EC-3, an inhibitor of α4β1 and α4β7 integrins
AssigneeTemple University of the Commonwealth System of Higher Education
InventorsStefan Niewiarowski, Cezary Marcinkiewicz
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Abstract
This invention relates to the identification, purification, and characterization of a novel heterodimeric disintegrin, EC-3, from Echis carinatus viper venom. EC-3 inhibits α4 integrins in an RGD-independent manner. The invention further relates to methods of using EC-3, or a biologically active fragment or derivative thereof, to inhibit the interaction between cells expressing α4 integrins and cellular ligands.
Claims24
A substantially purified EC3 protein isolated from Echis carinatus venom, characterized by:
- (a) an apparent molecular mass of about 14,762 Da, as determined by electrospray ionization mass spectrometry;
- (b) elution from a C-18 high performance liquid chromatography column at about 40% acetonitrile;
- and
- (c) the ability to inhibit adhesion of Jurkat cells to vascular cell adhesion molecule-1.
A substantially purified EC-3A peptide isolated from EC-3 protein which has been reduced and alkylated, characterized by:
- (a) a molecular mass of about 8478 Da in its ethylpyridylated form, as determined by electrospray ionization mass spectrometry;
- (b) elation from a C-18 high performance liquid chromatography column at about 42% acetonitrile;
- and
- (c) the ability to inhibit adhesion of K562 cells to fibronectin.
A substantially purified EC-3B peptide isolated from EC-3 protein which has been reduced and alkylated with vinylpyridine, characterized by:
- (a) a molecular mass of about 7950 Da in its carboxymethylated form, as determined by electrospray ionization mass spectrometry;
- (b) elution from a C-18 high performance liquid chromatography column at about 46% acetonitrile;
- and
- (c) the ability to inhibit adhesion of Jurkat cells to vascular cell adhesion molecule-1.
A substantially purified echistatin polypeptide represented by SEQ ID NO:
- 9, in which the Arg-Gly-Asp residues at positions 24-26 are replaced by M et-Leu-Asp.
A method of isolating a peptide from a venom, wherein the peptide binds to an integrin of interest, comprising:
- (a) dissolving venom in a solvent,
- (b) centrifuging the dissolved venom to remove high molecular weight proteins,
- (c) fractionating the supernatant from step (b),
- (d) immobilizing the fractions from step (c) on a solid support,
- (e) adding detectably labeled cells that express the integrin of interest to the immobilized fractions,
- (f) detecting the number of cells bound to each immobilized fraction, and
- (g) isolating peptide from those fractions which showed enhanced cell binding in step(f).
A substantially purified EC-3A peptide characterized by:
- (a) a sequence having greater than 90% sequence identity with SEQ ID NO:2;
- and
- (b) the ability to inhibit adhesion of K562 cells to fibronectin.
A substantially purified EC-3B peptide characterized by:
- (a) a sequence having greater than 90% sequence identity with SEQ ID NO:3;
- and
- (b) the ability to inhibit adhesion of Jurkat cells to VCAM-1.
Dependencies are inferred from the claim text.