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Title:  Retroinverso polypeptides that mimic or inhibit thrombospondin activity

United States Patent:  6,339,062

Inventors:  Williams; Taffy (Lansdale, PA); Tuszynski; George (Pittsgrove, NJ); Actor; Paul (Phoenixville, PA)

Assignee:  Inkine Pharmaceutical Company, Inc. (Blue Bell, PA)

Appl. No.:  197770

Filed:  November 23, 1998

Abstract

The present invention relates generally to polypeptides that mimic or inhibit the biological activity of thrombospondin, and particularly to polypeptides in retroinverso form. These polypeptides may be used for their biological and pharmaceutical applications such as: (a) inhibiting the invasive and metastatic activity of melanoma cells, (b) promoting and inhibiting cellular attachment to tissue culture flacks, (c) promoting wound healing, angiogenesis, and implant acceptance, (d) agents for anti-platelet aggregation, (e) agents for antimalarial activity, and (f) diagnostic reagents in different therapeutic applications, as well as other related areas.

SUMMARY OF THE INVENTION

The present invention provides thrombospondin fragments and analogs that mimic or inhibit the biological activity of intact thrombospondin and are, thus, useful in a variety of biological, prophylactic or therapeutic areas. These peptides are capable of modifying and inhibiting tumor cell metastasis, cell adhesion and platelet aggregation in mammals in vivo. The peptides are also useful in wound healing, for antimalarial activity, atherosclerosis, thrombotic, and thrombolytic conditions, angiogenesis, and as cell attachment promoters, complement modulators, and diagnostic reagents and in other related areas.

Analogs based on the type I repeat of thrombospondin described by Lawler et al., Seminars in Thrombosis & Hemostasis, 13:245-254 (1987), Robson et al., Nature, 335:79-82 (1988), and Groundis et al., Nature, 335:82-85 (1988) have been shown to have thrombospondin-like activity. Specifically, analogs based around and including at least a portion of the sequence motif Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 2) have been shown to have thrombospondin-like activity.

This invention is directed to polypeptide compounds of formula (I):

Z1 -Xaa2 -Xaa3 -Xaa4 -Xaa5 -Xaa6 -Xaa7 -Xaa8 -Xaa9 -Xaa10 -Z2 (SEQ ID NO: 3)

wherein:

Xaa2 is a neutral/non-polar/large/cyclic amino acid residue;

Xaa3 is a neutral/polar/small or neutral/polar/large/non-cyclic or acidic amino acid residue;

Xaa4 is a neutral/nonpolar/large/cyclic or neutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic or neutral/polar/small amino acid residue;

Xaa5 is a neutral/polar/small amino acid residue

Xaa6 is a neutral/polar/small or neutral/polar/large/non-cyclic amino acid residue;

Xaa7 is a neutral/nonpolar/large/non-cyclic or neutral/polar/large/non -cyclic amino acid residue;

Xaa8 is a neutral/polar/large/non-cyclic or neutral/polar/small amino acid residue;

Xaa9 is a neutral/polar/small amino acid residue;

Xaa10 is a neutral/polar/small amino acid residue;

Z1 is hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof;

Z2 is hydroxyl, carboxyl, non-amino acids such as agmatine, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof.

Preferably, the polypeptide compounds of this invention have formula (II):

R1 -Cys-Xaa11 -Xaa12 -Xaa13 -Cys-R2 (SEQ ID NO: 4)

wherein:

R1 is a protected or unprotected terminal amino group, including hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof;

Xaa11, Xaa12, and Xaa13 are the same or different neutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic or neutral/polar/small or basic/non-cyclic amino acid residues, preferably selected from the group consisting of valine, threonine, serine, and arginine;

R2 is a protected or unprotected terminal carboxyl group including hydroxyl, carboxyl, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof, preferably selected from the group consisting of lysine, glycine, and arginine;

wherein the structure of the polypeptide is optionally cyclized through a bond between the cysteines, such as a disulfide bond, or a bond between R1 and R2.

This invention also includes polypeptides having the retroinverso form of L-amino acid polypeptides of formulae (I) and (II), i.e, polypeptides comprising D-amino acids in reverse order. In particular, retroinverso peptides of formula Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1), i.e., d-Gly-Cys-Thr-Val-Ser-Cys (SEQ ID NO: 5) are preferred. Preferably, the cysteine residues are modified by a sulfhydral blocking group, such as --CH2 --NH--COCH3, that is adhesive toward melanoma cells. Alternatively, the cysteine residues may be conservatively substituted with another amino acid, e.g., methionine.

Also provided in accordance with aspects of the invention are pharmaceutical compositions, which contain the above-recited polypeptide compounds together with a pharmaceutically acceptable liquid, gel or, solid carrier. Administration of therapeutically effective doses of these compositions can provide effective enhancement or inhibition of thrombospondin-like activity to animals, particularly vertebrates such as mammalian and avian hosts.

Claim 1 of 14 Claims

We claim:

1. A polypeptide having the retroinverso form of a polypeptide of formula (I)

Z1 -Xaa2 -Xaa3 -Xaa4 -Xaa5 -Xaa6 -Xaa7 -Xaa8 -Xaa9 -Xaa10 -Z2 (SEQ ID NO: 3)

wherein:

Xaa2 is a neutral/non-polar/large/cyclic amino acid residue;

Xaa3 is a neutral/polar/small or neutral/polar/large/non-cyclic or acidic amino acid residue;

Xaa4 is a neutral/nonpolar/large/cyclic or neutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic or neutral/polar/small amino acid residue;

Xaa5 is a neutral/polar/small amino acid residue

Xaa6 is a neutral/polar/small or neutral/polar/large/non-cyclic amino acid residue;

Xaa7 is a neutral/nonpolar/large/non-cyclic or neutral/polar/large/non-cyclic amino acid residue;

Xaa8 is a neutral/polar/large/non-cyclic or neutral/polar/small amino acid residue;

Xaa9 is a neutral/polar/small amino acid residue;

Xaa10 is a neutral/polar/small amino acid residue;

Z1 is hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof;

Z2 is hydroxyl, carboxyl, non-amino acids such as agmatine, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof; and

wherein said polypeptide mimics or inhibits the biological activity of thrombospondin.

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