WO2000044931A2

WO2000044931A2 - High-throughput screening assays for modulators of atpase

Info

Publication number: WO2000044931A2
Application number: PCT/US2000/002241
Authority: WO
Inventors: Sheila G. Magil; Jason N. Labutti
Original assignee: Bion, Inc.
Priority date: 1999-01-28
Filing date: 2000-01-28
Publication date: 2000-08-03
Also published as: AU2743800A; WO2000044931A3

Abstract

The present invention relates to rapid, specific, high through-put assays for screening test substances for their ability to modulate activity of an ATPase involved in a variety of biological processes such as regulation of osmotic balance and cell volume, maintenance of the resting membrane potential, establishment of the ionic composition of cerebrospinal fluid and aqueous humor, electrical activity of muscle and nerve, and receptor-mediated endocytosis. The assays can be used to identify compounds for use in therapeutic applications to disease processes in which dysfunction of the ATPase contributes to a pathological process. The present invention also provides kits that can be used in the assays.

Description

HIGH-THROUGHPUT SCREENING ASSAYS FOR MODULATORS OF Na⁺-K⁺ATPase

1. FIELD OF THE INVENTION

The present invention relates to screening assays and kits, and methods of employing them for the detection of modulators of ATPases, particularly Na⁺-K⁺- ATPases. Further, the present invention relates to rapid, high- throughput assays to screen for agents that modulate ATPase activity and that may have utility in treating diseases and/or disorders associated with ATPase activity, particularly aberrant ATPase activity.

2. BACKGROUND OF THE INVENTION The main cellular source of energy for all cell processes is ATP. Adenosine triphosphatases (ATPases) are enzymes which catalyze the hydrolysis of adenosine triphosphate (ATP) while releasing energy and one or more inorganic phosphate groups. ATPases are expressed by viruses, prokaryotes and eukaryotes. Many viruses and bacteria including the HCV virus contain a viral or bacterial specific ATPase which is required to catalyze the hydrolysis of an ATP. Thus, the discovery of anti-viral and anti-bacterial substances which would inhibit these ATPases provides new methods of treating and preventing viral and bacterial infections.

Eukaryotic ATPases include Ca²⁺- ATPase, H⁺-K⁺-ATPase, and Na"-K⁺- ATPase, which provide ion gradients that are essential for maintaining homeostasis. For example, Ca^2"- ATPase regulates the concentration of Ca²⁺ in muscle and H⁺-K⁺-

ATPase regulates the acid concentration in the stomach. Numerous diseases and/or disorders have been associated with aberrant ATPase activity including, but not limited to, ischemic injury, heart disease, stroke, Wilson disease, Menkes syndrome, hyponatremia and hypertension. Thus, the discovery of ATPase modulators (i.e., inhibitors and activators) would provide new methods for the prevention and treatment of diseases and/or disorders resulting from aberrant regulation of ATPase activity (i.e., ischemic injury).

2.1. Na⁺-K⁺-ATPase Na⁺-K⁺-ATPase is a plasma membrane-associated enzyme which is encoded by a multigene family. Activity of the Na⁺-K⁺- ATPase provides gradients of Na⁺ and K7 that are essential for maintaining cellular homeostasis. (Levenson, Rev. Physiol. Biochem. Pharmacol, 123:1-45. 1994). The ion gradients established by the Na⁺-K^τ- ATPase play a central role in regulating osmotic balance, cell volume, and maintaining the resting membrane potential. Na⁺-coupled transport of nutrients, establishment of the ionic composition of cerebrospinal fluid and aqueous humor, electrical activity of muscle and nerve, and receptor-mediated endocytosis are all processes which depend on the activity of the enzyme.

The Na⁺-K⁺- ATPase consists of an and a β subunit present in equimolar amounts. There are four known isoforms of the subunit, l, 2, α3 and 4 (Shull et al, Biochemistry 25:8125-8132, 1986; Herral et al., J. Cell Biol 105:1055-1065, 1987; Takeyasu et al., Am. J. Physiol. 259:C619-C630 1990; and Blanco et al, Am. J. Physiol, 275:F633-F650, 1998) and two isoforms of β subunit, βl and β2 (Mercer et al., Mol. Cell Biol. 6:3884-3890, 1986; Young et al., J. Biol. Chem. 262:4905-4910. 1987; Martin-Vasallo et al., J. Biol. Chem. 264:4613-4618, 1989; Gloor et al., J. Cell Biol. 110:165-174, 1990; and Lemas et al, Kaplan and De Peer (eds.) The Sodium Pump: Recent Developments, Rockefeller University Press, New York, ppl 17-123, 1991). In kidney tissue another subunit, the γ subunit, has been identified (Blanco et al., Am. J. Physiol, 275:F633-F650, 1998). Several lines of evidence suggest that each of the four α subunit isoforms may be capable of association with each of the two β subunit isoforms to form a functional holoenzyme (Levenson, Rev. Physiol. Biochem. Pharmacol, 123: 1-45. 1994). Each α and β subunit isoform is encoded by a separate gene mapped to discrete chromosomal locations in both mouse (Kent et al., Proc. Natl. Acad. Sci. USA 84:5369-5372, 1987; and Malo et al, Genomics 6:697-699, 1990) and human (Yang-Feng et al., Genomics 8:128-138, 1988) genomes.

A number of Na⁺-K⁺- ATPase inhibitors are known in the art. Digitalis, digoxin, digitoxin, deslanoside, ouabain and related substances are cardiac glycosides derived from plants. It is known that these substances are capable of inhibiting the activity of Na⁺-K⁺- ATPase (U.S. Patent Nos. 5,716,937 and 5,153,178; Hoffman and Bigger, The Pharmacological Basis of the Therapeutics, eds. Goodman and Gilman, p. 732, (1980)).

A physiological Na⁺-K⁺- ATPase regulator has been isolated from bovine hypothalamus and has been named hypothalamic inhibitory factor (HIF) (Haupert, Jr. et al., Am. J. Physiol, 247:F919, 1984). Several other ouabain-like compounds have been reported in mammalian tissues.

U.S. Patent No. 5,240,714 discloses a substantially pure non-digoxin-like Na⁺- K⁺- ATPase inhibitory factor wherein said factor: 1) has a molecular weight of less than 1000 daltons; 2) does not substantially cross-react with anti-digoxin antibody; 3) exhibits maximal UN absorbance at approximately 202-210 nm and at approximately 274-280 nm; 4) is non-peptidic as determined by its resistance to acid hydrolysis; 5) is non-lipidic as determined by its resistance to incubation with BSA or phospholipase; and 6) loses substantial activity following alkaline hydrolysis.

U.S. Patent No. 5,667,811 discloses a substantially pure isolated low molecular weight plasma inhibitor of Na⁺-K⁺- ATPase occurring in human plasma wherein said inhibitor: 1) has vasoconstrictive and natriuretic activity; 2) displaces ouabain from its receptor; 3) lacks reactivity with anti-digoxin antibody; and 4) has a molecular weight of less than 500 daltons.

Beltowski et al., J. Physiol. Pharmacol, 49{2):271-283, 1998 discloses that infusion of atrial natriuretic factor (ANF) caused dose-dependent inhibition of medullary Na⁺-K⁺- ATPase activity without affecting cortical Na⁺-K⁺- ATPase. Beltowski et al. also discloses that this inhibition was mimicked by synthetic analogue of cyclic guanosine 3',5' monophosphate (e.g., 8-bromo-cGMP), inhibitors of phosphodiesterase (e.g., papaverine and IBMX) and the activator of soluble guanylate cyclase (e.g., sodium nitroprusside).

Bhattacharyya and Sen, Eur. J. Biochem., 2440): 829-834, 1997 discloses purification and characterization of a number of low-molecular mass (12-13 kDa) Na⁺- Y - ATPase inhibitor proteins from rat brain cytosol by gel filtration followed by FPLC fractionation on a Mono Q anion-exchange column.

Budzikowski et al., Clin. Exp. Hypertens., 20(2 : 119-140. 1998 discloses the existence of "brain ouabain", an endogenous inhibitor of brain Na⁺-K⁺- ATPase, in hypothalamic and medullary neurons.

Calderaro et al., Life Sc , 6JXI5): 1457-1468, 1997 discloses that one fraction (FI) isolated from pre- and post-dialysis sera from chronic dialysis patients displays significant effects on electrophysiological and transepithelial 22Na flux pattern of rabbit distal colon mucosa mounted in Ussing type chambers. Calderaro et al., also discloses that incubation of highly purified basolateral membranes with FI produced a approximately 26% inhibition of Na^{^}-K⁺- ATPase.

Foley, Biochem. Biophys. Res. Commun., 235(21:374-376. 1997 discloses that arachidonic acid hydroperoxide product of 5-lipoxygenase (5-HPETE) is a potent inhibitor of neuronal Na⁺-K⁺- ATPase activity.

Han et al, Chung Kuo Chung Yao Tsa Chih, 21(51:299-302. 1996 shows that gypenoside (Gyp) inhibits the enzyme activity of the microsomal Na⁺-K⁺- ATPase from rat hearts and brains rapidly and reversibly in vitro.

Inada et al., Diabetologia, 4JX12): 1451-1458, 1998 discloses that a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), inhibited Na⁺-K⁺- ATPase activity at 5 mmol/1 glucose.

Matsukawa et al., Chem. Pharm. Bull. (Tokyo), 45(21:249-254. 1997 identified a novel cardiac steroid, l l,19-epoxy-19-methoxytelocinobufagin, named marinosin (Compound 1), in the skin of the toad, Bufo marinus (L.) Schneider. The treatment of compound 1 with 50% CH₃CN containing 0.1 % trifluoroacetic acid yielded a l l alpha-hydroxyhellebrigenin (Compound 2). Both compounds 1 and 2 inhibit Na⁺-K⁺- ATPase enzymatic activity and the binding of [³H]ouabain to Na⁺-K^{^}- ATPase.

Pena et al., Neurochem. Res., 22(41:379-383. 1997 discloses that a cerebral cortex soluble fraction (II-E) is an endogenous brain Na⁺-K⁺- ATPase inhibitor which is structurally different from ouabain.

Tao et al., Hypertension 29:815-821, 1996 discloses that a labile, specific Na⁺- K⁺- ATPase inhibitor was isolated from the peritoneal dialysate of volume-expanded renal failure patients. This inhibitor, unlike ouabain, inhibits all three isoforms of rat Na⁺-K⁺- ATPase, including the ouabain-resistant rat αl Na⁺-K⁺- ATPase.

