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    Disclosed are high throughput assay systems and methods for identifying agents that alter the level of surface expression of integral membrane proteins, such as cardiac ion channels, in mammalian cells. Also disclosed are therapeutic methods of using agents identified using such methods.
    公开号:AU2013200842A1
    申请号:U2013200842
    申请日:2013-02-15
    公开日:2013-03-07
    发明作者:Arthur M. Brown；Eckhard Ficker；Barbara A. Wible
    申请人:ChanTest Corp；
    IPC主号:A61K31-495
    专利说明:
    Regulation 3.2 AUSTRALIA PATENTS ACT, 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT DIVISIONAL Name of Applicant: CHANTEST CORPORATION Actual Inventors: BROWN, Arthur M, FICKER, Eckhard, WIBLE, Barbara A Address for service in A J PARK, Level 11, 60 Marcus Clarke Street, Canberra ACT Australia: 2601, Australia Invention Title: High Throughput Assay Systems And Methods For Identifying Agents That Alter Surface Expression Of Integral Membrane Proteins The following statement is a full description of this invention, including the best method of performing it known to us. fA"n7) I WO 2005/009441 PCT/US2004/022751 HIGH THROUGHPUT ASSAY SYSTEMS AND METHODS FOR IDENTIFYING AGENTS THAT ALTER SURFACE EXPRESSION OF INTEGRAL MEMBRANE PROTEINS 5 This application is a continuation-in-part of Application Serial No. 10/619,184, filed July15, 2003. BACKGROUND OF THE INVENTION 1. Field of the Invention 10 The present invention relates to high throughput assay systems and methods for identifying agents that alter the level of surface expression of integral membrane proteins, such as cardiac ion channels, in mammalian cells. The present invention also relates to therapeutic methods of using agents identified using such methods. 15 2. Background of the Related Art A. Assays Human Ether-a-go-go Related Gene (bERG) is the pore-forming potassium channel subunit that underlies the cardiac repolarizing current Iy, and consists of six transmembrane segments (S1-S6) and cytoplasmic amino- and carboxyl-termini. HERG has been linked to 20 both congenital and drug-induced long QT syndrome, a serious and potential fatal heart condition. Mutations in hERG produce functionally impaired channels and/or trafficking defective channels, both of which reduce I currents. Mutations spanning most of the molecule have been identified in different long QT families. This suggests that hERG plays 25 a critical role in cardiac physiology. Most of the drugs associated with long QT syndrome (drug-induced) are hERG blockers. See, e.g., Vandenberg et al, Trends Pharmacol Sd. 22:240-246 (2001). Since the cardiotoxicity of the non-sedating antihistamine terfenadine (Seldane) was linked to bERG block in 1996 (see Roy et al., Circmlation 94:817-823 (1996)), a wide variety of drugs having 30 diverse structures, including antiarrhythmics, antibiotics, antipsychotics as well as antihistamines, have been shown to be potent hERG blockers. la WO 2005/009441 PCT/US20041022751 Accordingly, hERG has become an important target for cardiac safety testing of new therapeutic agents. The US Food & Drug Administration currently recommends that pharmaceutical companies seeking approval for novel therapeutic compounds have them screened for potential hERG blocking. 5 Presently, hERG cardiac safety testing involves eletrophysiology and consits of patch clamp recording of hERG currents in HEK 293 cells which stably overexpress hERG. This assay, however, is expensive, time-consuming and requires considerable expertise. Consequently, it is usually done relatively late in the drug development process. Unfortunately, at that time, discovery that a novel therapeutic compound is a potent hERG blocker would be 10 potentially devastating to the prospects of that compound being approved and used therapeutically. As a result, there is considerable interest in the pharmaceutical industry for assays for hERG blockers that are both less expensive and faster, and that can be employed much earlier in the drug development process. The limitations of the patch clamp assay has led to alternative methods for preclinical 15 screening of drugs for potential hERG interactions. Several methods have been described, but are limited, for example, in sensitivity, throughput capacity and/or false-postive rates (see Xu et al, Drug Discovey Today 6:1278-1287 (2001)). For example, one type of assay uses membrane potential sensitive fluorescent dyes, such as DiBAC 4 or FMP. Since these assays measure changes in membrane potential and not hERG 20 activity, the risk of false positives (i.e. drugs which change membrane potential but do not block hERG) is great. A recent evaluation of such assays (Tang et al, J. Biomol Semen. 