Methods for Identifying Modulators of Ion Channels

• US 20090137422A1
• Filed: 12/15/2008
• Published: 05/28/2009
• Est. Priority Date: 07/11/2007
• Status: Active Grant

• Current Assignee: X-Body Incorporated (Bristol-Myers Squibb Company)
• Original Assignee: Sru Biosystems Incorporated
• Inventors: Yuzakov, Alexander, Laing, Lance G., Wagner, Rick, Fernandez, Rafael
• Granted Patent: US 9,134,307 B2
• Time in Patent Office: Days
• US Class Current: 506/10
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 21/78 producing a change of colour
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

The invention provides methods for identifying modulators of ion channels without the use of recombinant cell lines over-expressing the ion channel proteins or the use of detection labels.

Claims

1. A method of determining ion channel modulating properties of a test reagent comprising:

(a) applying cells to a surface of a calorimetric resonant reflectance optical biosensor;

(b) substantially blocking the functional activity of one or more first types of ion channels, wherein the activity of one or more second types of ion channels is not blocked;

(c) detecting a calorimetric resonant reflectance optical first peak wavelength value

(PWV) for the cells;

(d) applying a test reagent to the cells;

(e) detecting a calorimetric resonant reflectance optical second PWV for the cells; and

(f) determining ion channel modulation properties of the test reagent based on a comparison of the first PWV and second PWV.

2. The method of claim 1, wherein the functional activity of one or more types of ion channels is substantially blocked by an antibody, protein, or small organic molecule.

3. The method of claim 1, wherein the one or more first type of ion channels are voltage-gated sodium channels, voltage-gated calcium channels, mechano-sensitive channels, potassium channels, inward-rectifier potassium channels, calcium-activated potassium channels, voltage-gated potassium channels, two pore domain potassium channels, transient receptor potential channels, cation channels of sperm, cyclic nucleotide gated channels, hyperpolaraization activated cyclic nucleotide gated channels, two pore channels, ligand gated channels, and light-gated channels.

4. The method of claim 1, wherein the one or more second type of ion channels are voltage-gated sodium channels, voltage-gated calcium channels, mechano-sensitive channels, potassium channels, inward-rectifier potassium channels, calcium-activated potassium channels, voltage-gated potassium channels, two pore domain potassium channels, transient receptor potential channels, cation channels of sperm, cyclic nucleotide gated channels, hyperpolaraization activated cyclic nucleotide gated channels, two pore channels, ligand gated channels, and light-gated channels.

5. The method of claim 1, wherein the cells are incubated for a period of time after:

(a) their application to the surface;

(b) the blocking the functional activity of one or more first types of ion channels;

(c) after the detecting the first PWV for the cells;

(d) after the application of the test reagent to the cells;

(e) after the detecting the first PWV for the cells, or (f) a combination thereof.

6. The method of claim 1, wherein the surface of the calorimetric resonant reflectance optical biosensor is an internal surface of a vessel selected from the group consisting of a microtiter well, microtiter plate, test tube, Petri dish, microfluidic channel, and microarray.

7. The method of claim 1, wherein the first and second PWVs are detected using a scanner with a lens having a lower limit pixel size of about 2 micrometers to about 200 micrometers.

8. The method of claim 1, wherein the cells and test reagent do not comprise detection labels.

9. The method of claim 1, wherein the method is performed at a temperature of about 2, 10, 15, 25, 30, or 37 degrees Celsius.

10. The method of claim 1, further comprising the steps of:

(g) washing the cells;

(h) equilibrating the cells;

(i) optionally repeating steps (a)-(f).

11. A method of identifying a modulator of an ion channel comprising:

(a) applying cells in a serum-free medium to a surface of a calorimetric resonant reflectance optical biosensor, wherein one or more extracellular matrix (ECM) ligands are immobilized to the surface of the biosensor;

(b) detecting a calorimetric resonant reflectance optical first peak wavelength value (PWV) for the cells;

(c) applying one or more test ion channel modulators to the surface of the biosensor;

(d) effecting a change in the activity of one or more ion channels of the cells;

(e) detecting a calorimetric resonant reflectance optical second PWV for the cells; and

(f) determining if the one or more test ion channel modulators modulated the one or more ion channels of the cells.

12. The method of claim 11, further comprising the steps of:

(g) washing the cells;

(h) equilibrating the cells;

(i) optionally repeating steps (a)-(f).

13. A method of identifying a modulator of an ion channel comprising:

(a) applying cells in a serum-free medium to a surface of a calorimetric resonant reflectance optical biosensor, wherein one or more extracellular matrix (ECM) ligands are immobilized to the surface of the biosensor;

(b) applying one or more test ion channel modulators to the surface of the biosensor;

(c) effecting a change in one or more ion channels of the cells;

(d) detecting a calorimetric resonant reflectance optical first PWV for the cells;

(e) effecting a change in one or more ion channels of the cells;

(f) detecting a calorimetric resonant reflectance optical second PWV for the cells; and

(g) determining if the one or more test ion channel modulators modulated the one or more ion channels of the cells.

14. The method of claim 13, further comprising the steps of:

(h) washing the cells;

(j) equilibrating the cells;

(k) optionally repeating steps (a)-(g).