Younes-Ibrahim et al., Acad. Sci. Paris, Ser. Ill, 318:619-625, 1995 showed that a glycolipoprotein fraction prepared from Leptospira interrogans inhibited Na⁺- K^"- ATPase purified from brain or kidney. Burth et al., Infect. Immun., 65(41:1557-1560. 1997 further demonstrated that unsaturated fatty acids such as oleic and palmitoleic acids, which are adsorbed to the glycolipoprotein fraction, are effective inhibitors of Na⁺-K⁺- ATPase.

The sensitivity of the Na⁺-K^τ- ATPase to ouabain and other cardiac glycosides lies within α subunit of the enzyme. To date, all α2 and 3 isoforms and αl isoform from most mammals and other organisms tested have been shown to be sensitive to ouabain, i.e., can be inhibited at a relatively low ouabain concentrations. However, αl isoforms, isolated from rats and other rodents, certain species of amphibians, such as the toad Bufo marinus, and certain species of butterflies, such as Monarch (Danaus plexippus), are resistant to ouabain (Lingrel et al, The Sodium Pump (Bamberg and Schoner, Ed.) pp276-286, 1994; Holzinger et al., FERS, 314:477-480, 1992; and Jaisser et al, J. Biol. Chem., 267:16895-16903, 1992). In order to inhibit these ouabain-resistant Na⁺-K⁺- ATPases, 100 fold or higher concentration of ouabain than is capable of inhibiting ouabain sensitive Na⁺-K⁺- ATPase is needed. In addition, a number of mutations have been found to be able to convert an ouabain-sensitive Na⁺-

K -ATPase into an ouabain-resistant one (Lingrel et al., The Sodium Pump (Bamberg and Schoner, Εd.) pp276-286, 1994). Most of these mutations are located within the extracellular domains or at the borders of the extracellular domains. In particular, it has been found that changing one or both border amino acid residues of the H1-H2 extracellular domain of α subunit of an ouabain-sensitive Na^{^}-K^"1"- ATPase to a charged amino acid residue, either a positively or a negatively charged residue, always confers ouabain resistance (Price et al., J. Biol. Chem., 265:6638-6641, 1990). The investigation surrounding the various Na⁺-K⁺- ATPase inhibitors alerted the skilled artisan to the potential utility for a potent, non-toxic cardiac glycoside. The art is in need of techniques to facilitate this search. To the Applicants' knowledge, a rapid screening assay which is highly specific, quantifiable and amenable to testing a large number of test substances in either aqueous or non-aqueous solutions for modulators of the ATPase, particularly Na⁺-K⁺- ATPase, has not been developed. The present invention addresses this and other needs in the art.

2.2. ATPase Assays

A variety of ATPase assays have been described in the literature for detecting ATPase activity. Traditionally, ATPase assays used radioactive isotopes to measure the generation of free phosphate. These assays require the separation of the ATP and the resultant ADP and free phosphate, which is time consuming. Further, radiolabeled ATP poses a health risk to the individual performing the assay.

ATPase activity has been detected by colorimetric assays (see, e.g., Chan, KM et al., 1986, Anal Biochem 157(21:375-801. fluorescent assays, and chemiluminescent assays (see, e.g., U.S. Patent No. 5,759,795). These assays are indirect methods for detecting the ATPase activity. The colorimetric, fluorescent and chemiluminescent assays described in the literature require additional enzymes to convert products of the ATP hydrolysis (i.e., ADP or free phosphate) into other products which are then quantitated by a spectrophotometer, spectrofluorimeter or luminometer, respectively.

Thus, there is a need for efficient high-throughput assays to screen for inhibitors ATPases which do not require additional enzymes or the separation of the substrate and resultant products.

3. SUMMARY OF THE INVENTION

The invention provides a rapid method to measure activity of ATP hydrolyzing enzymes. Further, the invention provides rapid assays for the detection and identification of agents that modulate ATPase activity. In particular, the present invention encompasses rapid, high-throughput assays for screening test substances such as drugs, ligands (natural or synthetic), ligand antagonists, polypeptides, peptides, polysaccharides, saccharides, small organic molecules and the like, for their ability to modulate activity of an ATPase. The rapid, high-throughput screening assays are particularly useful for the detection and identification of modulators of Na⁺-K⁺- ATPases.

The invention is based, in part, on the Applicants' design of sensitive, rapid, homogenous assay systems that permit detection of modulators of ATPases in samples, including but not limited to biological samples. The homogenous assay systems of the invention utilize robust detection systems that do not require separation steps for detection of ATPase activity. The invention provides rapid methods for the detection of ATPase activity, which makes the screening of modulators of ATPases particularly amenable to high through-put formats.

In one embodiment of the present invention, a test substance is screened for its ability to modulate (i.e., inhibit or activate) the activity of an ATPase in an assay, comprising: (a) contacting the ATPase with a test substance in the presence of ATP or an analog thereof for a time sufficient to measure ATPase activity; (b) contacting the ATPase with ATP or an analog thereof for a time sufficient to measure ATPase activity; (c) measuring the ATPase activity in steps (a) and (b); and (d) comparing the activity measured in steps (a) and (b) to determine the ability of the test compound to modulate (e.g., inhibit or activate) the activity of the ATPase. In accordance with this embodiment, a difference in the level of ATPase activity in step (a) from the level of ATPase activity in step (b) indicates that the test substance modulates the activity the ATPase.

In another embodiment of the present invention, a test substance is screened for its ability to modulate (e.g., inhibit or activate) the activity of an ATPase in an assay, comprising: (a) contacting the ATPase with a test substance in the presence of ATP or analog thereof for a time sufficient to measure ATPase activity; (b) contacting the ATPase with a control substance (e.g., ouabain) in the presence of ATP or analog thereof for a time sufficient to measure ATPase activity; (c) contacting the ATPase with ATP or an analog thereof for a time sufficient to measure ATPase activity; (d) measuring the ATPase activity in steps (a), (b) and (c); and (e) comparing the ATPase activity measured in step (a) with the activity measured in steps (b) and (c) to determine the ability of the test compound to modulate the activity of the ATPase. In accordance with this embodiment, a difference in the level of ATPase activity in step (a) from the levels of ATPase activity in steps (b) and/or (c) indicates that the test substance modulates the activity the ATPase.

In a preferred embodiment, the analog of ATP in the assays of the present invention is a fluorogenic or chemiluminogenic ATP analog, and the ATPase activity is measured by the generation of a fluorescent or chemiluminescent product, respectively. In another preferred embodiment, the assays of the present invention are incorporated into high-throughput assays to screen and identify modulators of ATPase activity.

The invention also encompasses kits for detecting modulators of ATPase activity. The invention further encompasses the novel agents identified by the screening assays described herein or known agents for which ATPase activity was not known. The invention also relates to therapeutic modalities and pharmaceutical compositions for the treatment of diseases and/or disorders associated with ATPase activity, including, but not limited to stroke, cardiovascular diseases, Wilson disease, Menkes syndrome, kidney disorders, hypotension, hypertension, ischemic injury, neurological disorders, muscle disorders, pulmonary disorders, hepatic disorders, and microbial infections.

3.1. Definitions

The term "agonist" as used herein refers to a compound that increases or enhances the activity of an ATPase as determined by assays known to those skilled in the art or described herein.

The term "antagonist" as used herein refers to compound that decreases or inhibits the activity of an ATPase as determined by assays known to those skilled in the art or described herein.

The terms "analog of ATP" or "ATP analog" as used herein refer to any substance or compound that has the ability to undergo hydrolysis by an ATPase as determined by assays known to those skilled in the art or described herein. In particular, analogs of ATP have an accessible phosphate moiety which the ATPase hydrolyzes. In a preferred embodiment, an analog of ATP is a chemiluminogenic or fluorogenic analog of ATP. The hydrolysis of a chemiluminogenic analog of ATP or a fluorogenic analog of ATP results in the generation of a product which is chemiluminescent or fluorescent, respectively. It is the chemiluminescent or fluorescent product generated by hydrolysis of the chemiluminogenic or fluorogenic analog of ATP that is detected in assays of the present invention.

The term "baseline ATPase activity" as used herein refers to the enzymatic activity of ATPase in the presence of ATP or an analog thereof as measured/quantitated in an assay of the invention.

The term "biological sample" as used herein refers to samples obtained from any biological organism (e.g., samples obtained from an animal, a virus, a bacteria, a fungus, a parasite or a unicellular organism). Biological samples may be obtained from a mammal, preferably a human and most preferably a human with a disease or disorder, and include bodily fluids, blood, serum, mucous, including oral, rectal or intestinal mucosa, urine, feces, etc. Biological samples may include tissue samples, biopsy tissue, cell samples, including muscle cells, bone marrow cells, lymphocytes, immune cells, mucosal cells obtained from oral, rectal or intestinal mucosal linings, etc. Biological samples or mixtures may encompass cell lysates or portions thereof, carbohydrates including lectins; proteins including glycoproteins, cell surface receptors, and peptides; nucleic acids including DNA or RNA, etc.

The term "control substance" as used herein refers to a substance which is capable of increasing or decreasing the activity of an ATPase. Preferably, a control substance that increases and decreases the activity of an ATPase is included in an assay of the present invention. Control substances include, but are not limited to, drugs, ligands (natural or synthetic), ligand antagonists, polypeptides, peptides, peptide mimics, polysaccharides, saccharides, glycoproteins, nucleic acids, DNA and RNA strands oligonucleotides and small organic molecules that inhibit, decrease, activate or increase the activity of an ATPase.

The term "isolated or "purified" as used herein refers to an ATPase or biologically active portion thereof that is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is extracted, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.

The term "modulate" as used herein in reference to ATPase activity refers to the inhibition or activation of an ATPase activity.

The term "modulator" as used herein refers to a substance, compound or agent which inhibits, decreases, activates or increases the activity of an ATPase. Modulators include, but are not limited to, drugs, ligands (natural or synthetic), ligand antagonists, polypeptides, peptides, peptide mimics, polysaccharides, saccharides, glycoproteins, nucleic acids, DNA and RNA strands oligonucleotides and small organic molecules that inhibit, decrease, activate or increase the activity of an ATPase.

The term "test substance" as used herein refers to a chemically defined compound, agent, or mixture of compounds whose effect on an ATPase is determined in an assay of the present invention. Test substances include, but are not limited to, drugs, ligands (natural or synthetic), ligand antagonists, polypeptides, peptides, peptide mimics, polysaccharides, saccharides, glycoproteins, nucleic acids, DNA and RNA strands oligonucleotides and small organic molecules. In one embodiment, test substances comprise an existing library of compounds or agents. In another embodiment, test substances comprise a novel library of compounds or agents. In another embodiment, test substances used in assays of the present invention are purified, partially purified, or unpurified (i.e., not purified from other components).

