Method for identifying modulators of human orexin-2 receptor
The present invention provides a new method for identifying modulators of human orexin-2 receptor without utilizing recombinantly produced nucleic acid molecules encoding human orexin receptor protein. This method combines and utilizes known methods and cell lines selected for their natural expression of orexin-2 receptors to carry out the methods of the present invention. Exemplary methods of the present invention utilize PFSK-1 cells to produce non-recombinant human orexin-2 receptor protein.
1. A method for identifying compounds that modulate human orexin-2 receptor activity, comprising:
a) combining a putative modulator of human orexin-2 receptor activity with human orexin-2 receptors contained within membranes of PFSK-1 cells non-recombinantly possessing the human orexin-2 receptor; and
b) measuring the effect of the modulator on activity of the human orexin-2 receptor.
2. The method of claim 1 , wherein the effect measured in step (b) is binding of the putative modulator to the orexin-2 receptors.
3. The method of claim 1 , wherein the effect measured in step (b) is competition of the putative modulator with a known ligand of the human orexin-2 receptor for binding to the receptors.
4. The method of claim 1 , wherein the effect measured in step (b) is modulation of a human orexin-2 receptor intracellular second messenger.
5. The method of claim 4 , wherein the intracellular second messenger is selected from a group consisting of cAMP, Ca ++ , and a reporter gene product.
6. The method of claim 5 , wherein the cells are transfected with a Gα-protein DNA construct.
7. The method of claim 5 , wherein the intracellular second messenger is Ca ++ , detected with a fluorescent Ca ++ indicator.
8. The method of claim 1 , adapted to distinguish the putative modulator as an agonist, antagonist or inverse agonist of the orexin-2 receptor.