Recognition of cellular target binding by a bioactive agent using intracellular bioluminescence resonance energy transfer

A method for detection of an interaction between a bioactive agent and a cellular target comprising:

• (a) contacting:
• i) a fusion of said cellular target and a bioluminescent reporter, said bioluminescent reporter having a first emission spectrum with a first peak emission, and
• ii) a conjugate of said bioactive agent and a fluorophore, said fluorophore having an excitation spectrum that overlaps said first emission spectrum, and said fluorophore having second emission spectrum with a second peak emission;
• (b) contacting said bioluminescent reporter with a substrate for said bioluminescent reporter;
• and
• (c) detecting fluorescence within said second emission spectrum, wherein the presence of said fluorescence within said second emission spectrum indicates the interaction of said bioactive agent with said cellular target.

A method for detection of an interaction between a bioactive agent and a cellular target comprising:

• (a) expressing in a cell a fusion of said cellular target protein and a bioluminescent reporter, wherein said bioluminescent reporter comprises a polypeptide with at least 70% sequence identity with SEQ ID NO.:
• 1 and has a first emission spectrum with a first peak emission;
• (b) contacting said cell with a conjugate of said bioactive agent and a fluorophore, wherein the conjugate of a bioactive agent and a fluorophore is added extracellularly and enters the cell, wherein said fluorophore has an excitation spectrum that overlaps said first emission spectrum, and said fluorophore has a second emission spectrum with a second peak emission;
• (c) contacting said cell with a substrate for said bioluminescent reporter, wherein the substrate for the bioluminescent reporter is selected from the list consisting of coelenterazine, a coelenterazine derivative, a structural or functional equivalent of coelenterazine, or a molecule substantially equivalent to coelenterazine;
• (d) detecting fluorescence within said second emission spectrum, wherein the presence of said fluorescence within said second emission spectrum indicates the interaction of said bioactive agent with said cellular target protein.