The term "time sufficient to measure ATPase activity" as used herein refers to the time needed to generate a detectable amount of product. The time needed to generate a detectable amount of product will vary depending on the assay system. A sensitive assay system will require less time to generate a detectable amount of product than a less sensitive assay system. For example, a fluorescent or chemiluminescent assay system will generally require less time to generate a detectable amount of product than a colorimetric assay system. One of skill in the art will know the amount of time sufficient to measure ATPase activity based upon the assay system.

In one embodiment, the time needed to generate a detectable amount of product in a colorimetric assay is between 5 minutes and 3 hours. In another embodiment, the time needed to generate a detectable amount in a colorimetric assay is at least 1 minute,

5 minutes, 15 minutes, 30 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes,

135 minutes, 150 minutes, 175 minutes or 180 minutes. In another embodiment, the time needed to generate a detectable amount of product in a fluorescent assay is between 1 second and 480 minutes. In another embodiment, the time needed to generate a detectable amount in a fluorescent assay is at least 1 millisecond, 15 milliseconds, 30 milliseconds, 45 milliseconds, 1 second, 15 seconds, 30 seconds, 1 minute, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes, 135 minutes, 150 minutes, 175 minutes, 180 minutes, 195 minutes, 210 minutes, 225 minutes, 240 minutes, 255 minutes, 270 minutes, 285 minutes, 300 minutes, 315 minutes, 330 minutes, 445 minutes, 360 minutes, 375 minutes, 400 minutes, 415 minutes, 430 minutes, 445 minutes, 460 minutes, 475 minutes or 480 minutes. In another embodiment, the time needed to generate a detectable amount of . product in a chemiluminescent assay is between 1 second and 480 minutes. In yet another embodiment, the time needed to generate a detectable amount in a chemiluminescent assay is at least Imillisecond, 15 milliseconds, 30 milliseconds, 45 milliseconds, 1 second, 15 seconds, 30 seconds, 1 minute, 5 minutes, 15 minutes, 30 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes, 135 minutes, 150 minutes,

175 minutes, 180 minutes, 195 minutes, 210 minutes, 225 minutes, 240 minutes, 255 minutes, 270 minutes, 285 minutes, 300 minutes, 315 minutes, 330 minutes, 445 minutes, 360 minutes, 375 minutes, 400 minutes, 415 minutes, 430 minutes, 445 minutes, 460 minutes, 475 minutes or 480 minutes.

4. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows inhibition of the dog kidney Na⁺-K⁺- ATPase by various control substances.

Figure 2 shows inhibition of the rat fetal brain Na⁺-K⁺- ATPase by various control substances.

Figure 3 shows inhibition of the rat kidney Na⁺-K⁺- ATPase by various control substances. Figure 4 depicts a timecourse of methylumbelliferyl phosphate hydrolysis by dog kidney Na⁺-K⁺- ATPase.

Figure 5 depicts a timecourse of the inhibition of dog kidney Na⁺-K⁺- ATPase.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses rapid, specific, high-throughput assays for screening test substances or libraries of test substances such as drugs, ligands (natural or synthetic), ligand antagonists, polypeptides, peptides, saccharides, polysaccharides, small organic molecules and the like, for their ability to modulate the activity of an ATPase. In particular, the present invention encompasses rapid, high-throughput screening assays for identifying modulators of ouabain-sensitive and ouabain-resistant Na⁺-K^T-ATPases.

In one embodiment of the present invention, the ability of a test substance to modulate ATPase activity is determined in an assay, comprising: (a) contacting a test substance dissolved in an aqueous or non-aqueous solution under buffered conditions with the ATPase in the presence of ATP or an analog thereof for a time sufficient to measure the activity of the ATPase; (b) contacting the ATPase with ATP or an analog thereof for a time sufficient to measure the activity of the ATPase; (c) measuring the ATPase activity in steps (a) and (b); and (d) comparing the activity measured in steps (a) and (b) to determine the ability of the test compound to modulate (e.g., inhibit or activate) the activity of the ATPases. In accordance with this embodiment, a difference in the level of ATPase activity in step (a) from the level of ATPase activity in step (b) indicates that the test substance modulates the activity the ATPase. An increase in the level of ATPase activity in step (a) compared to the level of ATPase activity in step (b) indicates that the test substance is an agonist of the ATPase. A decrease in the level of ATPase activity in step (a) compared to the level of ATPase activity in step (b) indicates that the test substance is an antagonist of the ATPase. The ATPase activity in steps (a) and (b) can measured separately. Preferably, the ATPase activity in steps (a) and (b) are measured simultaneously.

In another embodiment of the present invention, the ability of a test substance to modulate ATPase activity is determine in an assay, comprising: (a) contacting a test substance dissolved in an aqueous or non-aqueous solution under buffered conditions with the ATPase in the presence of ATP or an analog thereof for a time sufficient to measure ATPase activity; (b) contacting a control substance (e.g. , ouabain) dissolved in an aqueous or non-aqueous solution under buffered conditions with the ATPase in the presence of ATP or an analog thereof for a time sufficient to measure ATPase activity; (c) contacting the ATPase with ATP or an analog thereof for a time sufficient to measure ATPase activity; (d) measuring the ATPase activity in steps (a), (b) and (c); and (e) comparing the ATPase activity measured in step (a) with the activity measured in steps (b) and (c) to determine the ability of the test compound to modulate the activity of the ATPases. In accordance with this embodiment, a difference in the level of ATPase activity in step (a) from the levels of ATPase activity in steps (b) and (c) indicates that the test substance modulates the activity the ATPase. An increase in the level of ATPase activity in step (a) compared to the level of ATPase activity in step (c) indicates that the test substance is an agonist of the ATPase. An increase in the level of ATPase activity in step (a) compared to the level of ATPase activity in steps (b) and (c) when the control substance in step (b) is a known agonist indicates that the test substance is an agonist of the ATPase. A decrease in the level of ATPase activity in step (a) compared to the level of ATPase activity in step (c) indicates that the test substance is an antagonist of the ATPase. A decrease in the level of ATPase activity in step (a) compared to the level of ATPase activity in steps (b) and (c) when the control substance in step (b) is a known antagonist indicates that the test substance is an antagonist of the ATPase. The ATPase activity in steps (a), (b) and (c) can be measured separately. Preferably, the ATPase activity in steps (a) , (b) and (c) are measured simultaneously.

In another embodiment, once it is determined that an ATPase used in an assay of the invention is resistant to a control substance (e.g., ouabain), any inhibition of such a control substance-resistant ATPase by a test substance indicates that the test substance has the ability to inhibit the control substance-resistant ATPase.

In a preferred embodiment, the assays of the present invention are incorporated into high-throughput assays to screen and identify novel compounds that modulate ATPase activity or known compounds previously unknown to modulate ATPase activity. In a specific embodiment, the ATPases added to the assays of the present invention are isolated. In a preferred embodiment, the activity of a Na⁺-K⁺- ATPase is quantified/measured in the assays of the present invention. In another preferred embodiment, more than one test substance is tested simultaneously in an assay of the present invention. In another embodiment, the assays of the invention are conducted in a multi-well plate such as a 96-, 384- or 1536-well plate.

In another embodiment, the enzymatic activity of an ATPase (i.e., the ability of the ATPase to hydrolyze ATP or an analog thereof) in the absence of a control substance and a test substance (i.e., baseline ATPase activity) is determined once per plate. In another embodiment, the enzymatic activity of an ATPase in the absence of a control substance and a test substance (i.e., baseline ATPase activity) is determined once per analytical assay, regardless of the number of multi-well plates used or the number of test substances screened. In yet another embodiment, a control comprising ATPase in absence of ATP is included in the assays of the present invention.

In accordance with the present invention, ATPase activity is detected by an ATP hydrolysis method. Preferably, the ATP hydrolysis is assayed without separating the ATP and the resultant ADP and free phosphate. ATP hydrolysis can be assayed by measuring a decrease in the amount of ATP or by measuring an increase in the amount of the resultant ADP. Alternatively, ATP hydrolysis can be assayed by measuring the production of a fluorescent product or chemiluminescent product which results from the hydrolysis of a fluorogenic substrate or chemiluminogenic substrate, respectively. In a preferred embodiment, ATPase hydrolysis is detected by measuring the generation of a fluorescent product. In another preferred embodiment, ATPase hydrolysis is detected by measuring the generation of a chemiluminescent product.

The present invention also encompasses kits for detecting modulators of ATPase activity. The invention further encompasses methods of identifying modulators of the ATPases, the modulators of the ATPase identified by above screening assays and the use of such modulators in treating diseases and/or disorders associated with ATPase activity, particularly aberrant ATPase activity, including, but not limited to, cardiovascular diseases, kidney disorders, neurological disorders, and hepatic disorders. A substance, compound and/or agent that modulates ATPase activity may function as an agonist or antagonist.

In a preferred embodiment, the invention encompasses the use of endogenous or recombinant cardenolide compounds, which are identified in the assays as decreasing ATPase activity greater than that of ouabain. In accordance with this embodiment, a cardenolide compound can be used in a therapeutic or pharmaceutical composition for the treatment, inhibition or prevention of a disease or disorder associated with ATPase activity. In another preferred embodiment, the invention encompasses the use of 5[l-(4-trifluoromethoxyphenoxy)ethyl]-l,3,4-oxadiazole- 2(3H)-thione, a previously unknown inhibitor of ATPase that was identified in the assays of the present invention. In accordance with this embodiment, 5[l-(4- trifluoromethoxyphenoxy)ethyl]-l,3,4-oxadiazole-2(3H)-thione can be used in a therapeutic or pharmaceutical composition for the treatment, inhibition or prevention of a disease or disorder associated with ATPase activity, particularly aberrant ATPase activity.

For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections which follow. 5.1. ATPases

Naturally occurring and recombinantly expressed ATPases can be used in the assays of the present invention. ATPases can be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. The functional properties can be evaluated using any suitable assay (see Section 5.3.). Alternatively, the protein can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller, M., et al., 1984. Natwe. 310:105-1111.

