High-throughput high-information content label-free cell biology screening methods

• US 8,846,575 B2
• Filed: 09/29/2009
• Issued: 09/30/2014
• Est. Priority Date: 03/05/2008
• Status: Expired due to Fees

• Current Assignee: Corning Incorporated
• Original Assignee: Corning Incorporated
• Inventors: Fang, Ye, Frutos, Anthony Glenn, Tran, Elizabeth, Randle, David H
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US12/569,407
• Publication Number:
  US 20100087332A1
• Time in Patent Office: 1,827 Days
• Field of Search: None
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/025 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics antimicrobial activity C12Q1/18
• CPC Class Codes: G01N 2333/71 for growth factors; for growth regulators
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

A method for hit compound identification in a high throughput, label-free biosensor cellular assay, one method including, for example:

• • generating at least one pathway-specific kinetic profile for a target receptor;
  • generating a relational table that integrates each kinetic profile;
  • determining a delta response with the relational table for candidate compounds screened against the target receptor; and
  • identifying one or more hit compounds based upon the separation of the delta response from a threshold.

Claims

1. A method for hit compound identification in a high throughput, label-free biosensor cellular assay, the method comprising:

(a) generating at least one signaling pathway-specific kinetic profile for a target cell membrane receptor in a live-cell, wherein the at least one signaling pathway-specific kinetic profile for the target cell membrane receptor is obtained by;

(i) providing an optical biosensor having a single type of live-cell immobilized on a surface of the biosensor, the biosensor being situated in a well of a microtiter plate, and the live-cell having a first and second target, wherein the first target is a cell membrane receptor, wherein the second target is a cellular protein, wherein the cellular protein is a member of the signaling pathway triggered by activation of the cell membrane receptor,

(ii) separately stimulating the live-cell expressing target cell membrane receptor with a plurality of candidate compounds after the live-cell is attached to the surface of the biosensor;

(iii) contacting the ligand candidate-treated live-cell with a mixture containing two markers; and

(iv) determining the effect of the ligand candidate on the marker mixture-induced biosensor output;

(b) generating a relational table, which table incorporates each signaling pathway-specific kinetic profile for the plurality of candidate compounds;

(c) determining a delta response based on the relational table for each of the plurality of candidate compounds screened against the target cell membrane receptor; and

(d) identifying hit compounds from the plurality of candidate compounds based upon the separation of the delta response from a threshold.

2. The method of claim 1 wherein the threshold comprises the upper and lower limits of the response region for positive and negative control compounds.

3. The method of claim 1 wherein the separation comprises a data point outside an upper threshold value or a lower threshold value.

4. The method of claim 3 wherein the target cell membrane receptor is a G protein-coupled receptor (GPCR), wherein a data point outside an upper threshold value represents a G_s hit compound, and a data point outside a lower threshold value represents a G_q/G_i hit compound in the GPCR target cell membrane receptor.

5. The method of claim 2 wherein a threshold value is 50% of the positive control value.

6. The method of claim 1 further comprising plotting the delta response determined for each compound screened against the target cell membrane receptor.

7. The method of claim 1 wherein the live-cell comprises at least one of:

a eukaryote, a prokaryote, a hybrid, a synthetic construct, or a combination thereof.

8. The method of claim 1 wherein the target cell membrane receptor comprises a G protein-coupled receptor (GPCR), a tyrosine kinase, an ion channel, or a combination thereof.

9. The method of claim 1 wherein the candidate compound comprises an agonist, an antagonist, an inverse agonist, a positive control, a negative control, or a combination thereof.

10. The method of claim 1 wherein the signaling pathway-specific kinetic profile is accomplished in high throughput screening (HTS) format in about 8 hours with from 2 to about 3 data points compared to a full signaling pathway-specific kinetic profile about 80 hours and greater than about 50 data points.

12. The method of claim 1, further comprising:

wherein the candidate compound is an antagonist,

adding a single second compound to each assay well after contacting with a first antagonist candidate compound to create an additional response point R₄ measured at from about 10 mins to about 60 mins after R₃, wherein R₃ is a third time domain response and calculating a Δ

R_1antagonist delta response of zero according to the formula;

Δ

R_1antagonist=R₄−

R₃.

11. A method for classifying active G protein-coupled receptor (GPCR) responses, the method comprising:

(a) generating at least one signaling pathway-specific kinetic profile for a target GPCR in a live-cell, wherein the at least one signaling pathway-specific kinetic profile for the target cell membrane receptor is obtained by;

(i) providing an optical biosensor having a single type of live-cell immobilized on a surface of the biosensor, the biosensor being situated in a well of a microtiter plate, and the live-cell having a first and second target, wherein the first target is a GPCR, wherein the second target is a cellular protein, wherein the cellular protein is a member of the signaling pathway triggered by activation of the GPCR,

(ii) separately stimulating the live-cell expressing target cell membrane receptor with a plurality of candidate compounds after the live-cell is attached to the surface of the biosensor;

(iii) contacting the ligand candidate-treated live-cell with a mixture containing two markers; and

(iv) determining the effect of the ligand candidate on the marker mixture-induced biosensor output;

(b) generating a relational table that includes each signaling pathway-specific kinetic profile for the plurality of candidate compounds;

(c) determining a delta response from the relational table for each of the plurality of candidate compounds screened against the GPCR; and

(d) classifying the delta response for each of the plurality of candidate compounds as a Gs hit compound, a Gq/Gi hit compound, or a non-hit compound.