# US 2014/0194307 A1 · Recognition of cellular target binding by a bioactive agent using intracellular bioluminescence resonance energy transfer

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2014/0194307 A1
- Document ID: US20140194307A1
- Status: Published application
- Kind: A1
- Application number: 14/104,860
- Filed: 2013-12-12
- Published: 2014-07-10
- Earliest claimed: 2012-12-12
- Assignees: Promega Corporation
- Applicants: Promega Corporation
- Inventors: Carolyn W. Hitko; Thomas Kirkland; Thomas Machleidt; Rachel Friedman Ohana; Matt Robers; Keith Wood
- Web page: https://patentgrep.com/patent/US20140194307A1

## CPC

- G01N 33/582: {with fluorescent label}
- G01N 33/502: {for testing non-proliferative effects}
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention provides compositions and methods for detection and analysis of intracellular binding of a bioactive agent to a cellular target. In particular, provided herein are bioactive agents tethered to fluorophores, cellular targets fused to bioluminescent reporters, or portions, components, or subunits of bioluminescent reporters, and methods of detecting and analyzing the interaction of bioactive agents with cellular targets therewith.

## Claims

### Claim at position 1

-16. (canceled)

### Claim 17

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.

### Claim 18 (depends on 17)

The method of claim 17, wherein detecting fluorescence within said second emission spectrum comprises detecting said second peak emission.

### Claim 19 (depends on 17)

The method of claim 17, wherein the bioactive agent is a small molecule, peptide, or nucleic acid.

### Claim 20 (depends on 19)

The method of claim 19, wherein the bioactive agent is a small molecule.

### Claim 21 (depends on 17)

The method of claim 17, wherein the fluorophore is a carboxy rhodamine analog.

### Claim 22 (depends on 17)

The method of claim 17, wherein the fluorophore is a cyanine derivative.

### Claim 23 (depends on 17)

The method of claim 17, wherein the fluorophore is 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY).

### Claim 24 (depends on 17)

The method of claim 17, wherein the bioluminescent reporter comprises a polypeptide with at least 70% sequence identity with SEQ ID NO.: 1.

### Claim 25 (depends on 17)

The method of claim 17, 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.

### Claim 26 (depends on 25)

The method of claim 25, wherein the substrate is 2-furanylmethyl-deoxy-coelenterazine.

### Claim 27 (depends on 17)

The method of claim 17, wherein step (a) is occurs intracellularly.

### Claim 28 (depends on 27)

The method of claim 27, wherein step (b) is occurs intracellularly.

### Claim 29 (depends on 27)

The method of claim 27, wherein the fusion of a cellular target and a bioluminescent reporter is expressed intracellularly.

### Claim 30 (depends on 27)

The method of claim 27, wherein the conjugate of a bioactive agent and a fluorophore is added extracellularly and enters the cell.

### Claim 31 (depends on 30)

The method of claim 30, wherein the conjugate of a bioactive agent and a fluorophore is cell permeable.

### Claim 32 (depends on 30)

The method of claim 30, further comprising a step of contacting said cell with a permeabilization agent to potentiate entry of the conjugate of a bioactive agent and a fluorophore into the cell.

### Claim 33 (depends on 17)

The method of claim 17, wherein the cellular target is a binding partner of the bioactive agent.

### Claim 34 (depends on 17)

The method of claim 17, wherein the fusion of a cellular target and a bioluminescent reporter is one of a plurality of different cellular targets fused to bioluminescent reporters.

### Claim 35 (depends on 17)

The method of claim 17, wherein the bioactive agent or the bioactive agent tethered to a fluorophore is produced by non-natural chemical synthesis.

### Claim 36 (depends on 17)

The method of claim 17, wherein said second peak emission is substantially separated from said first peak emission.

### Claim 37 (depends on 36)

The method of claim 36, wherein substantially separated is at least 80 nm.

### Claim 38 (depends on 17)

The method of claim 17, wherein the Forster distance of the fluorophore and bioluminescent reporter pair is less than 20 nm.

### Claim 39 (depends on 38)

The method of claim 38, wherein the Forster distance of the fluorophore and bioluminescent reporter pair is less than 5 nm.

### Claim 40 (depends on 17)

The method of claim 17, wherein upon binding of the bioactive agent to the cellular target, conversion of the substrate to a reaction product by the bioluminescent reporter results in excitation of the fluorophore by BRET and fluorescence emission from the fluorophore.

### Claim 41 (depends on 17)

The method of claim 17, further comprising a step of: contacting the cellular target with unlabeled bioactive agent.

### Claim 42 (depends on 41)

The method of claim 41, wherein said contacting the cellular target with unlabeled bioactive agent is performed concurrently with said detecting step.

### Claim 43 (depends on 41)

The method of claim 41, further comprising a step of: determining the binding affinity of the bioactive agent to the cellular target.

### Claim 44 (depends on 43)

The method of claim 43, wherein determining the binding affinity comprises competition with a labeled bioactive agent for the cellular target.

### Claim 45 (depends on 41)

The method of claim 41, further comprising measuring the decrease in the emission of the fluorophore on addition of the unlabeled bioactive agent to determine the binding affinity of said unlabeled bioactive agent for the cellular target.

### Claim 46

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.

### Claim 47 (depends on 46)

The method of claim 46, wherein the fluorophore is a carboxy rhodamine analog.

### Claim 48 (depends on 46)

The method of claim 46, wherein the substrate is 2-furanylmethyl-deoxy-coelenterazine.

### Claim 49 (depends on 46)

The method of claim 46, wherein the bioactive agent is a small molecule, peptide, or nucleic acid.

### Claim 50 (depends on 46)

The method of claim 46, wherein the bioactive agent is a small molecule.

### Claim 51 (depends on 46)

The method of claim 46, wherein the conjugate of a bioactive agent and a fluorophore is cell permeable.

### Claim 52 (depends on 46)

The method of claim 46, further comprising a step between steps (a) and (b) of contacting said cell with a permeabilization agent to potentiate entry of the conjugate of a bioactive agent and a fluorophore into the cell.