Techniques known to those of skill in the art can be used to clone an ATPase and express the recombinant ATPase (e.g., see, U.S. Patent No. 5,989,822). Any animal cell potentially can serve as the nucleic acid source for the molecular cloning of an ATPase gene. The nucleic acid sequences encoding ATPase subunits can be isolated from any organism that expresses an ATPase, including but not limited to, viruses, bacteria, vertebrates, mammals, humans, porcines, bovines, felines, avians, equines and canines. Cells from various tissues, including kidney, pineal gland, skeletal muscle, heart, retina and brain, can serve as the nucleic acid source for the molecular cloning of ATPase subunits (Levenson, Rev. Physiol. Biochem. Pharmacol, 123:1-45. 1994). For example, the nucleotide sequence for human αl, α2, α3, βl and β2, which can be used in molecular cloning of Na⁺-K⁺- ATPase subunits from other animal species, have been reported (Sverdlov et al., FEBS Lett., 217:275-278, 1987; Kano et al., FEBS Lett., 250:91098, 1989; Tam et al., Mol. Cell. Biol, 10:6619-623, 1990; Shull et al., J. Biol. Chem., 28:4531-4535, 1989; Ocvhinnikov et al., FEBS Lett., 233:87-94, 1988; Pathak et al., Genomics, 8:641-647, 1990; Lane et al., Genomics, 5:445-453, 1989; Shyjan et al., The sodium pump: Recent developments. Rockefeller University press, New York, pp 131-136, 1991; and Magyar and Schahner, Nucleic Acid Res., 18:6695-6696, 1991). The contents of the above references are incorporated by reference herein.

Cells of certain animal origin, particularly rodent species, such as mouse or rat, amphibians such as toad Bufo marinus, and butterfly species such as Monarch (Danaus plexippus) can serve as a nucleic acid source for the isolation of ouabain-resistant Na⁺- K⁺- ATPase nucleic acids. The nucleotide sequence for rat, butterfly Monarch (Danaus plexippus) and toad Bufo marinus αl Na⁺-K⁺- ATPase is reported in Shull et al., Biochemistry, 25:8125-8132, 1986, Jaisser et al., J. Biol. Chem., 267:16895-16903. 1992 and Holzinger et al., EE5S, 3J4:477-480, 1992, respectively, the contents of which are incorporated herein by reference. 5.1.1. Na⁺-K⁺ ATPase

Both ouabain-sensitive and ouabain-resistant Na⁺-K⁺- ATPases can used in the assays described herein. Whether a Na⁺-K⁺- ATPase is sensitive or resistant to ouabain can be determined by methods known in the art. For example, one can measure activity of the Na⁺-K⁺- ATPase in the presence or absence of ouabain, and determine whether the activity of the Na⁺-K⁺- ATPase is inhibited by ouabain. A Na⁺-K⁺- ATPase is sensitive to ouabain if the IC₅₀ for ouabain is about equal to or is less than 10^"4 M. A Na⁺-K⁺- ATPase is resistant to ouabain if the IC₅₀ for ouabain is about more than 10^"4M.

Any ouabain-sensitive Na⁺-K⁺- ATPase can be used in the assays of the present invention. In one specific embodiment, the ouabain-sensitive Na⁺-K⁺-ATPase is derived from mammalian or avian species. In another specific embodiment, the ouabain-sensitive Na⁺-K⁺- ATPase is derived from mammalian and avian kidney, pineal gland, skeletal muscle, heart, retina or brain cells. In still another specific embodiment, the ouabain-sensitive Na⁺-K⁺- ATPase is derived from adult, juvenile or fetal tissues. In a preferred embodiment, the ouabain-sensitive Na⁺-K⁺-ATPase is an αl, α2, α3 or α4 Na⁺-K⁺- ATPase. In another preferred embodiment, the ouabain-sensitive Na⁺-K⁺- ATPase is the dog kidney αl Na⁺-K⁺- ATPase, the rat fetal brain α3, or the human αl or α3 Na⁺-K"- ATPase.

Any ouabain-resistant Na⁺-K⁺- ATPase can be used in the assays of the present invention. In one specific embodiment, the ouabain-resistant Na⁺-K⁺- ATPase is derived from a rodent, an amphibian, or a butterfly species. Preferably, the ouabain- resistant Na⁺-K^τ- ATPase is derived from a mouse, a rat, a toad Bufo marinus, or a Monarch (Danaus plexippus). Alternatively, the ouabain-resistant Na⁺-K⁺- ATPase can be obtained by converting an ouabain-sensitive Na⁺-K⁺- ATPase into an ouabain- resistant one. Preferably, the ouabain-resistant Na⁺-K⁺- ATPase is obtained by changing one or both border amino acid residues of the H1-H2 extracellular domain of α subunit of an ouabain-sensitive Na⁺-K⁺- ATPase to a charged amino acid residue, including both a positively charged and negatively charged residue. Additional examples of mutations that confer ouabain resistance to otherwise an ouabain-sensitive Na⁺-K⁺-ATPase is disclosed in Lingrel et al., The Sodium Pump (Bamberg and Schoner, Ed.) pp276-286, 1994, the contents of which is incorporated by reference.

5.2. Test Substances and Control Substances

Test substance means a chemically defined compound (organic molecules, inorganic molecules, organic/inorganic complexes, polypeptides, peptides, peptide mimics, polysaccharides, saccharides, glycoproteins, nucleic acids, DNA and RNA strands and oligonucleotides, etc.) or mixtures of compounds (such as in the case of a library of test substances or natural extract or tissue culture supernatant) whose effect on an ATPase is determined by the assay of the invention. Preferably, the test substance is an organic molecule such as a steroid , a cardenolide based compound, or cardiac glycoside or structurally similar compound or combinatorial libraries of such compounds. Ouabain and other control substances, as described below, are not considered "test substances" as that term used herein.

The test substances can be dissolved in aqueous, non-aqueous or aqueous/non- aqueous solvent systems. Preferably, the test substance is dissolved in a solution containing an organic solvent. More preferably, the solution contains up to 20% DMSO.

Control substance is a substance, compound or agent that either increases or decreases activity of an ATPase. Preferably, a control substance that increases and decreases the activity of an ATPase is included in an assay of the present invention. Examples of the control substance include, but are not limited to: digitalis, digoxin, digitoxin, deslanoside, and ouabain (U.S. Patent Nos. 5,716,937 and 5,153,178; Hoffman and Bigger, The Pharmacological Basis of the Therapeutics, eds. Goodman and Gilman, p. 732, (1980))); digitoxigenin (U.S. Patent No. 5,478,817); acetylstrophanthidin (Powis and Madsen, Biochim. Biophys. Acta, 86:251-258, 1986); convallatoxin and cymarin (Ozaki et al., Eur. J. Biochem., 152(21:475-480. 1985; Ozaki et al., FEBS Lett., 173(11:196-198. 1984); helveticocide; strophanthidin (Mishra et al., Neurochem. Res., 14(91:845-851. 1989); "brain ouabain" (Budzikowski et al, Clin. Exp. Hypertens., 20(21:119-1401: HIF (U.S. Patent No. 5,716,937; Tymiak et al., Proc. Natl. Acad. Sc , 90:8189-8193, 1993; and Haupert, Jr. et al, Am. J. Physiol, 247:F919, 1984); the non-digoxin-like Na⁺-K⁺- ATPase inhibitory factor (U.S. Patent No. 5,240,714); the low molecular weight Na⁺-K⁺-ATPase inhibitor (U.S. Patent No. 5,667,811); atrial natriuretic factor, 8-bromo-cGMP, papaverine, IBMX, and sodium nitroprusside (Beltowski et al., J. Physiol. Pharmacol, 49(21:271-283. 1998); the low-molecular mass (12-13 kDa) Na⁺-K⁺- ATPase inhibitor proteins (Bhattacharyya and Sen, Ewr. J. Biochem., 244(31:829-8341: the fraction FI (Calderaro et al, Life Scl, 61(151:1457-1468. 1997); arachidonic acid hydroperoxide product of 5-lipoxygenase (Foley, Biochem. Biophys. Res. Commun., 235(21:374-376. 1997); gypenoside (Han et al, Chung Kuo Chung Yao Tsa Chih, 21(5):299-302, 1996); NG-nitro-L-arginine methyl ester (Inada et al., Diabetologia, 41(121:1451-1458. 1998); marinosin and 11 alpha-hydroxyhellebrigenin (Matsukawa et al., Chem. Pharm. Bull. (Tokyo), 45(2):249-254, 1997); the fraction II-Ε (Pena et al., Neurochem. Res., 22(41:379-383. 1997); the labile, specific Na⁺-K⁺-ATPase inhibitor (Tao et al., Hypertension,29:8l5- 821, 1996); and oleic acids or palmitoleic acids (Younes-Ibrahim et al., Acad. Sci. Paris, Ser. Ill 318:619-625, 1995; and Burth et al., Infect. Immun., 65(41:1557-1560, 1997). The contents of the above references are incorporated herein by reference.

5.3. Detection of ATPase Activity

Activity of an ATPase can be measured and detected by methods known in the art, which methods include, but are not limited to, coupled-enzyme assay (Haupert et al., Am. J. Physiol, 247:F919-F924, 1984), ATP hydrolysis assay (Doucet et al., Am. J. Physiol, 237:F105-F113, 1979), and ion transport assay (Crabos et al, Am. J. Physiol, 254-.F912-F917, 1988; Anner et al., Am. J. Physiol, 258:F144-F153,1990). The assay can be qualitative or quantitative.

In a preferred embodiment, ATPase activity is detected by an ATP hydrolysis method. Preferably, ATP hydrolysis is assayed without separating the ATP and the resultant ADP and free phosphate. ATP hydrolysis can be assayed by measuring a decrease in the amount of ATP or by measuring an increase in the amount of the resultant ADP. Alternatively, ATP hydrolysis can be assayed by measuring the production of a fluorescent product or chemiluminescent product which results from the hydrolysis of a fluorogenic substrate or chemiluminogenic substrate, respectively.

5.3.1. Colorimetric Assay

In one embodiment, ATP hydrolysis is assayed by measuring an increase in the amount of free phosphate using a colorimetric assay. In accordance with this embodiment, the colorimetric assay is based on the detection of a complex formed between the free phosphate and a dye. One skilled in the art would appreciate the dyes that form a complex with free phosphate. In particular, a molybdate dye such as ammonium molybdate is used in the colorimetric assay. Preferably, the amount of phosphate-ammonium molybdate complex is quantified by colorimetry at an OD between 650 and 750 nm.

