Screening systems utilizing RTP801
View Patent ↗RTP801 represents a unique gene target for hypoxia-inducible factor-1 (HIF-1). Down-regulation of the mTOR pathway activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of RTP801. The present invention relates to screening systems utilizing RTP801 and/or RTP801 interactors to and/or RTP801 biological activity, to drug candidates identified by such screening systems, and to the use of such drug candidates in the treatment of various disorders.
1. A process for determining whether a test compound modulates the binding of an RTP801 polypeptide to a second polypeptide comprising the following steps:
(a) separately contacting the RTP801 polypeptide (SEQ ID NO:2 or a fragment thereof) with the second polypeptide, which is selected from the group consisting of a TSC1 polypeptide (SEQ ID NO:4 or a fragment thereof), a TSC2 polypeptide (SEQ ID NO:6 or a fragment thereof) and an alpha-tubulin polypeptide (SEQ ID NO:8 or a fragment thereof), both in the presence and in the absence of the test compound; and
(b) determining the amount of complex formed between the RTP801 polypeptide or fragment thereof and the second polypeptide in the presence and in the absence of the test compound,
wherein a difference in the amounts determined indicates that the test compound modulates the binding of the RTP801 polypeptide to the second polypeptide.
2. The process of claim 1 , wherein the RTP801 polypeptide or fragment thereof or the second polypeptide or both are substantially purified.
3. The process of claim 1 , wherein the RTP801 polypeptide or fragment thereof comprises a tag.
4. The process of claim 1 , wherein the second polypeptide comprises a second tag.
5. The process of claim 1 , wherein the RTP801 polypeptide comprises a first tag and the second polypeptide comprises a second tag.
6. The process of claim 1 , wherein one of the RTP801 polypeptide or fragment thereof or the second polypeptide is attached to a solid support.
7. The process of claim 1 , wherein the RTP801 polypeptide or fragment thereof or the second polypeptide is expressed in a cell.
8. The process of claim 1 , wherein the fragment of the RTP801 polypeptide consists of amino acids 1-88 of SEQ ID NO:2.
9. The process of claim 1 , wherein the fragment of the RTP801 polypeptide consists of amino acids 89-232 of SEQ ID NO:2.
10. The process of claim 1 , wherein the fragment of the RTP801 polypeptide consists of amino acids 1-161 of SEQ ID NO:2.
11. The process of claim 1 , wherein the fragment of the RTP801 polypeptide consists of amino acids 1-195 of SEQ ID NO:2.
12. The process of claim 1 , wherein the fragment of the RTP801 polypeptide consists of amino acids 161-232 of SEQ ID NO: b 2 .
13. The process of claim 1 , wherein the fragment of the RTP801 polypeptide consists of amino acids 161-195 of SEQ ID NO:2.
14. A process for determining whether a test compound modulates the binding of an RTP801 polypeptide to a second polypeptide comprising the following steps:
(a) separately contacting a cell which expresses the RTP801 polypeptide (SEQ ID NO:2 or a fragment thereof) with a second polypeptide, which is selected from the group consisting of TSC1 polypeptide (SEQ ID NO; 4 or a fragment thereof), a TSC2 polypeptide (SEQ ID NO:6 or a fragment thereof) and an alpha-tubulin polypeptide (SEQ ID NO:8 or a fragment thereof), both in the presence and in the absence of the test compound; and
(b) determining the amount of complex formed between the RTP801 polypeptide or fragment thereof and the second polypeptide in the presence and in the absence of the test compound,
wherein a difference in the amounts determined indicates that that the test compound modulates the binding of the RTP801 polypeptide to the second polypeptide.
15. The process of claim 14 , wherein a lysate is prepared from the cell expressing the RTP801 polypeptide or fragment thereof after step (a) and the determination in step (b) is performed on the lysate.
16. The process of claim 14 , wherein the RTP801 polypeptide or fragment thereof comprises a tag.
17. The process of claim 14 , wherein the second polypeptide comprises a tag.
18. The process of claim 14 , wherein the RTP801 polypeptide or fragment thereof comprises a first tag and the second polypeptide comprises a second tag.