Screening systems utilizing RTP801L
RTP801L represents a unique gene target for hypoxia-inducible factor-1 (HIF-1) that may regulate hypoxia-induced pathogenesis; down-regulation of the mTOR pathway activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of RTP801L. The present invention relates to screening systems utilizing RTP801L and/or RTP801L interactors and/or RTP801L biological activity, and to the potential drugs and methods of treatment identified by such screening systems.
1 . A process for determining whether a test compound modulates the activity of an RTP801L polypeptide comprising the following steps:
a) providing an RTP801L polypeptide and a second polypeptide selected from the group consisting of RTP801, RTP801L, TSC1, TSC2 and alpha-tubulin;
(b) treating or contacting the polypeptides of a) with the test compound;
(c) determining the amount of a complex comprising the RTP801L polypeptide and the second polypeptide; and
(d) comparing the amount of such complex determined in step c) with the amount determined for control polypeptides not treated or contacted with the test compound.
2 . The process of claim 1 wherein a difference in the amount determined in step c) with the amount determined for the control polypeptides indicates that the test compound modulates the activity of RTP801L.
3 . The process of claim 1 wherein one or both of the polypeptides are substantially purified.
4 . The process of claim 1 wherein the RTP801L polypeptide is a form of RTP801L comprising a tag.
5 . The process of claim 1 wherein the second polypeptide is a form of the second polypeptide comprising a tag.
6 . The process of claim 1 wherein the RTP801L polypeptide is a form of RTP801L comprising a first tag and the second polypeptide is a form of the second polypeptide comprising a second tag.
7 . The process of claim 1 wherein one of the polypeptides is attached to a solid support.
8 . A process for determining whether a test compound modulates the activity of an RTP801L polypeptide comprising the following steps:
a) providing a cell which expresses
(i) an RTP801L polypeptide and
(ii) a second polypeptide selected from the group consisting of RTP801, RTP801L, TSCl, TSC2 and alpha-tubulin;
(b) treating or contacting the cell of (a) with the test compound;
(c) determining the amount of a complex comprising the RTP801L polypeptide and the second polypeptide present in the cell; and
(d) comparing the amount of such complex determined in step c) with the amount determined in a control cell not treated or contacted with the test compound.
9 . The process of claim 8 wherein a difference in the amount determined in step c) with the amount determined in the control cell indicates that the test compound modulates the activity of RTP801L.
10 . The process of claim 8 wherein a lysate is prepared from the cell of step (b) and the detection of step (c) is performed on the lysate.
11 . The process of claim 8 wherein a lysate is prepared from the cell of step (a) and the treatment of step b) and detection of step (c) are performed on the lysate.
12 . A process for determining whether a test compound modulates the activity of RTP801L comprising the following steps:
a) providing a cell which expresses
(i) a form of RTP801L comprising a first tag; and
(ii) a form of a second polypeptide selected from the group consisting of RTP801, RTP801L, TSC 1, TSC2 and alpha-tubulin, wherein the second polypeptide comprises a second tag;
(b) treating or contacting the cell of (a) with the test compound;
(c) determining the amount of a complex comprising the tagged form of RTP801L and the tagged form of the second polypeptide present in the cell; and
(d) comparing the amount of such complex determined in step c) with the amount determined in a control cell not treated or contacted with the test compound.
13 . The process of claim 12 wherein a difference in the amount determined in step c) with the amount determined in the control sample indicates that the test compound modulates the activity of RTP801L.
14 . The process of claim 12 wherein a lysate is prepared from the cell of step (b) and the detection of step (c) is performed on the lysate.
15 . The process of claim 12 wherein a lysate is prepared from the cell of step (a) and the treatment of step b) and detection of step (c) are performed on the lysate.
16 . The process of claim 12 wherein the first tag and the second tag interact to produce a moiety, the amount of which can be determined.
17 . A process for determining whether a test compound modulates the activity of an RTP801L polypeptide comprising the following steps:
a) providing an RTP801L polypeptide;
(b) treating or contacting the polypeptide of a) with the test compound;
(c) determining the amount of an RTP801L polypeptide complex; and
(d) comparing the amount of such complex determined in step c) with the amount determined for a control RTP801L polypeptide not treated or contacted with the test compound.
18 . The process of claim 17 wherein a difference in the amount determined in step c) with the amount determined for the control polypeptides indicates that the test compound modulates the activity of RTP801L.
19 . The process of claim 17 wherein the RTP801L polypeptide is substantially purified.
20 . The process of claim 17 wherein a portion of the RTP801L polypeptide is a form of RTP801L comprising a tag.
21 . The process of claim 17 wherein a first portion of the RTP801L polypeptide is a form of RTP801L comprising a first tag and the second portion of the RTP801L polypeptide is a form of RTP801L comprising a second tag.
22 . The process of claim 17 wherein a portion of the RTP801L polypeptide is attached to a solid support.
23 . The process of claim 17 wherein the complex is a dimer.
24 . A process for obtaining a compound which modulates apoptosis in a cell comprising:
a) providing cells which express the human RTP801L polypeptide;
b) contacting the cells with a plurality of compounds;
c) determining which of the plurality of compounds modulates apoptosis in the cells; and
d) obtaining the compound determined to modulate apoptosis in step c).
25 . The process according to claim 24 comprising:
a) providing cells which express the human RTP801L polypeptide at a level such that about 50% of the cells undergo apoptosis in the presence of a known apoptosis-stimulating agent;
b) contacting the cells with the plurality of compounds;
c) treating the cells with an amount of the known apoptosis-stimulating agent so as to cause apoptosis in the cells;
d) determining which of the plurality of compounds modulates apoptosis in the cells; and
e) obtaining the compound determined to modulate apoptosis in step d).
26 . A process for obtaining a compound which modulates the activity of the RTP801L polypeptide comprising:
a) measuring the activity of the RTP801L polypeptide;
b) contacting the RTP801L polypeptide with a plurality of compounds;
c) determining which of the plurality of compounds modulates the activity of the RTP801L polypeptide; and
d) obtaining the compound determined to modulate the activity of the RTP801L polypeptide in step c).
27 . A process for obtaining a compound which modulates the activity of the RTP801L polypeptide comprising:
a) measuring the binding of the RTP801L polypeptide to a species with which the RTP801L polypeptide interacts;
b) contacting the RTP801L polypeptide with a plurality of compounds;
c) determining which of the plurality of compounds modulates the binding of the of the RTP801L polypeptide to the species; and
d) obtaining the compound determined to modulate the binding of the RTP801L polypeptide to the species in step c).
28 . A kit for obtaining a compound which modulates the biological activity of RTP801L comprising:
(a) RTP801L; and
(b) an interactor with which RTP801L interacts.
29 . The kit of claim 28 wherein the interactor is selected from the group consisting of an RTP801 polypeptide, a TSC1 polypeptide, a TSC2 polypeptide and an alpha-tubulin polypeptide.