ATP hydrolysis can be assayed after the release of the terminal phosphate from ATP, as previously described in Henkel et al., 1988, Anal Biochem. 169(21:312-318. In a preferred embodiment, this ATP hydrolysis technique has been modified and incorporated into a high-throughput screening assay to identify modulators of ATPases, preferably Na⁺-K⁺- ATPases, regardless of whether the enzyme is sensitive or resistant to ouabain. The activity of an ATPase, preferably a Na⁺-K⁺- ATPase, in this novel assay is quantified simultaneously in the presence of numerous test substances and in the absence of any test substance. Alternatively, the activity of an ATPase, preferably a Na^{^}-K⁺- ATPase, in this novel assay is quantified simultaneously in the presence of numerous test substances, in the presence of a control substance such as ouabain, and in the absence of both a control and test substance. Preferably, a control substance which functions as an agonist and antagonist is included in the novel assay.

In one embodiment, the ATPase activity in a colorimetric assay system is quantified/measured using a nonautomated system (e.g., a manual system). In another embodiment, the ATPase activity in a colorimetric assay assay system of the invention is quantified/measured using an automated system or other means for measuring/detecting the signal that results from the ATP hydrolysis, such means are known to those of skill in the art. In accordance with this embodiment, the automated system may comprise a computer program or other means for analyzing the data. In a specific embodiment, the ATPase activity in a colorimetric assay system of the invention is quantified/measured using a microtiter plate spectrophotometer.

For example, the following assay procedure can be used. A control substance such as ouabain can be diluted with an aqueous solution, water or a solution containing up to 20% DMSO from lxlO^'2 M to lxl 0^"9 M in 1:10 dilution steps. Purified, partially purified, or unpurified (i.e., not purified from other components of a sample) test compounds can be diluted with an aqueous solution, water, or a solution containing up to 20%) DMSO to the desired concentrations. Test compounds, control substances and negative control solutions can be pipetted into wells of a 96-, 384-, or 1536-well plate. The contents of the plate can then be dried. Dried material can be reconstituted in a buffer such as TRIS-MgCl₂-EGTA (pH 7.4). The assay can also proceed without drying the samples. Na⁺-K^"-ATPase solution is added to wells containing test compounds, control substances and negative control solutions. The contents in wells are vortexed and ATP is added to each well. The ATP hydrolysis reaction can be allowed to proceed at a temperature between 5° C and 37° C for between 5 minutes and 180 minutes. The incubation time is dependent on the purity and form of the enzyme and the reaction temperature, and it may affect the sensitivity of the assay at low concentrations of test substance. The reaction can be stopped by adding detergent solution into each well. Preferably, the stopping detergent is SDS. Solution containing ammonium molybdate and ascorbic acid can be added to each well and the plate can be incubated at room temperature, i.e., from about 20° C to about 30° C, for about 3-5 minutes. After the incubation, solution containing sodium ascorbic meta-arsenate, sodium citrate and acetic acid can be added to each well and the plate can be incubated at room temperature, i.e., from about 20° C to about 30 ° C, for additional 5 minutes to about 3 hours. After the incubation, the color intensity of each well can be measured by colorimetry at an OD between 650 and 750 nm. The color resulting from the reaction between the liberated free phosphate and the molybdate dye is stable up to three hours. If test compounds are dissolved in solutions containing DMSO, the following changes are made in the above assay procedure: 1) the test compounds are diluted to the desired molarity with H₂O or a solution containing DMSO; 2) the final DMSO concentration is kept at or less than 20% (V/N); and 3) the drying step is omitted.

5.3.2. Fluorescent And Chemiluminescent Assays

In a preferred embodiment, ATPase activity is assayed by measuring the fluorescent product produced by the hydrolysis of a fluorogenic substrate. In accordance with this embodiment, the hydrolysis of a fluorogenic analog of ATP (e.g., 4-methylumbelliferyphosphate) results in the production of a fluorescent product (e.g., 0 7-hydroxy-4-methylcoumarin) which can be detected by a spectrofluorimeter. One skilled in the art would know the fluorogenic ATP analogs that can be used in the assay (e.g., see , Haugland, R.P. Handbook of Fluorescent Probes and Research Chemicals, 6^tn Ed., 1996) . Fluorescent and fluorogenic analogs of ATP include, but are not limited to, 4-methylumbelliferyphosphate (MUP), dimethylacridanone phosphate, ELF 5 97 phosphate, and fluoescein diphosphate (e.g., see, the Sigma catalog). In another preferred embodiment, ATPase activity is assayed by measuring the chemiluminescent product produced the hydrolysis of a chemiluminogenic substrate. In accordance with this embodiment, the hydrolysis of a chemiluminogenic analog of ATP results in the emission of a photon which can be detected by a luminometer, a photomultipler tube or 0 a charged coupled device (CCD) camera. Chemiluminogenic analogs of ATP can be generated by conjugating ATP or a derivative of ATP to a compound such as hydroxyphenyldioxetane which becomes chemiluminescent upon enzymatic activity. One skilled in the art would know the chemiluminogenic ATP analogs that can be used in the assay. 5

In a preferred embodiment, this ATP hydrolysis technique has been modified and incorporated into a high-throughput screening assay to identify modulators of ATPases, preferably Νa⁺-K⁺- ATPases, regardless of whether the enzyme is sensitive or resistant to ouabain. The activity of an ATPase, preferably a Na⁺-K"- ATPase, in this

.„ novel assay is quantified simultaneously in the presence of numerous test substances and in the absence of any test substance. Alternatively, the activity of an ATPase, preferably a Na⁺-K⁺- ATPase, in this novel assay is quantified simultaneously in the presence of numerous test substances, in the presence of a control substance such as ouabain, and in the absence of both a control and test substance. Preferably, a control

-^ substance which functions as an agonist and antagonist is included in the novel assay.

In one embodiment, the ATPase activity in a fluorescent or chemiluminescent assay system is quantified/measured using a nonautomated system (e.g., a manual system). In another embodiment, the ATPase activity in a fluorescent or chemiluminescent assay system of the invention is quantified/measured using an automated system or other means for measuring/detecting the signal that results form the ATP hydrolysis, such means are known to those of skill in the art. In accordance with this embodiment, the automated system may comprise a computer program or other means for analyzing the data. In a specific embodiment, the ATPase activity in a fluorescent assay system of the invention is quantified/measured using a micro titer plate fluorimeter such as manufactured by Tecan (i.e., Tecan spectrofluor).

For example, the following assay procedure can be used. A control substance such as ouabain can be diluted with 20% DMSO or water from lxlO^"2M to lxlO^"9M in

1:10 dilution steps. Test compounds can be diluted with 20% DMSO or water to desired concentration. Test compounds, control substances and negative control solutions can be pipetted into wells of a 96-, 384- or 1536- well high throughput assay plate. A buffered Na⁺-K⁺- ATPase solution is added to wells containing test compounds, control substances and negative control solutions. The buffer system that is used may vary with the pH chosen for the reaction. The plate is then incubated at a specified temperature from 5° C to 50° C for between 5 minutes and 480 minutes. The incubation time is dependent on the purity and form of the enzyme and the reaction temperature, and it may affect the sensitivity of the assay at low concentrations of test substance. After incubation, a buffered solution of pH 4.0 to 10.0 of MUP is added, incubated at a specified temperature from 5° C to 50° C for a specified time from less than 1 second to 480 minutes, and the fluorescent product, 7-hydroxy-4- methylcoumarin, is measured. The pH of the buffered MUP solution and the temperature of the incubation step have significant effects on the velocity of the reaction. The reaction may be stopped in an endpoint measurement, or allowed to proceed in a kinetic measurement .

5.4. Kits

The present invention also provides kits that can be used in the above assays. In one embodiment, a kit comprises: (a) an isolated or purified ATPase and (b) means for detection of ATP hydrolysis. Preferably, the kit further comprises a control substance (e.g., ouabain) and ATP. In a preferred embodiment, the ATPase included in the kit is a Na⁺-K⁺- ATPase.

In a specific embodiment, a kit comprises: (a) an isolated or purified ATPase and (b) a fluorogenic analog of ATP. In another specific embodiment, a kit comprises: (a) an isolated or purified ATPase and (b) a chemiluminogenic analog of ATP. Preferably, the kits described in these embodiments further comprises a control substance (e.g., ouabain). In a preferred embodiment, the ATPase included in the kits is a Na⁺-K⁺- ATPase.

In a specific embodiment, a kit comprises: (a) an isolated or purified Na⁺-K⁺- ATPase; (b) a control substance; (c) ATP; and (d) means for a colorimetric detection of ATP hydrolysis. In accordance with this embodiment, the means for a colorimetric detection of ATP hydrolysis comprises a dye such as ammonium molybdate dye that forms a complex with free phosphate. In a particular embodiment, the Na⁺-K⁺- ATPase is the dog kidney αl Na⁺-K⁺- ATPase, the rat kidney αl Na⁺-K⁺- ATPase, the rat fetal brain α3 Na⁺-K⁺- ATPase or the human αl or α3 Na⁺-K⁺- ATPase.

In a specific embodiment, kits of the invention further comprise instructions and/or multiple well plates for performing the assay.

5.5. Use of the Modulators of Na⁺-K⁺-ATPase

Modulators of an ATPase identified by the screening assays disclosed above can be to treat, inhibit or prevent certain diseases and disorders associated with ATPase activity, particularly aberrant ATPase activity, including, but are not limited to, stroke, cardiovascular diseases (e.g., angina pectoris, myocardial infarction, chronic ischemic heart disease, hypertensive heart disease, pulmonary heart disease, valvular heart disease, rheumatic fever, rheumatic heart disease, endocarditis, mitral valve prolapse, aortic valve stenosis, congenital heart disease, valvular and vascular obstructive lesions, atrial or ventricular septal defect, patent ductus arteriosus, and myocardial disease), Wilson disease, Menkes syndrome, kidney disorders (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, systemic lupus erythematosus, Goodpasture's syndrome, multiple myeloma, diabetes, neoplasia, sickle cell disease, chronic inflammatory diseases, acute tubular necrosis, acute renal failure, polycystic renal diseasemedullary sponge kidney, medullary cystic disease, nephrogenic diabetes, renal tubular acidosis, tubulointerstitial diseases, acute and rapidly progressive renal failure, chronic renal failure, and nephrolithiasis), hypotension, hypertension, ischemic injury, neurological disorders (e.g., Alzheimer's disease and Parkinson's disease), muscle disorders, pulmonary disorders (e.g., emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, Goodpasture's syndrome, idiopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis, desquamative interstitial pneumonitis, chronic interstitial pneumonia, fibrosing alveolitis, hamman-rich syndrome, pulmonary eosinophilia, diffuse interstitial fibrosis, Wegener's granulomatosis, lymphomatoid granulomatosis, and lipid pneumonia), hepatic disorders (e.g., hepatic vein thrombosis, portal vein obstruction, thrombosis, hepatitis, and cirrhosis), proliferative disorders (e.g., neoplasms or tumors such as carcinomas, sarcomas, adenomas, and myeloid leukemia), and disorders in which a positive ionotropic effect is desired. Modulators of an ATPase identified by the screening assays disclosed above can also be used to treat, inhibit or prevent microbial infections (e.g., viral infections such as hepatitis C virus (HCV)), fungal infections and bacterial infections such as Heliobacter pylori) since many microbes contain specific microbial ATPases. In a specific embodiment, cardiac disorders can be treated as taught in U.S. Patent No. 5,716,937, which the contents of which are incorporated herein in their entirety.

