Modulation of Anergy and Methods for Isolating Anergy-Modulating Compounds
AssigneeThe CBR Institute for Biomedical Research, Inc., a Massachusetts corporation
InventorsAnjana Rao, Patrick G. Hogan, Vigo Heissmeyer
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Abstract
The present invention provides methods for identifying compounds capable of modulating anergy by inhibiting the production or activity of anergy associated E3 ubiquitin ligases or by altering the interaction between a ligase and its substrate.
Claims26
A method of identifying an anergy modulating agent, comprising:
- (a) providing an E3 ubiquitin ligase polypeptide, E3 ubiquitin ligase substrate polypeptide, and a test compound;
- (b) contacting the test compound, the ligase polypeptide, and the ligase substrate polypeptide together under conditions that allow the ligase polypeptide to bind or ubiquitinate the substrate polypeptide;
- and
- (c) determining whether the test compound decreases the level of binding or ubiquitination of the substrate polypeptide by the ligase polypeptide, relative to the level in the absence of the test compound, wherein a decrease indicates that the test compound is an anergy modulating agent.
7-9. (canceled)
14-16. (canceled)
A method of identifying an anergy modulating agent, comprising:
- (a) providing a test compound and a polypeptide selected from the group consisting of:
- Itch, Aip4, GRAIL, Cbl, Cbl-b, Cbl-b3, Nedd4, PLC-γ and PLCθ, or a biologically active fragment thereof;
- (b) contacting the test compound and the polypeptide or fragment thereof under conditions that allow the test compound to bind the polypeptide or fragment thereof;
- (c) determining whether the test compound binds the polypeptide or fragment thereof;
- and
- (d) determining whether the test compound reduces anergy in an immune cell in vivo or in vitro, wherein a test compound that reduces anergy is an anergy modulating agent.
18-19. (canceled)
A method of identifying an anergy modulating agent, comprising:
- (a) providing a test compound and a polypeptide comprising Itch, Aip4, or a HECT fragment of Itch or Aip4;
- (b) contacting the test compound and the polypeptide under conditions that allow the test compound to interact with the polypeptide;
- (c) contacting the polypeptide with a reaction mix comprising E1, E2, tagged ubiquitin, and ATP;
- and
- (d) determining whether the test compound prevents the autoubiquitination of the polypeptide in the presence of the reaction mix;
- wherein a test compound that prevents the autoubiquitination of the polypeptide is an anergy modulating agent.
(canceled)
24-28. (canceled)
A method of identifying an anergy modulating agent, the method comprising:
- (a) contacting a test compound and an E3 ubiquitin ligase polypeptide under conditions that allow the test compound to interact with the ligase polypeptide;
- (b) contacting the ligase polypeptide with a reaction mix comprising E1, E2, tagged ubiquitin, ATP, and an E3 ubiquitin ligase substrate polypeptide;
- and
- (c) determining whether the test compound inhibits the ligase polypeptide from transubiquitinating the substrate polypeptide in the presence of the reaction mix, wherein a test compound that inhibits transubiquitination is an anergy modulating agent.
32-38. (canceled)
A method of identifying an agent that inhibits protein-protein interaction between an anergy associated E3 ubiquitin ligase and an E3 ubiquitin ligase substrate, the method comprising:
- (a) providing an E3 ubiquitin ligase polypeptide, E3 ubiquitin ligase substrate polypeptide, and a test compound, wherein the ligase polypeptide or the substrate polypeptide is labeled;
- (b) contacting the ligase polypeptide, the substrate polypeptide, and the test compound, with each other;
- and
- (c) determining the amount of label bound to the unlabeled polypeptide, wherein a reduction in the amount of label that binds the unlabeled polypeptide indicates that the test compound is an agent that inhibits protein-protein interaction between an anergy associated E3 ubiquitin ligase and an E3 ubiquitin ligase substrate.
A method of identifying an agent that inhibits protein-protein interaction between an anergy associated E3 ubiquitin ligase and an E2 ubiquitin ligase, the method comprising:
- (a) providing E3 ubiquitin ligase polypeptide, E2 ubiquitin ligase polypeptide, and a test compound, wherein the E3 ligase polypeptide or the E2 ubiquitin ligase polypeptide is labeled;
- (b) contacting E3 ubiquitin ligase polypeptide, the E2 ubiquitin ligase polypeptide, and the test compound with each other;
- and
- (c) determining the amount of label bound to the unlabeled ligase polypeptide, wherein a reduction in the amount of label that binds the unlabeled ligase indicates that the test compound is an agent that inhibits protein-protein interaction between an anergy associated E3 ubiquitin ligase and an E2 ubiquitin ligase.
41-54. (canceled)
Dependencies are inferred from the claim text.