6:325-331 (2001)) indicates a signficant problem with false positives and, to a lesser extent, false negatives. In addition, sensitivity is reduced about 100-fold. Moreover, the rank order of hERG blocker potency differs with membane potential assays relative to patch clamp measurements, limiting 25 the use of such fluorescent assays to identifying potential hERG channel blockers without providing useful information as to their potency. Finally, dye/druginteractions have also caused problems in this assay. A second assay suggested as a potential high throughput preclinical screen for hERG interactions is [ 3 H]-dofelitide binding to membranes for hERG transfected cells. (See Finlayson -2- WO 2005/009441 PCT/US2004/022751 et al, Eur. J. Pbarm 430:147-148 (2001)). This binding assay is nonfunctional and relies on the ability of drugs to compete with [ 3 H]-dofelitide for binding to hERG channels. In preliminary experiments, however, the rank order of hERG blockers identified by patch clamp methods was not replicated in the [H]-dofeitide binding assay. Also, the requirements for purified cell 5 membranes as binding substrate and radio-labelled dofelitide limit the usefulness of this assay. A third assay that has been suggested involves the measurement of rubidium (Rb) flux through cells expressing hERG (see Terstappen, Anal Biochem. 272:149-155 (1999)). These cells are loaded with Rb channels and activated with high potassium levels, and Rb released into the medium is measured. The rank order of potency obtained by this method, however, does not 10 correlate with patch clamp data (see Tang et al,J. BiomoL Scnen., 6:325-331 (2001)). In addition, throughput is limited and sensitivity reduced 10-fold. Accordingly, there remains a need for assay systems for identifying blockers of integral membrane proteins, including cardiac ion channels such as hERG. 15 B. Cardiac Arrhythmias Atrial flutter and/or atrial fibrillation (AF) are the most commonly sustained cardiac arthythmias in clinical practice, and are likely to increase in prevalence with the aging of the population. Currently, AF affects more than 1 million Americans annually, represents over 5% of all admissions for cardiovascular diseases and causes more than 80,000 strokes each year in !0 the United States. While AF is rarely a lethal arrhythmia, it is responsible for substantial morbidity and can lead to complications such as the development of congestive heart failure or thromboembolism. Currently available Class I and Class III anti-arrhythmic drugs reduce the rate of recurrence of AF, but are of limited use because of a variety of potentially adverse effects, including ventricular proarrhythmia. Because current therapy is inadequate and fraught with side 25 effects, there is a clear need to develop new therapeutic approaches. Ventricular fibrillation (VF) is the most common cause associated with acute myocardial infarction, ischemic coronary artery disease and congestive heart failure. As with AF, current therapy is inadequate and there is a need to develop new therapeutic approaches. -3- WO 2005/009441 PCT/US2004/022751 Although various anti-arrhythmic agents are now available on the market, those having both satisfactory efficacy and a high margin of safety have not been obtained. For example, anti arrhythmic agents of Class I, according to the classification scheme of Vaughan-Williams ("Classification of antiarrhythmic drugs", Cardiac Arrhythnias, edited by: E. Sandoe, E. 5 Flensted-Jensen, K. Olesen; Sweden, Astra, Sodertalje, pp 449-472 (1981)), which cause a selective inhibition of the maximum velocity of the upstroke of the action potential (VrQj are inadequate for preventing ventricular fibrillation because they shorten the wave length of the cardiac action potential, thereby favoring re-entry. In addition, they have problems regarding safety, i.e. they cause a depression of myocardial contractility and have a tendency to induce 10 arrhythmias due to an inhibition of impulse conduction. The CAST (coronary artery suppression trial) study was terminated while in progress because the Class I antagonists had a higher mortality than placebo controls. B-adrenergenic receptor blockers and calcium channel (Ic) antagonists, which belong to Class II and Class IV, respectively, have a defect in that their effects are either limited to a certain type of arrhythmia or are contraindicated because of their cardiac 15 depressant properties in certain patients with cardiovascular disease. Their safety, however, is higher than that of the anti-arrhythmic agents of Class I. Anti-arrhythmic agents of Class III are drugs that cause a selective prolongation of the action potential duration (APD) without a significant depression of the maximum upstroke velocity (V.n). They therefore lengthen the save length of the cardiac action potential increasing 20 refractories, thereby antagonizing re-entry. Available drugs in this class are limited in number. Examples such as sotalol and aniodarone have been shown to possess interesting Class III properties (Singh B. N., Vaughan Williams E. M., "A third class of anti-arthythmic action: effects on attial and ventricular intracellular potentials and other pharmacological actions on cardiac muscle of MJ 1999 and AH 3747", BrJ. Pharmacol 39:675-689 (1970), and Singh B. N.,Vaughan 25 Williams E. M., "The effect of amiodarone, a new anti-anginal drug, on cardiac muscle", Br. J. Pharmaco39:657-667 (1970)), but these are not selective Class III agents. Sotalol also possesses Class II (B-adrenergic blocking) effects which