10

5.6 Demonstration of Therapeutic or Prophylactic Utility of a Modulator

The modulators identified in assays of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use 5 in humans. For example, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed. In one embodiment, the toxicity of a modulator identified in an assay of the

™ present invention is tested in vitro prior to in vivo testing.

In various specific embodiments, in vitro assays can be carried out with representative tissue samples or cells of cell types involved in a patient's disorder, to determine if a compound has a desired effect upon such cell types.

Compounds for use in therapy can be tested in suitable animal model systems

25 prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, prior to administration to humans, any animal model system known in the art may be used.

5.7. Administration of Modulators Identified in the 30 Screening Assays of the Invention

The invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a modulator of an ATPase identified using a screening assay described herein. In a preferred aspect, the modulator (i.e., a compound) is substantially purified (e.g., substantially free from

35 substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human. In a specific embodiment, a non-human mammal is the subject.

Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intratumoral, intradermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, and oral routes. The compounds may be administered by any convenient route, for example parenterally (e.g., by infusion or bolus injection), by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), by oral, or transdermal delivery and may be administered together with other biologically active agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.

In another embodiment, the compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al, N Engl J. Med. 321 :574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sc Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

In a specific embodiment where the compound to be administered to a subject is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g. , Joliot et al., 1991, Proc. Natl Acad. Sc USA 88:1864-1868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term

"pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compounds to be administered to a subject can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the compound administered to a subject which will be effective in the treatment of a disease or disorder disclosed herein can be determined by standard . . clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Oral formulations preferably contain 10% to 95% active ingredient.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

EXAMPLE: High-Throughput Colorimetric ATP Hydrolysis Assay

Materials Ammonium molybdate heptahydrate (#43134-6) was obtained from Aldrich.

Ascorbic acid (#AX 1772-1), KCl (#PX 1405-1) and sodium citrate (#SX 0444-1) were obtained from EM Sciences. MgCl₂ hexahydrate (#5958), NaCl (#7581) and TRIS (#H7523) were obtained from Mallinckrodt. SDS (electrophoresis purity reagent #161- 0301) was obtained from Bio-Rad. Sodium meta-arsenate (#3487-04) was obtained from Baker. The following reagents were obtained from Sigma: acetic acid (glacial), disodium ATP (#A), EDTA (#E9884), EGTA (#E0396), HC1 (36%=12N), HEPES (#H7523), imidazole (#12399), sucrose (#S7903), ouabain octahydrate (#03125), digoxin (D-6003), digitoxin (D-5878), digitoxigenin (D-9404), acetylstrophanthidin (A-3259), convallatoxin (C-9140), cymarin (C-0259), helveticoside (H-2634) and strophanthidin (S-5883 and S-6626).

Solutions

1. 20 mM Imidazole-250 mM Sucrose- 1 mM EDTA (pH 7.4)

340 mg imidazole, 21.3 g sucrose and 7.3 mg EDTA were completely dissolved in approximately 240 ml H₂O. The pH was adjusted to about 7.4 with 1 N HC1. The final volume of the solution was brought to 250 ml with H₂O. After the solution was thoroughly mixed, pH was verified, and the solution was stored at 25 °C.

2. 20 mM Imidazole-250 mM Sucrose-5 mM MgCl₂- 1 mM EGTA (pH 7.4)

340 mg imidazole, 21.3 g sucrose, 250 mg MgCl₂ and 9.5 mg EGTA were completely dissolved in approximately 240 ml H₂O. The pH was adjusted to about 7.4 with 1 N HC1. The final volume of the solution was brought to 250 ml with H₂O. After the solution was thoroughly mixed, pH was verified, and the solution was stored at 25°C. 3. 20 mM TRIS-5 mM MgCl₂-l mM EGTA (pH 7.4)

605 mg TRIS, 250 mg MgCl₂ and 9.5 mg EGTA were completely dissolved in approximately 240 ml H₂O. The pH was adjusted to about 7.4 with 1 N HCI. The final volume of the solution was brought to 250 ml with H₂O. After the solution was thoroughly mixed, pH was verified, and the solution was stored at 25 °C.

4. 30 mM HEPES-3 mM ATP-210 mM NaCl-30 mM KC1-5 mM MgC12 (PH 7.4)

3.6 g HEPES, 6 g NaCl, 1.11 g KCl, 0.5 g MgCl₂ and 1.23 g ATP were completely dissolved in approximately 490 ml H₂O. The pH was adjusted to about 7.4 with 1 N HCI. The final volume of the solution was brought to 500 ml with H₂O. After the solution was thoroughly mixed, pH was verified. The solution was aliquoted in 10 ml volumes and stored at -20 °C.

5. 1 N HCI

50 ml 12 N HCI was diluted in 550 ml H₂O.

6. 2% Ammonium Molybdate- 1 N HCI

2 g ammonium molybdate was completely dissolved in approximately 90 ml 1 N HCI. The final volume of the solution was brought to 100 ml with 1 N HCI. The solution was thoroughly mixed and stored at 4°C.

7. 12% Ascorbic Acid-1 N HCI

12 g ascorbic acid was completely dissolved in approximately 90 ml 1 N HCI. The final volume of the solution was brought to 100 ml with 1 N HCI. The solution was thoroughly mixed and stored at 4°C.

8. Solution E: 2% Sodium Ascorbic Meta-Arsenate-2%> Sodium Citrate- 2% Acetic Acid

5 g sodium meta-arsenate, 5 g sodium citrate and 5 ml glacial acetic acid were completely dissolved in approximately 240 ml H₂O. The final volume of the solution was brought to 250 ml with H₂O. The solution was thoroughly mixed and the solution was stored at 25 °C.

9. 10% SDS 25 g SDS was completely dissolved in approximately 240 ml H₂O. The final volume of the solution was brought to 250 ml with H₂O. The solution was thoroughly mixed and the solution was stored at 25 °C.

10. 10 mM Ouabain

58.4 mg ouabain octahydrate was completely dissolved in 10 ml H₂O by vortexing thoroughly. The solution was stored in plastic container wrapped in aluminum foil (keep out light) at 4°C.

Preparation of Rat and Dog Kidney Na⁺-K⁺- ATPases

1. Rat Kidney Na⁺-K⁺- ATPase

Rat kidney Na⁺-K⁺- ATPase was purified according to the procedures disclosed in Jorgensen, Biochimi. Biophys. Acta, 356:36-52. 1974. The purified enzyme can be used at different concentrations, typically at about 0.2U/mL. If test substances to be screened were dissolved in H₂O, the Na⁺-K⁺- ATPase was diluted in solution #1 (20 mM Imidazole-250 mM Sucrose-1 mM EDTA, pH 7.4) to 0.05 mg/ml. If test substances to be screened were dissolved in a DMSO solution, the Na"-K^τ- ATPase was diluted in solution #2 (20 mM Imidazole-250 mM Sucrose-5 mM MgCl₂-l mM EGTA, pH 7.4) to 0.05 mg/ml.

2. Rat Fetal Brain Na⁺-K⁺- ATPase

Rat fetal brain Na⁺-K⁺- ATPase was purified according to the procedures disclosed in Jorgensen, Biochimi. Biophys. Acta, 356:36-52, 1974. The purified enzyme can be used at different concentrations, typically at about 0.2U/mL. If test substances to be screened were dissolved in H₂O, the Na^ ^*- ATPase was diluted in solution #1 (20 mM Imidazole-250 mM Sucrose-1 mM EDTA, pH 7.4) to 0.05 mg/ml. If test substances to be screened were dissolved in a DMSO solution, the Na⁺-K⁺- ATPase was diluted in solution #2 (20 mM Imidazole-250 mM Sucrose-5 mM MgCl₂- 1 mM EGTA, pH 7.4) to 0.05 mg/ml.

3. Dog Kidney Na⁺-K⁺- ATPase

In some experiments, dog kidney Na⁺-K⁺- ATPase was purified according to the procedures disclosed in Jorgensen, Biochimi. Biophys. Acta, 356:36-52, 1974. The purified enzyme can be used at different concentrations. If test substances to be screened were dissolved in H₂O, the Na⁺-K⁺- ATPase was diluted in solution #1 (20 mM Imidazole-250 mM Sucrose-1 mM EDTA, pH 7.4) to 0.01 mg/ml. If test substances to be screened were dissolved in a DMSO solution, the Na⁺-K⁺- ATPase was diluted in solution #2 (20 mM Imidazole-250 mM Sucrose-5 mM MgCl₂-l mM EGTA, pH 7.4) to 0.01 mg/ml. In other experiments, dog kidney Na⁺-K⁺- ATPase was obtained from a commercial source such as Sigma Chemical Company, e.g., A7305 and A0142.

Efficacy of the Assay

Ouabain was dissolved and diluted with water from lxl 0^"2 M to lxl 0^"9 M in 1:10 dilution steps. Other control substances such as acetylstrophanthidin, convallatoxin, cymarin, digoxin, digitoxin, digitoxigenin, helveticoside and strophanthidin was dissolved in a solution containing DMSO and diluted with water or a water/DMSO mixture to desired concentration, respectively. As Na⁺-K⁺- ATPase negative controls, 10 μl of water were pipetted into wells B2 to D2 of a 96-well plate. As Na⁺-K⁺- ATPase positive controls, 10 μl of ouabain were pipetted in triplicate into adjacent wells. Ten (10) μl of acetylstrophanthidin, convallatoxin, cymarin, digoxin, digitoxin, digitoxigenin, helveticocide and strophanthidin were pipetted into remaining wells, receptively. As Na⁺-K⁺- ATPase negative controls, 10 μl water were pipetted into three wells after last compound again. The content in the 96-well plate was dried in speed- vacuum for approximately 20 minutes. Dried material was reconstituted in 10 μl of solution No. 3 (20 mM TRIS-5 mM MgCl₂-l mM EGTA, pH 7.4). Ten (10) μl of rat or dog kidney Na⁺-K⁺- ATPase solution or rat fetal brain Na⁺-K^τ- ATPase solution were added to wells containing water, ouabain and/or other control substances. The 96-well plate was vortexed briefly to mix the content in the wells. Forty (40) μl of solution No. 4 (30 mM HEPES-3 mM ATP-210 mM NaCl-30 mM KC1-5 mM MgC12, pH 7.4) was added to each of these wells and 50 μl water was added to the empty wells. The ATP hydrolysis reaction was allowed to proceed for 5 minutes for rat enzyme, and for 10 minutes for dog enzyme, both at 37 °C. The reaction was stopped by adding 40 μl 10% SDS to each well. Eighty (80) μl of solution "D" (50-50 mix of solutions No. 6 and No. 7) was added to each well and the 96-well plate was incubated at room temperature for 3 minutes. After the 3-minute incubation, 120 μl solution E (2% Sodium Ascorbic Meta-Arsenate-2% Sodium Citrate-2% Acetic Acid) were added to each well and the 96-well plate was incubated at room temperature for additional 10 minutes. After the 10 minute incubation, the color intensity of each well was measured by colorimetry at an OD between 650 and 750 nm. The color resulting from the reaction between the liberated free phosphate and the molybdate dye is stable up to three hours.

