
Biology
1(3), 125-29 (1994)).
There is a clear need in the art for easily synthesizable agonists with enhanced functional activity or in vivo efficacy than those currently available.
Citation or identification of any reference in Section 2 of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention provides a bivalent agonist having affinity for one or more G-protein coupled receptors, said agonist comprising two ligand domains, the ligand domains being agonists for a first and a second G-protein coupled receptor, respectively, wherein the distance between the ligand domains ranges from about 40 to about 250 Å, and further comprising a molecular backbone, said backbone being covalently bonded to the two ligand domains.
The invention further provides a bivalent agonist having affinity for one or more G-protein coupled receptors, said agonist comprising two ligand domains, the ligand domains being antagonists for a first and a second G-protein coupled receptor, respectively, wherein the distance between the ligand domains ranges from about 40 to about 250 Å, and further comprising a molecular backbone, said backbone being covalently bonded to the two ligand domains.
The invention further provides a bivalent agonist having affinity for one or more G-protein coupled receptors, said agonist comprising a first and second ligand domain, the first ligand domain being an agonist for a first G-protein coupled receptor, and the second ligand domain being an antagonist for a second G-protein coupled receptor, wherein the distance between the ligand domains ranges from about 40 to about 250 Å, and further comprising a molecular backbone, said backbone being covalently bonded to the first and second ligand domains.
The invention further provides a method for synthesizing a bivalent agonist dimer, comprising the step of treating an amount of a monomer with an amount of
Carrithers Michael D.
Lerner Michael Rush
Kaufman Claire M.
Pennie & Edmonds LLP
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