Methods for modulating signal transduction mediated by TGF-beta related proteins
AssigneeSignal Pharmaceuticals, LLC
InventorsMerl F. Hoekstra, Weilin Xie, Brion W. Murray, Frank M. Mercurio
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Abstract
Methods are provided for identifying agents that modulate signaling mediated by transforming growth factor beta (TGF-β) and members of the TGF-β family, such as bone morphogenic protein (BMP). Such agents may be identified using screens that evaluate candidate agents for the ability to modulate Smad protein degradation. Agents identified as described herein may be used to augment or inhibit signaling mediated by one or more TGF-β family members in a variety of cell types and for therapeutic purposes.
Claims54
A method for screening for an agent that modulates TGF-β- and/or BMP-mediated signaling, comprising the steps of:
- (a) contacting
- (i) a first polypeptide comprising a HECT E3 ubiquitin ligase WW domain, or a variant thereof in which the ability of the polypeptide to bind to a Smad protein is not substantially diminished relative to the HECT E3 ubiquitin ligase;
- (ii) a second polypeptide comprising a Smad PY motif, or a variant thereof in which the ability of the polypeptide to bind to an E3 ubiquitin ligase is not substantially diminished relative to a native Smad protein comprising the PY motif;
- and
- (iii) a candidate agent;
- under conditions that permit a detectable level of binding of the first polypeptide to the second polypeptide in the absence of candidate agent;
- (b) determining a level of binding of the first polypeptide to the second polypeptide;
- and
- (c) comparing the level of binding to a control level of binding of the first polypeptide to the second polypeptide in the absence of candidate agent, and therefrom determining whether the candidate agent modulates TGF-β- and/or BMP-mediated signaling.
A method for screening for an agent that modulates TGF-β- and/or BMP-mediated signaling, comprising the steps of:
- (a) contacting
- (i) a candidate agent;
- (ii) a ubiquitinated HECT E3 ubiquitin ligase;
- and
- (iii) a Smad protein or a variant thereof that comprises a PY motif;
- wherein the contact takes place under conditions and for a time sufficient to permit ubiquitination of the Smad protein or variant thereof by the HECT E3 ubiquitin ligase in the absence of candidate agent;
- (b) determining a level of ubiquitination of the Smad protein or variant thereof;
- and
- (c) comparing the level of ubiquitination to a control level of ubiquitination in the absence of candidate agent, and therefrom determining whether the candidate agent modulates TGF-β- and/or BMP-mediated signaling.
A method for screening for an agent that modulates BMP-mediated signaling, comprising the steps of:
- (a) contacting a cell that expresses a BMP receptor with a bone morphogenic protein and a candidate agent;
- and
- (b) detecting a level of a Smad protein in the cell, relative to a level of the Smad protein in a cell that is contacted with the bone morphogenic protein in the absence of the candidate agent, and therefrom determining whether the candidate agent is a modulator of BMP-mediated signaling.
A method for screening for an agent that modulates BMP-mediated signaling, comprising the steps of
- (a) contacting a cell that expresses a BMP receptor with a bone morphogenic protein and a candidate agent;
- and
- (b) detecting a level of ubiquitination of a Smad protein in the cell, relative to a level of the Smad protein ubiquitination in a cell that is contacted with the bone morphogenic protein but is not contacted with the candidate agent, and therefrom determining whether the candidate agent modulates BMP-mediated signaling.
A method for screening for an agent that modulates TGF-β-mediated signaling, comprising the steps of:
- (a) contacting a cell that expresses a TGF-β receptor with TGF-β and a candidate agent;
- and
- (b) detecting a level of a Smad protein in the cell, relative to a level of the Smad protein in a cell that is contacted with the bone morphogenic protein in the absence of the candidate agent, and therefrom determining whether the candidate agent is a modulator of TGF-β-mediated signaling.
A method for screening for an agent that modulates TGF-β-mediated signaling, comprising the steps of:
- (a) contacting a cell that expresses a TGF-βreceptor with TGF-β and a candidate agent;
- and
- (b) detecting a level of ubiquitination of a Smad protein in the cell, relative to a level of the Smad protein ubiquitination in a cell that is contacted with the bone morphogenic protein but is not contacted with the candidate agent, and therefrom determining whether the candidate agent modulates TGF-β-mediated signaling.
A method for screening for an agent that modulates BMP-mediated signaling, comprising the steps of:
- (a) contacting a cell that expresses a BMP receptor with bone morphogenic protein and a candidate agent;
- and
- (b) detecting a level of a HECT E3 ubiquitin ligase activity in the cell, relative to a level of HECT E3 ubiquitin ligase activity in a cell that is contacted with the bone morphogenic protein in the absence of the candidate agent, and therefrom determining whether the candidate agent modulates BMP-mediated signaling.
A method for screening for an agent that modulates TGF-β-mediated signaling, comprising the steps of:
- (a) contacting a cell that expresses a TGF-β receptor with TGF-β and a candidate agent;
- and
- (b) detecting a level of a HECT E3 ubiquitin ligase activity in the cell, relative to a level of HECT E3 ubiquitin ligase activity in a cell that is contacted with the bone morphogenic protein in the absence of the candidate agent, and therefrom determining whether the candidate agent modulates TGF-β- mediated signaling.
A method for augmenting TGF-β- and/or BMP-mediated signaling in a cell, comprising contacting a cell with an agent that inhibits binding of a HECT E3 ubiquitin ligase WW domain to a Smad PY motif.
A method for augmenting TGF-β and/or BMP-mediated signaling in a cell, comprising contacting a cell with an agent that inhibits ubiquitination of a Smad protein.
A method for stimulating bone formation in a patient, comprising administering to a patient a therapeutically effective amount of an agent that inhibits binding of a HECT E3 ubiquitin ligase WW domain to a Smad PY motif.
A method for stimulating bone formation in a patient, comprising administering to a patient a therapeutically effective amount of an agent that inhibits ubiquitination of a Smad protein.
A method for preventing or treating a condition associated with insufficient TGF-β and/or BMP-mediated cell signaling, comprising administering to a patient a therapeutically effective amount of an agent that inhibits binding of a HECT E3 ubiquitin ligase WW domain to a Smad PY motif.
A method for preventing or treating a condition associated with insufficient TGF-β and/or BMP-mediated cell signaling, comprising administering to a patient a therapeutically effective amount of an agent that inhibits ubiquitination of a Smad protein.
Dependencies are inferred from the claim text.