Cell surface receptors for the detection and identification of compounds

• US 6,475,733 B1
• Filed: 10/04/2000
• Issued: 11/05/2002
• Est. Priority Date: 04/06/1998
• Status: Expired due to Fees

• Current Assignee: Lerner Pharmaceuticals, Inc.
• Original Assignee: Lerner Pharmaceuticals, Inc.
• Inventors: Lerner, Michael R.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/647,653
• Time in Patent Office: 762 Days
• Field of Search: 435/6, 435/7.1, 435/69.1
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• 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 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• 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 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• 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

The invention exploits the evolutionary principles responsible for the development of the broad spectrum general odorant detector system, to create a cell-surface receptor based system capable of detecting and discriminating between thousands of chemicals. This is accomplished by subjecting a defined set of cell-surface receptors such as G-protein coupled receptors, tyrosine kinase receptors, and/or ion channels, to the types of evolutionary forces that have created the array of approximately 1,000 natural receptors used in general olfaction by higher animals. This goal is further accomplished by a ‘directed evolution-in-a-test-tube’ method, imposing very high rates of mutation and extremely strict selection criteria to create a sensor. The novel sensor of the present invention is selected using a sensitive melanophore-based functional bioassay.

Claims

1. A method for identifying a plurality of mutated receptors that bind a ligand to produce an identifying fingerprint for the ligand, comprising:

generating a plurality of nucleic acids that encode a plurality of mutated receptors,

introducing the plurality of nucleic acids into a plurality of cells, there being different nucleic acids in different of the plurality of cells,

expressing the plurality of nucleic acids in the plurality of cells to produce a plurality of mutated receptors on the surface of the cells,

contacting the plurality of cells with the ligand,

detecting an intracellular signal in a cell generated by a ligand binding to one of the plurality of mutated receptors, the signal being indicative of the presence of a mutated receptor that binds the ligand, wherein the mutated receptor is selected from the group consisting of mutated G-protein coupled receptors, tyrosine kinase receptors and ion channels, and

selecting a plurality of mutated receptors based upon the generated intracellular signals to produce an identifying fingerprint for the ligand.

2. The method of claim 1, wherein the signal detected is compared to a signal generated by a cell expressing a non-mutated receptor of the same type as the mutated receptor, thereby permitting the identification of a mutated receptor with an altered binding specificity for the ligand versus the non-mutated receptor.

3. The method of claim 1, wherein the signal is compared to a cell expressing a different mutated receptor which binds the ligand.

4. The method of claim 1, wherein the ligand is not the natural ligand for the receptor.

5. The method of claim 1, wherein the ligand is selected from the group consisting of chemical warfare agents, explosives, drugs, fragrances, impurities, environmental toxins and pollutants.

6. The method according to any of claims 1-5, wherein the signals generated are second messenger signals.

7. The method of claim 6, wherein the second messenger signals result in pigment dispersion or aggregation.

8. The method of claim 7, wherein the cells are melaniferous.

9. The method of claim 8, wherein the cells are lower animal pigment cells.

10. The method of claim 6, wherein the second messenger signals cause alterations in calcium levels in the cell and the signal is calcium mediated fluorescence.

11. A method for testing a sample for the presence of a ligand comprising:

contacting the sample with a plurality of recombinant cells expressing a plurality of exogenous cell surface receptors, each receptor mutated to have altered binding to its natural ligand, and

determining the presence of a preselected signal generated if the ligand binds to the plurality of exogenous cell surface receptors.

12. The method of claim 11, wherein the recombinant cell is at least two recombinant cells, each expressing a respective exogenous cell surface receptor mutated differently to have differently altered binding to the natural ligand.

13. The method of claim 11, wherein the recombinant cell is an array of recombinant cells expressing an array of differently mutated exogenous cell surface receptors.

14. The method of claim 11, wherein the mutated exogenous cell surface receptor is selected from the group consisting of mutated G-protein coupled receptors, tyrosine kinase receptors and ion channels.

15. The method of claim 11, wherein the signal is compared to a cell expressing a different mutated receptor which binds the ligand.

16. The method of claim 11, wherein the ligand is not the natural ligand for the receptor.

17. The method of claim 11, wherein the ligand is selected from the group consisting of chemical warfare agents, explosives, drugs, fragrances, impurities, environmental toxins and pollutants.

18. The method according to any of claims 11-17, wherein the signals generated are second messenger signals.

19. The method of claim 18, wherein the second messenger signals result in pigment dispersion or aggregation.

20. The method of claim 19, wherein the cells are melaniferous.

21. The method of claim 20, wherein the cells are lower animal pigment cells.

22. The method of claim 11, wherein the second messenger signals cause alterations in calcium levels in the cell and the signal is calcium mediated fluorescence.

23. A method for generating an identifying fingerprint for a ligand comprising:

contacting an army of recombinant cells expressing an array of exogenous mutated cell surface receptors with a ligand to generate a plurality of signals, each of said receptors having a different selectivity or specificity for the ligand, and

selecting a plurality of the generated signals to produce an identifying fingerprint for the ligand.

24. The method of claim 23, wherein the fingerprint is a fluorescence read-out, a spatial pattern, or a graph.

25. The method of claim 23, wherein the ligand is selected from the group consisting of chemical warfare agents, explosives, drugs, fragrances, impurities, environmental toxins and pollutants.

26. A method for determining relatedness of a sample to a standard known to be authentic or known to have at least one selected characteristic of authentic material, the method comprising:

generating a plurality of signals for a ligand-containing standard by contacting an array of recombinant cells expressing an array of exogenous mutated cell surface receptors with a ligand-containing standard, each of said receptors having a different selectivity or specificity for a ligand in the ligand-containing standard, the plurality of signals comprising a standard-fingerprint for the ligand-containing standard,

generating a plurality of signals for a ligand-containing sample by contacting an array of recombinant cells expressing an array of exogenous mutated cell surface receptors with a ligand-containing sample, each of said receptors having a different selectivity or specificity for a ligand in the ligand-containing sample, the plurality of signals comprising a sample-fingerprint for the ligand-containing sample, wherein the array of recombinant cells expressing an array of exogenous mutated cell surface receptors contacted by the ligand-containing sample is identical to the array of recombinant cells expressing an array of exogenous mutated cell surface receptors contacted by the ligand-containing standard, and

comparing the sample-fingerprint with the standard-fingerprint to determine whether the ligand-containing sample is authentic.

27. A computer-implemented method for determining the identity of a product, comprising:

receiving standard-fingerprint data produced by generating a plurality of signals for a ligand-containing standard by contacting an array of recombinant cells expressing an array of exogenous mutated cell surface receptors with a ligand-containing standard, each of said receptors having a different selectivity or specificity for a ligand in the ligand-containing standard, the plurality of signals comprising a standard-fingerprint for the ligand-containing standard;

receiving sample-fingerprint data produced by generating a plurality of signals for a ligand-containing sample by contacting an array of recombinant cells expressing an array of exogenous mutated cell surface receptors with a ligand-containing sample, each of said receptors having a different selectivity or specificity for a ligand in the ligand-containing sample, the plurality of signals comprising a sample-fingerprint for the ligand-containing sample, wherein the sample-fingerprint data is generated using the same array of recombinant cells expressing an array of exogenous mutated cell surface receptors contacted by the ligand-containing standard to generate the standard-fingerprint data;

comparing sample-fingerprint data from the ligand-containing sample to standard-fingerprint data from the ligand-containing standard, and wherein identity requires the sample-fingerprint data from the ligand-containing sample to be within a pre-selected confidence limit defining a range of values calculated from the standard-fingerprint data.