EC-3, an inhibitor of α4β1 and α4β7 integrins
First Claim
Patent Images
1. 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.
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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.
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Citations
24 Claims
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1. A substantially purified EC3 protein isolated from Echis carinatus venom, characterized by:
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(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. - View Dependent Claims (8, 15, 16, 17, 18, 19, 20, 21, 22)
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2. A substantially purified EC-3A peptide isolated from EC-3 protein which has been reduced and alkylated, characterized by:
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(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. - View Dependent Claims (4, 5)
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3. A substantially purified EC-3B peptide isolated from EC-3 protein which has been reduced and alkylated with vinylpyridine, characterized by:
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(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. - View Dependent Claims (6, 7, 9, 10, 11, 12)
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13. 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.
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14. A method of isolating a peptide from a venom, wherein the peptide binds to an integrin of interest, comprising:
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(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).
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23. A substantially purified EC-3A peptide characterized by:
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(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.
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24. A substantially purified EC-3B peptide characterized by:
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(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.
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Specification