The results of the above testing experiments are shown in Figures 1-3 and Tables I-III. These results indicate that ouabain and other control substances such as acetylstrophanthidin, convallatoxin, cymarin, digoxin, digitoxin, digitoxigenin, helveticoside and strophanthidin inhibit various forms of Na⁺-K⁺- ATPase under the present assay procedures and can be used as control substances in the screening assays for the Na⁺-K⁺- ATPase modulators.

EXAMPLE: High-Throughput Fluorescent ATPase Assay

The following example demonstrates the utility of the fluorescent ATP hydrolysis assay described herein for high-throughput format.

Solutions

1. 20 mM Imidazole-250 mM Sucrose-1 mM EDTA (pH 7.4)

340 mg imidazole, 21.3 g sucrose and 7.3 mg EDTA were completely dissolved in approximately 240 ml H₂O. The pH was adjusted to about 7.4 with 1 N HCI. The final volume of the solution was brought to 250 ml with H₂O. After the solution was thoroughly mixed, pH was verified, and the solution was stored at 25 °C.

2. 20 mM Imidazole-250 mM Sucrose-5 mM MgCl₂-l mM EGTA (pH 7.4) 340 mg imidazole, 21.3g sucrose, 250 mg MgCl₂ and 9.5 mg EGTA were completely dissolved in approximately 240 ml. The pH was adjusted to 7.4 with IN HCI. The final volume of solution was brought up to 250 ml with H₂O. After the solution was thoroughly mixed, pH was verified, and it was stored at 25° C.

3. 30mM TAPS-2mM MUP-5mM MgC12-5mM KCl, (pH 8.0):

84.4 mg of methylumbelliferyl phosphate, 61.8 mg CC1, 101.5 mg MgC12. and 729 mg TAPS were completely dissolved in approximately 90 ml H2O. The pH was adjusted to 8.0 with IN HCI. The final volume of the solution was brought up to 100 ml with H2O. After the solution was thoroughly mixed, pH was verified. The solution was divided into 10 ml aliquots and immediately frozen at -20° C.

4. lO mM Ouabain

58.4 mg ouabain octahydrate was completely dissolved in 10 ml 20% DMSO,

80%) H2O by vortexing. The solution was stored at 4° C protected from light.

Preparation of Rat and Dog Kidney Na⁺-K⁺- ATPases 1. Rat Kidney Na⁺-K⁺- ATPase

Rat kidney Na⁺-K⁺- ATPase was purified according to the procedures disclosed in Jorgensen, Biochimi. Biophys. Acta, 356:36-52, 1974. The purified enzyme can be used at different concentrations, typically at about 0.2 U/mL. If test substances to be screened were dissolved in H₂O, the Na⁺-K⁺- ATPase was diluted in solution #1 (20 mM Imidazole-250 mM Sucrose-1 mM EDTA, pH 7.4) to 0.05 mg/ml. If test substances to be screened were dissolved in a DMSO solution, the Na⁺-K⁺- ATPase was diluted in solution No. 2 (20 mM Imidazole-250 mM Sucrose-5 mM MgCl₂-l mM EGTA, pH 7.4) to 0.05 mg/ml.

2. Rat Fetal Brain Na⁺-K⁺- ATPase

Rat fetal brain Na⁺-K⁺- ATPase was purified according to the procedures disclosed in Jorgensen, Biochimi. Biophys. Acta, 356:36-52. 1974. The purified enzyme can be used at different concentrations, typically at about 0.2U/mL. If test substances to be screened were dissolved in H₂O, the Na⁺-K⁺- ATPase was diluted in solution No. 1 (20 mM Imidazole-250 mM Sucrose-1 mM EDTA, pH 7.4) to 0.05 mg/ml. If test substances to be screened were dissolved in a DMSO solution, the Na⁺- K⁺- ATPase was diluted in solution No. 2 (20 mM Imidazole-250 mM Sucrose-5 mM MgCl₂-l mM EGTA, pH 7.4) to 0.05 mg/ml.

3. Dog Kidney Na⁺-K⁺- ATPase

In some experiments, dog kidney Na⁺-K⁺- ATPase was purified according to the procedures disclosed in Jorgensen, Biochimi. Biophys. Acta, 356:36-52. 1974. The purified enzyme can be used at different concentrations. If test substances to be screened were dissolved in H₂O, the Na⁺-K⁺-ATPase was diluted in solution No. 1 (20 mM Imidazole-250 mM Sucrose-1 mM EDTA, pH 7.4) to 0.01 mg/ml. If test substances to be screened were dissolved in a DMSO solution, the Na⁺-K⁺- ATPase was diluted in solution No. 2 (20 mM Imidazole-250 mM Sucrose-5 mM MgCl₂-l mM EGTA, pH 7.4) to 0.01 mg/ml. In other experiments, dog kidney Na^τ-K^~- ATPase was obtained from a commercial source such as Sigma Chemical Company, e.g., A7305 and A0142.

Efficacy of the Assay

A kinetic timecourse assay was performed to determine the reaction velocity and percent inhibition by ouabain. Dog kidney Na⁺-K⁺- ATPase was diluted in a solution of 30 mM imidazole, 250mM sucrose, and ImM EDTA. Then 2 mU or 5 mU of dog kidney Na⁺-K⁺- ATPase was added to a 96 well opaque plate (Costar Corning). Simultaneously, various concentrations of ouabain (Sigma Chemical Co.) were added to some of the wells containing the Na⁺-K⁺- ATPase. The plate was incubated at 37° C for different amounts of time and methylumbelliferylphosphate (MUP; Molecular Probes) solution (1 mM MUP, 5 mM MgCl₂, 10 mM KCl, 30 mM TAPS, pH 8.0) was added to the wells. The plate was incubated at 37° C for two to five minutes and the presence of the fluorescent product 7-hydroxy-4-methylcoumarin was analyzed by a spectrofluorimeter (Tecan, Inc.). The results from the timecourse are depicted in Figures 4 and 5.

Figure 4 demonstrates that the assay detects ATPase activity and that the higher the concentration of ATPase added to the reaction mixture and the longer the reaction time, the more reaction product generated. Figure 5 demonstrates that lxl 0⁶ M ouabain is sufficient to inhibit Na⁺-K⁺- ATPase activity.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

TABLE I. Inhibition of Dog Kidney Na⁺-K⁺- ATPase (inhibition %)

Concentration Acetylstrophanthidin Convallatoxin Cymarin Digoxin Digitoxin Digitoxigenin Helveticoside Strophanthidin Ouabain

10-⁹ M 0.6 1.6 1.2 0.7 10.6 -0.9 4.5 2.5 2.5

10^'8 M 1.8 2.9 -0.1 0.2 14.7 7.8 0 10.6

10^"7 M 20.4 12.1 8.3 3.9 28.9 8.6 1.2 29.7 9.4

10^"6 M 73.3 49.0 45 8.1 71.8 2.2 2.4 68

10^"5 M 92.6 95.3 95.4 70.9 94 79.4 19.9 91.5 94.7

10^'4 M 96.3 97.5 96.6 95.3 97.3 95.5 87.9 99.6

TABLE II. Inhibition of Rat Kidney Na⁺-K⁺-ATPase (inhibition %)

Cone. Acetylstrophanthidin Convallatoxin Cymarin Digoxin Digitoxin Digitoxigenin Helveticoside Strophanthidin Ouabain

10-" M 0.3 5.3 2.4 -4.1 -2.7 -3.1 10.5 1.9 2.3

10^'8 M -1.3 5 0.9 -2.1 -0.6 2.6 5.3

10^"7 M 0.3 2.5 2.9 1 -1.6 1.2 2.5 -2.1 2.6

10^"6 M 0 4.8 4.9 -1.9 -2.3 2.3 4 1.2

10 ⁵ M 1 6.7 9.2 1.9 -0.6 2.6 2.6 1.5 6.5

10^"4 M 4.7 24.3 40.4 28.5 10.6 13.5 8.1 6.7

t-

TABLE III. Inhibition of Rat Fetal Na⁺-K⁺-ATPase (inhibition %)

10'M -3.9 0.6 10.4 -0.3 6.8 -4.3 4 -2 2.7

10^'8M 0.5 1.5 11.6 -2.4 4.3 -5.1 -3.5 11.1

10⁷M 6.7 3.9 14.2 -1.4 8.8 -3 -4.5 32.6 5

10° M 33.3 19.2 26.9 3.6 31.1 1.9 8.3 33

10^"5M 59 52 59.1 28.2 57.3 41.5 15.3 57.4 52.7

10^'4M 62 62.2 68.1 67.2 64.3 59.6 48.1 61.6

--

Claims

WHAT IS CLAIMED IS:

1. An assay for screening test substances for their ability to modulate activity of an ATPase, comprising: (a) contacting the ATPase with a test substance in the presence of a fluorogenic ATP analog for a time sufficient to measure activity of the ATPase; (b) contacting the ATPase with a fluorogenic ATP analog for a time sufficient to measure activity of the ATPase; (c) contacting the ATPase with a control substance in the presence of a fluorogenic ATP analog for a time sufficient to measure activity of the ATPase;

(d) measuring the ATPase activity in steps (a), (b) and (c); and

(e) comparing the activity in step (a) with the activity in steps (b) and (c) to determine whether the test substance modulates the activity of the

ATPase.

