Nuclear hormone receptor drug screens
View Patent ↗Methods for identifying modulators of nuclear hormone receptor function comprise the steps of (a) forming a mixture comprising a nuclear hormone receptor, a peptide sensor and a candidate agent, but not a natural coactivator protein of the receptor, wherein the sensor provides direct, in vitro binding to the receptor under assay conditions; (b) measuring an agent-biased binding of the sensor to the receptor; and (c) comparing the agent-biased binding with a corresponding unbiased binding of the sensor to the receptor. In particular embodiments, the sensor comprises an amphipathic alpha helix nuclear hormone interacting domain comprising a recited nuclear hormone transcriptional coactivator motif sequence, the sensor is present at sub-micromolar concentration, the binding reaction occurs in solution, the sensor comprises a fluorescent label and the measuring step comprises detecting fluorescence polarization of the label. Reagents include labeled sensor peptides and reaction mixtures consisting essentially of nuclear hormone receptor, a peptide and a candidate agent.
1. An in vitro solid-phase binding assay method for characterizing an agent as a ligand of a nuclear hormone receptor, comprising steps:
incubating an in vitro mixture comprising a first, purified nuclear hormone receptor, a peptide sensor and a candidate agent, but not a natural coactivator protein of the first receptor, wherein the sensor consists of a peptide comprising the sequence L 1 X 1 X 2 L 2 L 3 (SEQ ID NO:18) covalently coupled to a detectable label, wherein L 1 –L 3 are independently selected from hydrophobic amino acids and X 1 –X 2 are independently selected from any amino acid and wherein the peptide provides direct, in vitro ligand-dependent binding to the first receptor and is 24 or fewer residues in length, under conditions whereby the peptide of the sensor binds the first receptor to form a first receptor-sensor complex and the complex is immobilized on a solid-phase;
measuring an assay binding of the sensor to the first receptor by selectively detecting immobilized first receptor-sensor complexes; and
comparing the assay binding with a corresponding control binding, wherein the control binding provides an indication of sensor binding to the first receptor in the absence of the agent, and wherein a greater assay binding than control binding indicates that the agent is a ligand of the receptor.
2. A method according to claim 1 , wherein the measuring step comprises detecting the first receptor of immobilized first receptor-sensor complexes.
3. A method according to claim 1 , wherein the measuring step, the first receptor is immobilized through the sensor.
4. A method according to claim 1 , wherein the measuring step, the first receptor is immobilized through the sensor and the sensor is immobilized through the label.
5. A method according to claim 1 , wherein the measuring step, the first receptor is immobilized through the sensor, and the sensor is immobilized through the label by a second, label-specific receptor.
6. A method according to claim 1 , wherein the measuring step, the first receptor is immobilized through the sensor, and the sensor is immobilized through the label by a second, label-specific receptor and wherein the measuring step comprises detecting the immobilized first receptor.
7. A method according to claim 1 , wherein the measuring step, the first receptor is immobilized through the sensor, and the sensor is immobilized through the label by a second, label-specific receptor and wherein the measuring step comprises detecting the immobilized first receptor with a third, first receptor-specific receptor.
8. A method according to claim 1 , wherein the sensor comprises an epitope label, wherein the measuring step, the first receptor is immobilized through the sensor and the sensor is immobilized through the label by a second, label-specific receptor comprising an immobilized epitope label-specific antibody moiety.
9. A method according to claim 1 , wherein the sensor comprises a biotin label and wherein the measuring step, the first receptor is immobilized through the sensor and the sensor is immobilized through the label by a second, label-specific receptor comprising an immobilized avidin moiety.
10. A method according to claim 1 , wherein the measuring step, the sensor is immobilized through the first receptor.
11. A method according to claim 1 , wherein the measuring step, the sensor is immobilized through the first receptor and the first receptor is immobilized through a second, first-receptor specific receptor.
