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Metalloprotease activation of myostatin, and methods of modulating myostatin activity

a myostatin and metalloprotease technology, applied in the field of can solve the problems of diverse and deleterious effects, and achieve the effects of facilitating the action or activity of the agent, reducing or inhibiting metalloprotease mediated activation of latent myostatin, and increasing or decreasing cleavage activity

Inactive Publication Date: 2006-12-07
THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides agents that can modulate the activity of myostatin, a negative regulator of muscle growth and development, by targeting the protease that cleaves its pro peptide. The agents can increase or decrease the activity of the protease, which can lead to increased or decreased myostatin activation. The invention also provides methods for identifying and using these agents, as well as methods for inhibiting myostatin activation in an organism. The technical effect of the invention is the ability to control myostatin activity, which can lead to increased or decreased muscle mass or decreased fat content in an organism.

Problems solved by technology

Unfortunately, these proteases also can act on a variety of other proteins and, therefore, agents that target and inhibit these proteases, for example, signal peptidase, likely would have diverse and deleterious effects if administered to a living organism.

Method used

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  • Metalloprotease activation of myostatin, and methods of modulating myostatin activity
  • Metalloprotease activation of myostatin, and methods of modulating myostatin activity
  • Metalloprotease activation of myostatin, and methods of modulating myostatin activity

Examples

Experimental program
Comparison scheme
Effect test

example 1

BMP-1 / TLD Metalloprotease Family Members Cleave Myostatin Pro Peptide

[0090] This example demonstrates that the members of the bone morphogenic protein-1 / Tolloid (BMP-1 / TLD) family of metalloproteases cleave the myostatin pro peptide.

[0091] Five hundred ng of purified myostatin pro peptide or of purified latent myostatin complex comprising the pro peptide and C-terminal dimer (Lee and McPherron, supra, 2001) was incubated overnight at 37° C. with 100 ng purified BMP-1, mTLD, mTLL-1, or mTLL-2 (Scott et al., Devel. Biol. 213:283-300, 1999, which is incorporated herein by reference). Reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blot analysis using antiserum raised against the myostatin pro peptide (Lee and McPherron, supra, 2001).

[0092] A discrete proteolytic cleavage product of the pro peptide was detected in each reaction containing one of the four proteases, but not in control reactions that did not co...

example 2

Metalloprotease Cleavage of Myostatin Pro Peptide Activates Latent Myostatin

[0093] This example demonstrates that cleavage of the myostatin pro peptide by a BMP-1 / TLD metalloprotease activates latent myostatin.

[0094] Purified myostatin pro peptide and C-terminal dimer complex was incubated with mTLL-1, then examined using a reporter gene assay that specifically detects myostatin activity. A204 rhabdomyosarcoma cells were transfected with the pGL3-(CAGA)12 luciferase reporter gene construct, which comprises the luciferase coding sequence linked to the TGF-Θ responsive CAGA sequence from the promoter of the TGF-Θ inducible PAI-1 gene (Thies et al., supra, 2001). The transfected cells were contacted with either untreated pro peptide / C-terminal dimer complex or complex that had been pre-incubated with mTLL-1. Incubation of the complex with mTLL-1 dramatically increased the amount of luciferase activity detected in the reporter cell assay, whereas no change was observed in cells treate...

example 3

Peptide Substrates for Tolloid Family Members

[0096] A series of three peptides each of 10, 20, 30, 40, or 50 amino acid residues was synthesized based on the sequence of the myostatin pro peptide, and encompassing the BMP-1 / TLD metalloprotease cleavage site (amino acid residues “RD” as shown in bold, below, in wild type peptides; SEQ ID NOS:9, 12, 15, 18, and 21). Peptides in which the arginine residue at the P1 position just upstream of the cleavage site was changed to a glutamine residue (SEQ ID NOS: 10, 13, 16, 19, and 22; see bold), and peptides in which the aspartic acid at the P1′ position just downstream of the cleavage site was changed to an alanine (SEQ ID NOS: 11, 14, 17, 20, and 23; see bold), also were synthesized. The sequences of the peptides are shown below:

[0097] 50-mer

KDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDD(SEQ ID NO:9)DYHATTETIITMPTKDVIRQLLPKAPPLRELIDQYDVQQDDSSDGSLEDD(SEQ ID NO:10)DYHATTETIITMPTKDVIRQLLPKAPPLRELIDQYDVQRADSSDGSLEDD(SEQ ID NO:11)DYHATTETIITMPT

[0098]...

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Abstract

It has been determined that metalloprotease cleavage of a myostatin pro peptide results in activation of a latent inactive myostatin to an active form. Accordingly, methods of identifying agents that modulate metalloprotease mediated activation of myostatin are provided, as are agents identified using such methods. Also provided are methods of modulating muscle growth in an organism by increasing or decreasing metalloprotease mediated cleavage of a myostatin pro peptide.

Description

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Ser. No. 60 / 486,863, filed Jul. 10, 2003; U.S. Ser. No. 60 / 439,164, filed Jan. 9, 2003; and U.S. Ser. No. 60 / 411,133, filed Sep. 16, 2002; the entire content of each which is incorporated herein by reference.[0002] This invention was made in part with government support under Grant Nos. HD35887, AR47746, and GM63471 awarded by the National Institutes of Health. The United States government has certain rights in this invention.BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The invention relates generally to metalloprotease regulation of myostatin activity, and more specifically to methods of using agonists or antagonists of the BMP-1 / TLD family of metalloproteases to modulate myostatin activity including, for example, to regulate muscle development in an organism, to methods of identifying agonists and antagonists of such metalloproteases, and to agonists and antagonists so ide...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/46C12P21/06A61K38/00A61K38/16A61K38/18C07K14/475C12N9/64
CPCA61K38/00C07K2319/00C07K14/475A61P21/00A61P21/04A61P3/00A61P3/04A61P31/18A61P35/00A61P37/02A61P37/04A61P43/00A61P7/00A61P3/10
Inventor LEE, SE-JINMCPHERRON, ALEXANDRAGREENSPAN, DANIELPAPPANO, WILLIAMWOLFMAN, NEILTOMKINSON, KATHY
Owner THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE
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