Assays and Cell-Based Tests Using a Receptor Na/K-ATPase/Src Complex and Uses Thereof

1 . A method of identifying a compound that alters Src activity, the method comprising:

i) purifying α1 Na/K-ATPase to obtain Na/K-ATPase exhibiting specific activity higher than 800 μmol Pi/mg protein/h;

ii) mixing the purified Na/K-ATPase of step (i) with Src to form a receptor Na/K-ATPase/Src complex having inhibited Src activity;

iii) exposing the receptor Na/K-ATPase/Src complex of step (ii) to a compound, wherein binding of the compound releases the inhibited Src from the receptor Na/K-ATPase/Src complex, resulting in an increase in Src activity; and

iv) measuring the increase in Src activity, wherein the increased Src activity is indicative of the compound altering Src activity.

2 . The method of claim 1 , wherein the step of measuring comprises determining whether there is an enhanced level of Src activity as compared to a control level of Src activity,

wherein an increase in the level of Src activity indicates that the compound is an agonist, and,

wherein a decrease in the level of Src activity relative to the control level indicates that the compound is an antagonist.

3 . The method of claim 2 , wherein the agonist compound of receptor Na/K-ATPase/Src complex comprises at least one of:

Agonist Compound Name

Formula

Ouabain

C 29 H 44 O 12

Digoxin

C 41 H 64 O 14

Marinobufagenin (MBG)

C 24 H 32 O 5

Oleic acid

C 18 H 34 O 2

Docosahexaenoic (DHA)

C 22 H 32 O 2

Glutathione disulfide (GSSG)

C 20 H 32 N 6 O 12 S 2

Allyl isothiocyanate

C 4 H 5 NS.

4 . The method of claim 3 , wherein the agonist of receptor Na/K-ATPase/Src complex comprises at least two of: ouabain, digoxin, marinobufagenin (MBG), oleic acid, docosahexaenoic (DHA), glutathione disulfide (GSSG), and allyl isothiocyanate.

5 . The method of claim 2 , wherein the antagonist compound of receptor Na/K-ATPase/Src complex comprises at least one of:

Antagonist Compound Name

Formula

3,4,5-Trihydroxyxanthone (MB5)

C 13 H 8 O 5

3,4,5,6-Tetrahydroxyxanthone (MB7)

C 13 H 8 O 6

Curcumin

C 21 H 20 O 6

Bisdemethoxycurcumin

C 19 H 16 O 4

Tanshinone I

C 18 H 19 O 3

Sodium danshensu

C 9 H 9 O 5 Na

Astragaloside IV

C 41 H 68 O 14

Ferulic acid

C 10 H 10 O 4

Tanshinone IIA

C 19 H 18 O 3 .

6 . The method of claim 5 , wherein the antagonist of receptor Na/K-ATPase/Src complex comprises two or more of: 3,4,5-trihydroxyxanthone (MB5), 3,4,5,6-tetrahydroxyxanthone (MB7), curcumin, bisdemethoxycurcumin, tanshinone I, sodium danshensu, astragaloside IV, ferulic acid and tanshinone IIA.

7 . The method of claim 2 , wherein the antagonist of receptor Na/K-ATPase/Src complex, comprises a combination of: tanshinone I, sodium danshensu, astragaloside IV and ferulic acid.

8 . The method of claim 2 , wherein the agonist of the receptor Na/K-ATPase/Src complex comprises cardiotonic steroids.

9 . The method of claim 2 , wherein the agonist and/or antagonists of receptor Na/K-ATPase/Src complex is found in one or more of: tan seng, red sage root, radix salvia miltiorrhizae, dang gui, angelica sinensis, huang qi and astragalus.

10 . The method of claim 1 , wherein the compound is for the treatment of a disorder associated with one or more of: cardiac hypertrophy, tissue fibrosis, congestive heart failure, cancer, wound or skin lesion.

11 . The method of claim 1 , wherein the compound comprises a mixture of compounds.

12 . The method of claim 11 , wherein the cell based assay comprises a LLC-PK1 derived α1 knockdown PY-17 cell, a first control cell comprised of P11, and a second control cell comprised of AAC-19.

13 . The method of claim 12 , wherein the first control cell P11 comprises LLC-PK1transfected with empty vector, and wherein the second control cell AAC-19 comprises rat α1-rescued PY-17 cells.

14 . The method of claim 11 , wherein the cell based assay comprises a pair of cell lines, LL-A416P-4 and LL-A420P-20, wherein mutation of A420 to P results in inability of expressed Na/K-ATPase to bind and form a functional receptor complex as activated Src in A416P-rescued cells but not A420P mutant-rescued cells.

15 . The method of claim 11 , wherein the cell based assay comprises cell lines (LY-I279A-3, LY-F286A-19), wherein expressed I279A or F286A mutant Na/K-ATPase is defective in conformational transition.

16 . The method of claim 15 , wherein I279A and F286A mutants are defective in E1 to E2 and E2 to E1 conformational transition, respectively.

17 . An herbal medicinal preparation, comprising effective amounts one or more raw medicinal materials selected from: tan seng, red sage root, radix salvia miltiorrhizae, dang gui, angelica sinensis, huang qi and astragalus,

wherein an agonist or antagonist of receptor Na/K-ATPase/Src complex found in each of the raw medicinal materials is present in an effective amount ranging from about 0.1 nM to about 10 nM.

18 . The preparation of claim 17 , wherein the agonist comprises one or more of: ouabain, digoxin, marinobufagenin (MBG), oleic acid, docosahexaenoic (DHA), glutathione disulfide (GSSG) and allyl isothiocyanate.

19 . The preparation of claim 17 , wherein the antagonist comprises one or more of: 3,4,5-trihydroxyxanthone (MB5), 3,4,5,6-tetrahydroxyxanthone (MB7), curcumin, bisdemethoxycurcumin, tanshinone I, sodium danshensu, astragaloside IV, ferulic acid and tanshinone IIA.

20 . The preparation of claim 17 , wherein the antagonist comprises one or more of: tanshinone I, sodium danshensu, astragaloside IV and ferulic acid.

21 . An herbal medicinal preparation for treatment of a disorder associated with one or more of cardiac hypertrophy, tissue fibrosis, congestive heart failure, cancers, wound or skin lesion, comprising the herbal medicinal preparation of claim 17 .

22 . The herbal medicinal preparation of claim 21 , wherein the herbal composition is formulated for oral administration.