Methods for identifying candidate modulators of NOTUM activity
View Patent ↗The invention provides compositions and methods of use for identifying modulators of NOTUM, e.g., NOTUM inhibitors. In some aspects, identified compounds are useful for modulating Wnt signaling at sites of tissue damage. The invention further provides methods of promoting regeneration by inhibiting NOTUM.
1. A method of identifying a candidate modulator of NOTUM activity comprising: (a) providing a composition comprising: (1) an isolated NOTUM polypeptide; (2) a reporter molecule comprising a GPI anchor and a detectable moiety; and (3) a test compound, wherein the reporter molecule is attached to a membrane via the GPI anchor; (b) maintaining the composition for a suitable time period; (c) and measuring the amount of detectable moiety released from the membrane during the time period, wherein release of a greater amount of detectable moiety than would occur in the absence of the test compound indicates that the test compound is a candidate enhancer of NOTUM activity, and release of a lower amount of the detectable moiety as compared with the amount that would occur in the absence of the test compound indicates that the test compound is a candidate inhibitor of NOTUM activity.
2. The method of claim 1 , wherein the GPI anchor is a GPI anchor of a NOTUM substrate polypeptide.
3. The method of claim 1 , wherein the GPI anchor is a vertebrate glypican GPI anchor.
4. The method of claim 1 , wherein the GPI anchor is a vertebrate glypican 3 GPI anchor.
5. The method of claim 1 , wherein the GPI anchor is a human glypican GPI anchor.
6. The method of claim 1 , wherein the GPI anchor is a human glypican 3 GPI anchor.
7. The method of claim 1 , wherein the reporter molecule is a fusion protein comprising at least a portion of a mature NOTUM substrate polypeptide, a GPI anchor of the mature NOTUM substrate polypeptide, and a detectable polypeptide.
8. The method of claim 1 , wherein the reporter molecule comprises a fusion protein comprising a GPI anchor of a human glypican 3 polypeptide, at least a portion of a human glypican polypeptide, and a detectable polypeptide.
9. The method of claim 1 , wherein the reporter molecule comprises a fusion protein comprising a GPI anchor of a human glypican 3 polypeptide, at least a portion of a human glypican 3 polypeptide, and a detectable polypeptide.
10. The method of claim 1 , wherein the reporter molecule is a polypeptide, the composition comprises a cell that produces the polypeptide, and at least some of the polypeptide is attached to the cell membrane of the cell via the GPI anchor so that cleavage of the GPI anchor releases the detectable moiety from the cell.
11. The method of claim 10 , wherein the cell is in a liquid medium and step (c) comprises detecting the detectable moiety released from the cell.
12. The method of claim 1 , wherein the detectable moiety comprises a fluorescent polypeptide.
13. The method of claim 1 , wherein the NOTUM polypeptide is a vertebrate NOTUM polypeptide.
14. The method of claim 1 , wherein the NOTUM polypeptide is a human NOTUM polypeptide.
15. The method of claim 1 , wherein the test compound is a small molecule.
16. The method of claim 1 , comprising (i) comparing the amount of detectable moiety released from the membrane during the time period with a reference value; and (ii) determining whether the test compound is a candidate enhancer or inhibitor of NOTUM activity based at least in part on the result of step (i).
17. The method of claim 1 , further comprising: (d) providing a second composition substantially similar to the first composition but lacking active NOTUM polypeptide; and determining whether the test compound affects the amount of detectable moiety of the second composition released from the membrane, wherein if the test compound affects the amount of detectable moiety released in the composition comprising active NOTUM polypeptide but has little or no effect on the amount of detectable moiety released in the second composition, then the test compound is confirmed as a modulator of NOTUM activity, and if the test compound affects the amount of detectable moiety released in both compositions to approximately the same extent, the test compound is not confirmed as a modulator of NOTUM activity.
18. The method of claim 1 , wherein the membrane is in contact with a liquid medium and steps (c) comprises detecting the detectable moiety in the medium.