12. A method according to claim 1 , wherein the measuring step, the sensor is immobilized through the first receptor and the first receptor is immobilized through a second, first-receptor specific receptor and wherein the measuring step comprises detecting the immobilized sensor.
13. A method according to claim 1 , wherein the measuring step, the sensor is immobilized through the first receptor and the first receptor is immobilized through a second, first-receptor specific receptor and wherein the measuring step comprises detecting the immobilized sensor with a third, label-specific receptor.
14. A method according to claim 1 , wherein the measuring step, the sensor is immobilized through the first receptor and the first receptor is immobilized through a second, first-receptor specific receptor comprising a first receptor-specific antibody.
15. A method according to claim 1 , wherein the peptide comprises a sequence selected from the group consisting of: KLVQLLTTT (SEQ ID NO:1), IHRLLQE (SEQ ID NO:2), LLRYLLDK (SEQ ID NO:3), LLRYLLD (SEQ ID NO:4), LRYLLD (SEQ ID NO:5), LLRYLL (SEQ ID NO:6), LRYLL (SEQ ID NO:7), LLRYLLDKD (SEQ ID NO:8), QLLRYLLDKD (SEQ ID NO:9), HQLLRYLLDKD (SEQ ID NO:10), PQAQQKSLLQQLLT (SEQ ID NO:11), LLQQLLTE (SEQ ID NO:12), VTLLQLLLG (SEQ ID NO:13), ILRKLLQE (SEQ ID NO:14, ILKRLLQE (SEQ ID NO:15), ILRRLLQE (SEQ ID NO:16) and ILKKLLQE (SEQ ID NO:17).
16. A method according to claim 1 , wherein the peptide consists of a sequence selected from the group consisting of: KLVQLLTTT (SEQ ID NO:1), ILHRLLQE (SEQ ID NO:2), LLRYLLDK (SEQ ID NO:3), LLRYLLD (SEQ ID NO:4), LRYLLD (SEQ ID NO:5), LLRYLL (SEQ ID NO:6), LRYLL (SEQ ID NO:7), LLRYLLDKD (SEQ ID NO:8), QLLRYLLDKD (SEQ ID NO:9), HQLLRYLLDKD (SEQ ID NO:10), PQAQQKSLLQQLLT (SEQ ID NO:11), LLQQLLTE (SEQ ID NO:12), VTLLQLLLG (SEQ ID NO:13), ILRKLLQE (SEQ ID NO:14, ILKRLLQE (SEQ ID NO:15), ILRRLLQE (SEQ ID NO:16) and ILKKLLQE (SEQ ID NO:17).
17. A method according to claim 1 , wherein the peptide is 12 or fewer residues in length.
18. A method according to claim 1 , wherein the label is coupled to the N-terminus of the peptide.
19. A method according to claim 1 , wherein the label provides for indirect detection of the sensor.
20. A method according to claim 1 , wherein the label provides for indirect detection of the sensor, wherein the label is an epitope tag.
21. A method according to claim 1 , wherein the label provides for direct detection of the sensor.
22. A method according to claim 1 , wherein the label provides for direct detection of the sensor, wherein the label is a luminescent label.
23. A method according to claim 1 , wherein the label provides for direct detection of the sensor, wherein the label is a luminescent label, wherein the luminescent label is a fluorescent label.
24. A method according to claim 1 , wherein the label provides for direct detection of the sensor, wherein the label is a luminescent label, wherein the luminescent label is a fluorescent label, wherein the fluorescent label is coupled to the N-terminus of the peptide.
25. A method according to claim 6 , wherein the label is coupled to the N-terminus of the peptide.
26. A method according to claim 7 , wherein the label is coupled to the N-terminus of the peptide.
27. A method according to claim 8 , wherein the label is coupled to the N-terminus of the peptide.
28. A method according to claim 13 , wherein the label is coupled to the N-terminus of the peptide.
29. A method according to claim 14 , wherein the label is coupled to the N-terminus of the peptide.