may cause cardiac depression and is contraindicated in certain susceptible patients. -4- WO 2005/009441 PCTUS2004/022751 Amiodarone also is not a selective Class III antiarrhythmic agent because it possesses multiple electrophysiological actions and is severely limited by side effects. (Nademanee, K., "The Amiodarone Odyssey",J. Am. Coll Cardiol 20:1063-1065 (1992)) Drugs of this class are expected to be effective in preventing ventricular fibrillation. Selective Class III agents, by 5 definition, are not considered to cause myocardial depression or an induction of arrhythmias due to inhibition of conduction of the action potential as seen with Class I antiarrhythmic agents. Class III agents increase myocardial refractoriness via a prolongation of cardiac action potential duration (APD). Theoretically, prolongation of the cardiac action potential can be achieved by enhancing inward currents (i.e. Na+ or Ca 2 + currents; hereinafter INa and Ic, 10 respectively) or by reducing outward repolarizing potassium K+ currents. The delayed rectifier (Ix) K+ current is the main outward current involved in the overall repolarization process during the action potential plateau, whereas the transient outward (J) and inward rectifier (I) K+ currents are responsible for the rapid initial and terminal phases of repolarization, respectively. Cellular electrophysiologic studies have demonstrated that IK consists of two 15 pharmacologically and kinetically distinct K+ current subtypes, IK (rapidly activating and deactivating) and IK (slowly activating and deactivating). (Sanguinetti and Jurkiewicz, "Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by Class III anti-arrhytmic agents", JGen Physiol96:195-215 (1990)). Iis also the product of the human ether-a-go-go gene (hERG). Expression of hERG cDNA in cell lines leads to production of M0 the hERG current which is almost identical to IK (Curran et al., "A molecular basis for cardiac arrhythmia: hERG mutations cause long QT syndrome," Cell 80(5):795-803 (1995)). Class III anti-arthythmic agents currently in development, including d-sotalol, dofetilide (UK-68,798), almokalant (H234/09), E-4031 and methanesulfonamide--N--[I'-6-cyano-1,2,3,4 tetrahydro-2-naphthaleny)-3,4-dihydro-4-hydroxyspiro[2H-1-benzopyran-2, 4'-piperidin]-6y], 25 (+)-, monochloride (MK-499) predominantly, if not exclusively, block IK. Although, amiodarone is a blocker of IK, (Balser J. R. Bennett, P. B., Hondeghem, L. M. and Roden, D. M. "Suppression of time-dependent outward current in guinea pig ventricular myocytes: Actions of quinidine and amiodarone", Circ. Res. 69:519-529 (1991)), it also blocks IN. and IC, effects thyroid function, is as a nonspecific adtenergic blocker, acts as an inhibitor of the enzyme -5- WO 2005/009441 PCT/US2004/022751 phospholipase, and causes pulmonary fibrosis (Nademanee, K. "The Amiodarone Odessey". J. Am. Coi Cardioi 20:1063-1065 (1992)). Reentrant excitation (reentry) has been shown to be a prominent mechanism underlying supraventricular arthythmias in man. Reentrant excitation requires a critical balance between 5 slow conduction velocity and sufficiently brief refractory periods to allow for the initiation and maintenance of multiple reentry circuits to coexist simultaneously and sustain AF. Increasing myocardial refractoriness by prolonging APD, prevents and/or terminates reentrant arrhythmias. Most selective, Class III antiarrhythmic agents currently in development, such as d-sotalol and dofetilide predominantly, if not exclusively, block Iy, the rapidly activating component of IK 10 found both in atrium and ventricle in man. Since these I, blockers increase AF'D and refractoriness both in atria and ventricle without affecting conduction per se, theoretically they represent potential useful agents for the treatment of arrhythmias like AF and VF. These agents have a liability in that they have an enhanced risk of proarrhythmia at slow heart rates. For example, torsade de pointes, a specific 15 type of polymorphic ventricular tachycardia which is commonly associated with excessive prolongation of the electrocardigraphic QT interval, hence termed "acquired long QT syndrome", has been observed when these compounds are utilized (Roden, D. M. "Current Status of Class III Antiarrhythmic Drug Therapy", Am J. Cardio, 72:44B-49B (1993)). The exaggerated effect at slow heart rates has been termed "reverse frequency-dependence" and is 20 in contrast to frequency-independent or frequency-dependent actions. (Hondeghem, L. M., "Development of Class III Antiarrhythmic Agents",j. Cardiovasc. Cardiol 20 (Suppl. 