Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Chemical aftertreatment – e.g. – acylation – methylation – etc.
Reexamination Certificate
1998-08-31
2001-02-13
Russel, Jeffrey E. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Chemical aftertreatment, e.g., acylation, methylation, etc.
C530S356000, C530S402000
Reexamination Certificate
active
06187907
ABSTRACT:
BACKGROUND OF THE INVENTION
In general, a therapeutic molecule which targets an enzyme (e.g., collagenase), a receptor (e.g., G protein couple receptor), or an effector (e.g., antibody) must meet two requirements. First, it must contain a template with which one or more biologically active ligands are incorporated. Second, the conformation of the template must be further modified chemically so as to augment the specificity and potency of the therapeutic molecule. Unfortunately, chemical modification typically introduces excessive degrees of freedom into the initial template, thereby decreasing the probability of the interaction between the biologically active ligand and its target.
SUMMARY OF THE INVENTION
The present invention provides a triple helix coil template for presenting one or more biologically active ligands to a target.
More specifically, an aspect of this invention relates to a template-ligand conjugate which includes (1) a template made of three cross-linked polypeptide chains (i.e., covalently crossed-linked via a tri-crosslinker or otherwise), the three polypeptide chains each containing tripeptide or hexapeptide repeat sequences aligned to form a triple helix coil; and (2) at least a biologically active ligand attached to the template via covalent bonding with one of the three polypeptide chains. Preferably, at least a biologically active ligand is covalently bonded to each of the three polypeptide chains.
If the repeat sequences are located in one terminal region (either N- or C-terminal) of each of the three polypeptide chains, it is preferred that at least one biologically active ligand be attached to the terminal amino acid residue in the same terminal region of one polypeptide chain and that each terminal amino acid residue in the opposite region of each polypeptide chains be covalently bonded to a tri-crosslinker. Further, it is preferable that all three polypeptide chains in a template-ligand conjugate of this invention are identical.
As an example of a template-ligand conjugate of this invention, each of the three polypeptide chains may, independently, have repeat tripeptide sequences of the following formula:
(AA
1
-AA
2
-AA
3
)
m
in which each AA
1
, independently, is a Gly, or a D- or L-Ala; each AA
2
, independently, is a D- or L-Pro; each AA
3
, independently, is a D- or L-Hyp, or a D- or L-Cys; and m is 4-14 or, preferably, is 4-8. Note that the thiol group of a Cys residue of a tripeptide sequence can form a disulfide bond with the thiol group of a Cys residue of another tripeptide sequence. Also, if one of AA
1
's, AA
2
's and AA
3
's is a D-amino acid residue, then all the of others must also be of D-amino acid residues; and vice versa.
As another example of a template-ligand conjugate of this invention, each of the three polypeptide chains may, independently, have repeat hexapeptide sequences of the following formula:
(AA
1
-AA
2
-AA
3
-AA
4
-AA
5
-AA
6
)
n
wherein each AA
1
, independently, is a Gly; each AA
2
, independently, is a D- or L-Pro; each AA
3
, independently, is a D- or L-Hyp, or a D- or L-Cys; each AA
4
, independently, is a D- or L-Ala; each AA
5
, independently, is a D- or L-Pro; each AA
6
, independently, is a D- or L-Hyp, or a D- or L-Cys; and n is 2-7, or, preferably, 2-4. The thiol group of a Cys residue of a hexapeptide sequence can form a disulfide bond with the thiol group of a Cys residue of another hexapeptide sequence. Also, if one of AA
1
's, AA
2
's, AA
3
's, AA
4
's, AA
5
's and AA
6
's is a D-amino acid residue, then all the of others must also be of D-amino acid residues; and vice versa.
The above-described template has several advantages. For example, the template has a well-defined three-dimensional structure. Thus, a biologically active ligand attached to it can be studied immediately using molecular modeling. Additionally, such a template provides a greater surface region for chemical modifications to increase target specificity and affinity through “multi-domain” binding and to alter any undesirable chemical properties. In addition, a template-ligand conjugate of this invention enables one to reduce the conformational flexibility or entropy of the biologically active ligand in a selective manner. Further, such a conjugate can be used as a substrate-based drug screening tool in lieu of using traditional linear peptide-based combinatorial methods. Finally, a template-ligand conjugate of this invention can also be used in highly specific and efficient columns for the purification of enzymes.
Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a molecule having a template of a covalently crosslinked triple helix coil of three suitable polypeptide chains, each containing tripeptide or hexapeptide repeat sequences, and at least one biologically active ligand attached to the template via covalent bonding with one of the three polypeptide chains (e.g., covalently attached to a terminal or an internal residue of, or bound within, one of the polypeptide chain) without disrupting the triple helix coil. Preferably, the three polypeptide chains are covalently linked to each other via a tri-crosslinker (for example, see below or Goodman, et al., J. Am. Chem. Soc. 118, 5156, 1996). In any event, it is important that the triple helical conformation be maintained when the three polypeptide chains are linked together. Repeat sequences, as defined herein, include a cluster of amino acids which is repeated within a polypeptide chain, preferably forming a substantial portion of the polypeptide chain. Bound within, as defined herein, means that one or more ligands are used in lieu of amino acids within a polypeptide chain of the template. The three polypeptide chains can be identical or can differ by one or more amino acids, and can contain either D- or L-amino acids, or both (herein L-amino acid is intended unless specified). Suitable template-ligand covalent bonds include, for example, normal peptide bonds, modified peptide bonds, or non-peptide bonds. Indeed, each of the polypeptide chains itself may also contain one or more modified peptide bonds or non-peptide bonds.
A biologically active ligand, as defined herein, is a ligand which, when bound to the template, can bind to a target site, such as an enzyme, an effector, or a receptor, to act as an inhibitor, agonist, antagonist or mixed agonist/antagonist. It is also understood that the biologically active ligands also includes physiologically acceptable salts of the ligands. Examples of a suitable biologically active ligands include ligands which inhibit enzymatic action, such as N-[2-isobutyl-3(methoxycarbonyl)-propanoyl]-L-tryptophan, which can be used to inhibit metalloprotease enzymes (e.g., collagenase, gelatinase, and stromelysin). Other suitable ligands include those which bind to receptors, such as an anti-idiotic antibody against Angiotensin II receptor or an Arg-Gly-Asp tripeptide, which, when bound within a polypeptide chain, will also bind to integrin cell surface receptors, thereby inhibiting blood cell adhesion.
One method for synthesizing a template-ligand conjugate of this invention includes synthesizing a triple helix coil template, and then dissolving the template in a suitable solvent such as methanol. A suitable biologically active ligand is then mixed with a reagent, or a combination of reagents, suitable to activate the ligand to support subsequent covalent bonding of the ligand to the template. The activated ligand is then contacted with the template under conditions suitable to support covalent bonding of the ligand to the template.
After reaction, the product is precipitated, for example, by addition of anhydrous ether. The product is then mixed with an hydroxylamine solution to form the composition according to this invention. The syntheses of a composition of this invention is further described in Example
Chen James
Yeh Li-an
Fish & Richardson P.C.
Russel Jeffrey E.
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