2. The assay of Claim 1, wherein the ATPase activity is measured by the generation of fluorescent product.

3. The assay of Claim 1, wherein the fluorogenic ATP analog is methylumbelliferylphosphate.

4. An assay for screening test substances for their ability to modulate activity of an ATPase, comprising:

(a) contacting the ATPase with a test substance in the presence of a chemiluminogenic ATP analog and for a time sufficient to measure activity of the ATPase;

(b) contacting the ATPase with a chemiluminogenic ATP analog and for a time sufficient to measure activity of the ATPase;

(c) contacting the ATPase with a control substance in the presence of a chemiluminogenic ATP analog for a time sufficient to measure activity of the ATPase;

(d) measuring the ATPase activity in steps (a), (b) and (c); and (e) comparing the activity in step (a) with the activity in steps (b) and (c) to determine whether the test substance modulates the activity of the ATPase.

5. The assay of Claim 4, wherein the ATPase activity is measured by the generation of chemiluminescent product.

6. The assay of Claim 4, wherein the chemiluminogenic ATP analog is generated by conjugating ATP or a derivative of ATP to hydroxyphenyldioxetane.

7. A high-throughput assay for screening test substances for their ability to modulate activity of a Na⁺-K⁺- ATPase, comprising:

(a) contacting the Na⁺-K⁺- ATPase with a test substance in the presence of ATP or an analog thereof for a time sufficient to measure activity of the Na⁺-K⁺- ATPase;

(b) contacting the Na⁺-K⁺- ATPase with ATP or an analog thereof for a time sufficient to measure activity of the Na⁺-K⁺- ATPase;

(c) contacting the Na^"-K⁺- ATPase with a control substance in the presence of ATP of an analog thereof for a time sufficient to measure activity of the Na⁺-K⁺- ATPase;

(d) measuring the Na⁺-K⁺-ATPase activity in steps (a), (b) and (c); and

(e) comparing the activity in step (a) with the activity in steps (b) and (c) to determine whether the test substance modulates the activity of the Na^"-K"-ATPase.

8. The assay of Claim 1, wherein the ATPase is a Na^{^}-K⁺- ATPase.

9. The assay of Claim 4, wherein the ATPase is a Na⁺-K⁺-ATPase.

10. The assay of Claim 7, wherein the ATPase is an ouabain-sensitive Na⁺-K⁺-

ATPase.

11. The assay of Claim 8, wherein the ATPase is an ouabain-sensitive Na⁺-K⁺- ATPase.

12. The assay of Claim 9, wherein the ATPase is an ouabain-sensitive Na⁺-K⁺

ATPase.

13. The assay of Claim 10, 11 or 12, wherein the ouabain-sensitive Na⁺-K⁺-

ATPase is derived from mammalian and avian kidney, pineal gland, skeletal muscle, heart, retina and brain cells.

14. The assay of Claim 10, 11 or 12, wherein the ouabain-sensitive Na⁺-K⁺- ATPase is an αl, α2, α3 or α4 Na⁺-K⁺- ATPase.

15. The assay of Claim 10, 11 or 12, wherein the ouabain-sensitive Na⁺-K⁺-

ATPase is the dog kidney αl Na⁺-K⁺- ATPase or the rat fetal brain α3 Na⁺-K⁺- ATPase.

16. The assay of Claim 10, 11 or 12, wherein the ouabain-sensitive Na⁺-K⁺- ATPase is the human αl Na⁺-K⁺- ATPase or the human α3 Na⁺-K⁺- ATPase.

17. The assay of Claim 7, wherein the ATPase is an ouabain-resistant Na⁺-K⁺- ATPase.

18. The assay of Claim 8, wherein the ATPase is an ouabain-resistant Na⁺-K⁺- ATPase.

19. The assay of Claim 9, wherein the ATPase is an ouabain-resistant Na⁺-K⁺- ATPase.

20. The assay of Claim 17, 18 or 19, wherein the ouabain-resistant Na⁺-K⁺-

ATPase is selected from the group consisting of a rodent, toad and butterfly αl Na⁺-K"- ATPase.

21. The assay of Claim 17, 18 or 19, wherein the ouabain-resistant Na⁺-K^{^}- ATPase is obtained by substituting one or both border amino acid residues of the H1-H2 extracellular domain of αl subunit of an ouabain-sensitive Na⁺-K⁺- ATPase to a charged amino acid residue.

22. The assay of Claim 1, 4 or 7, wherein the test substance is selected from the group consisting of organic molecules, inorganic molecules, organic/inorganic complexes, proteins, peptides, peptide mimics, polysaccharides, saccharides, glycoproteins, nucleic acids, oligonucleotides and mixtures thereof.

23. The assay of Claim 1, 4 or 7, wherein the test substance is dissolved in a solution containing an organic solvent.

24. The assay of Claim 1 , wherein the control substance is selected from the - group consisting of digitalis, digoxin, digitoxin, digitoxigenin, deslanoside, ouabain, acetylstrophanthidin, convallatoxin, cymarin, helveticoside, strophanthidin, HIF, atrial natriuretic factor, 8-bromo-cGMP, papaverine, IBMX, sodium nitroprusside, arachidonic acid hydroperoxide product of 5-lipoxygenase, gypenoside, NG-nitro-L-arginine methyl ester, marinosin, 11 alpha-hydroxyhellebrigenin, oleic acids and palmitoleic acids.

25. The assay of Claim 4, wherein the control substance is selected from the group consisting of digitalis, digoxin, digitoxin, digitoxigenin, deslanoside, ouabain, acetylstrophanthidin, convallatoxin, cymarin, helveticoside, strophanthidin, HIF, atrial natriuretic factor, 8-bromo-cGMP, papaverine, IBMX, sodium nitroprusside, arachidonic acid hydroperoxide product of 5-lipoxygenase, gypenoside, NG-nitro-L-arginine methyl ester, marinosin, 11 alpha-hydroxyhellebrigenin, oleic acids and palmitoleic acids.

26. The assay of Claim 7, wherein the control substance is selected from the group consisting of digitalis, digoxin, digitoxin, digitoxigenin, deslanoside, ouabain, acetylstrophanthidin, convallatoxin, cymarin, helveticoside, strophanthidin, HIF, atrial natriuretic factor, 8-bromo-cGMP, papaverine, IBMX, sodium nitroprusside, arachidonic acid hydroperoxide product of 5-lipoxygenase, gypenoside, NG-nitro-L-arginine methyl ester, marinosin, 11 alpha-hydroxyhellebrigenin, oleic acids and palmitoleic acids.

27. The assay of Claim 24, 25 or 26, wherein the control substance is ouabain.

28. The assay of Claim 1 or 4, wherein the activity measured in step (a) is different from that measured in step (b) indicates that the test substance modulates the activity of the ATPase.

29. The assay of Claim 7, wherein the activity measured in step (a) is different from that measured in step (b) indicates that the test substance modulates the activity of the Na^T-K^T-ATPase.

30. The assay of Claim 1, 4 or 7, wherein steps (a) and (b) are conducted simultaneously.

31. The assay of Claim 1 , wherein more than one test substance is tested simultaneously.

32. The assay of Claim 4, wherein more than one test substance is tested simultaneously.

33. The assay of Claim 7, wherein more than one test substance is tested simultaneously.

34. The assay of Claim 31 , 32 or 33, wherein the simultaneous tests are conducted in a 96-well plate, a 384-well plate, or 1536-well plate.

35. The assay of Claim 1 or 4, wherein the assay is a high-throughput assay.

36. The assay of Claim 7, wherein the Na⁺-K⁺- ATPase activity is detected by an ATP hydrolysis method.

37. The assay of Claim 36, wherein the ATP hydrolysis is assayed without separating the remaining ATP and the resultant ADP and free phosphate.

38. The assay of Claim 36, wherein ATP hydrolysis is assayed by measuring a decrease in the amount of ATP.

39. The assay of Claim 36, wherein ATP hydrolysis is assayed by measuring an increase in the amount of the resultant ADP or free phosphate.

40. The assay of Claim 39, wherein the increase in the amount of the resultant free phosphate is measured by a colorimetric assay.

41. The assay of Claim 40, wherein the colorimetric assay is based on the detection of a complex formed between the free phosphate and a molybdate dye.

42. The assay of Claim 41, wherein the molybdate dye is ammonium molybdate.

43. The assay of Claim 42, wherein the amount of phosphate-ammonium molybdate complex is quantified by colorimetry at an OD between about 650 to about 750 nm.

44. The assay of Claim 7, wherein the Na⁺-K⁺- ATPase activity is detected by the hydrolysis of an ATP analog.

45. The assay of Claim 44, wherein the ATP analog is fluorogenic.

46. The assay of Claim 45, wherein the ATP analog is methylumbelliferyl- phosphate.

47. The assay of Claim 44, wherein the ATP analog is chemiluminogenic.

48. The assay of Claim 47, wherein the ATP analog is a derivative of hydroxyphenyldioxetane.

49. A kit comprising:

(a) an isolated or purified ATPase;

(b) a control substance; and

(c) a fluorogenic ATP analog.

50. A kit comprising:

(a) an isolated or purified ATPase;

(b) a control substance; and

(c) a chemiluminogenic ATP analog.

51. A kit comprising:

(a) an isolated or purified Na⁺-K⁺- ATPase;

(b) a control substance;

(c) ATP; and (d) means for a colorimetric detection of ATP hydrolysis.

52. The kit of Claim 49 or 50, wherein the ATPase is Na⁺-K"- ATPase.

53. The kit of Claim 49, 50 or 51 , wherein the Na⁺-K⁺- ATPase is selected from the group consisting of the dog kidney αl Na⁺-K⁺-ATPase, the rat kidney αl Na⁺-K⁺-

ATPase, the rat fetal brain α3 Na⁺-K⁺- ATPase, the human αl Na⁺-K^"- ATPase and the human α3 Na⁺-K⁺- ATPase.

54. The kit of claim 49, 50 or 51, wherein the control substance is ouabain.

55. The kit of Claim 51, wherein the means for a colorimetric detection of ATP hydrolysis comprises ammonium molybdate.

56. A high-throughput assay for screening test substances for their ability to - modulate activity of an ATPase, comprising:

(a) contacting the ATPase with a test substance in the presence of ATP or an analog thereof for a time sufficient to measure activity of the ATPase;

(b) contacting the ATPase with a control substance in the presence of ATP or an analog thereof for a time sufficient to measure activity of the ATPase;

(c) measuring the ATPase activity in steps (a) and (b); and (d) comparing the activity in step (a) with the activity in step (b) to determine whether the test substance modulates the activity of the ATPase, wherein baseline ATPase activity has been considered in comparing the activity in steps (a) and (b).