2):S17-S22). The pro-arrhythmic tendency led to suspension of the SWORD trial when d-sotalol had a higher mortality than placebo controls. The slowly activating component ofthe delayed rectifier (I) potentially overcomes some 25 of the limitations of IK, blockers associated with ventricular arrhythmias. Because of its slow activation kinetics, however, the role of IK, in atrial repolarization may be limited due to the relatively short APD of the atrium. Consequently, although I1c blockers may provide distinct advantage in the case of ventricular arrhythmias, their ability to affect supra-ventricular tachyarrhythmias (SVT) is considered to be minimal. -6- WO 2005/009441 PCT/US2004/022751 Another major defect or limitation of most currently available Class III anti-arrhythmic agents is that their effect increases or becomes more manifest at or during bradycardia or slow heart rates, and this contributes to their potential for proarrhythmia. On the other hand, during tachycardia or the conditions for which these agents or drugs are intended and most needed, 5 they lose most of their effect. This loss or diminishment of effect at fast heart rates has been termed "reverse use-dependence" (Hondeghem and Snyders, "Class III antiarrhythmic agents have a lot of potential but a long way to go: Reduced effectiveness and dangers of reverse use dependence", Grcu/aion, 81:686-690 (1990); Sadanaga et al., "Clinical evaluation of the use dependent QRS prolongation and the reverse use-dependent QT prolongation of class III anti 10 arrhythmic agents and their value in predicting efficacy" Amer. HeartJournaI126:114-121 (1993)), or "reverse rate-dependence" (Bretano, "Rate dependence of class III actions in the heart", Fundam. Clin. Pharmacol 7:51-59 (1993); Jurkiewicz and Sanguinetti, "Rate-dependent prolongation of cardiac action potentials by a methanesulfonanilide class III anti-arrhythmic agent- Specific block of rapidly activating delayed rectifier K+current by dofetilide", Circ. ReS. 15 72:75-83 (1993)). Thus, an agent that has a use-dependent or rate-dependent profile, opposite that possessed by most current class III anti-arrhythmic agents, should provide not only improved safety but also enhanced efficacy. In view of the problems associated with current class III anti-arrhythmic agents, there remains a need for an effective treatment of cardiac arrhythmias in mammals. 20 SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide assays and methods for identifying agents which alter the level of surface expression ofintegralmembrane proteins, such 25 as cardiac ion channels. It is also an object of the present invention to provides methods of preventing or treating cardiac arrhythmia. In accordance with these and other objects, a first embodiment of the present invention is directed to a method for identifying an agent that alters the level of surface expression of an integral membrane protein in a mammalian cell, comprising: a) preparing a first medium -7- WO 2005/009441 PCT/US20041022751 containing mammalian cells that express a first mutant form of a membrane protein of interest, wherein this first mutant form is expressed on the surface of said cells at a level less than a wild type form; b) adding an effective amount of a candidate agent; c) incubating the cells in the presence of the active agent for a sufficient period of time; d) adding an effective amount of at 5 least one antibody which binds to at least one extracellular epitope of the mutant form; and e) determining the level of binding, wherein a change in said level of binding indicates that the candidate agent alters the level of surface expression. A second embodiment of the present invention is directed to a method for preventing or treating cardiac arrhythmia comprising administering to a mammal in need thereof an 10 effective amount of an active agent which increases the level of surface expression of a first mutant form of hERG in a mammalian cell and does not increase the level of surface of a second mutant form of hERG in a mammalian cell as determined by the method comprising: a) preparing a first medium containing mammalian cells that express a first mutant form of hERG which is expressed on the surface of the cells at a level lower than that of a wild-type ,5 form of hERG; b) adding an effective amount of the active agent; c) incubating the cells in the presence of the active agent for a sufficient period of time; d) adding an effective amount of at least one antibody which binds to at least one extracellular epitope of this mutant form of hERG; e) determining the level of binding of the antibody; f) preparing a second medium containing mammalian cells that express a second mutant form of hERG which is different from 0) the first mutant form and is expressed on the surface of the mammalian cells at a level lower than that of a wild-type form of hERG; g) adding an effective amount of the active agent to the second medium; h) incubating the cells in the presence of the active agent for a sufficient period of time; i) adding an effective amount of at least one antibody which binds to at least one extracellular epitope of this second mutant form; and j) determining the level of binding of the 25 antibody. Additional advantages, objects and feature of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The -8- WO 2005/009441 PCT/US2004/022751 objects and advantages of the invention may be realized and attained as particularly pointed out in the appended claims. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 5 1. Assays and Methods of Use Preferred embodiments of the present invention include an assay system and method for identifying an agent that binds to an integral membrane protein, such as a membrane ion channel, and thereby increase or decrease its surface expression in mammalian cells. In certain 10 particularly preferred embodiments, the assay and system determine the ability of an agent to bind to a particular site on a mutant form of an integral membrane protein and thereby alter the surface expression thereof. Such an alteration in surface expression may result from the agent blocking a site on the mutant that corresponds to an active site on the wild-type membrane protein and/or by blocking intracellular trafficking and/or processing of the mutant membrane 5 protein. Alternatively, an alteration in surface expression may result from the agent improving intracellular trafficking of the mutant membrane protein. There are a wide variety of formats known and available to those skilled in the art for appropriate binding assays. According to certain embodiments of the present invention, one or more cells expressing a membrane protein of interest may be provided in a suitable liquid 0 medium and exposed to one or more candidate compounds, while in other embodiments the cells may be immobilized on a surface. Similarly, according to still other embodiments of the invention, one or more candidate compounds may be immobilized on a surface and exposed to a liquid medium containing one or more cells that express a membrane protein of interest or the candidate compound(s) may be included in a suitable liquid medium to which one or more cells 5 expressing a membrane protein of interest is added. Binding is often easier to detect in systems in which at least one of the candidate compound and the membrane protein of interest is labeled (e.g., with fluorescence, radioactivity, an enzyme, an antibody, etc., including combinations thereof, as known to those skilled in the art). After exposing the candidate compound to the cell expressing a membrane protein and -9- WO 2005/009441 PCT/US2004/022751 washing off or otherwise removing unbound reagents, the presence of the labeled moiety (i.e., bound to the unlabelled component of the test system) is measured. Methods for performing various binding assays are known in the art, including but not limited to the assay systems such as those described in PCT Application US98/18368. Various 5 references provide general descriptions of various formats for protein binding assays, including competitive binding assays and direct binding assays, (see e.g, Stites and Tert, Basic and Clinical Immnology, 7th ed. (1991); Maggio, Enymelmmunoassay, CRC Press, Boca Raton, FL (1980); and Tijssen, Practice and TheoLy of En.yme Immunoassqys, in Laboratory Teelmiques in Biochemistry and Molewlar Biology, Elsevier Science Publishers, B.V. Amsterdam, (1985)). 0 Particularly preferred embodiments of the present invention involve assay systems and methods to identify compounds that increase or decrease the surface expression of a membrane protein of interest by blocking the activity of the membrane protein and/or by blocking intracellular trafficking and/or processing thereof. Thus, according to certain particularly preferred embodiments, immunoassays are 5 provided in which one or more cells expressing a membrane protein of interest are generally bound to a suitable solid support (e.g the well of a microtiter plate, a microcard, or any other similar format known to those skilled in the art) and combined with a candidate agent, and observing changes in the level of surface expression of the membrane protein of interest. Thus, in these preferred embodiments, one or more of the assay components are attached to a solid .0 surface. In some embodiments, an assay system may used (as known in the art) to detect the change in the surface expression of the membrane protein due to the binding of the candidate agent. For example, if the membrane protein of interest is a membrane ion channel, a patch clamp assay may be employed to detect a change in the flux of ions across the membrane, thus 25 evidencing an increase in the level of surface expression of the ion channel. In alternative embodiments, an indirect immunoassay system is used in which the membrane protein on the surface of the cell(s) is detected by the addition of one or more antibodies directed against an extracellular epitope of the membrane protein, as known in the art. -10- WO 2005/009441 PCTIUS2004/022751 When using a solid support in the methods of the present invention, virtually any solid surface is suitable, as long as the surface material is compatible with the assay reagents and it is possible to attach the component to the surface without unduly altering the reactivity of the assay components. Those of skill in the art recognize that some components exhibit reduced 5 activity in solid phase assays, but this is generally acceptable, as long as the activity is sufficient to be detected and/or quantified. Suitable solid supports include, but are not limited, to any solid surface such as glass beads, planar glasses, controlled pore glasses, plastic porous plastic metals, or resins to which a material or cell may be adhered, etc.). Those of skill in the art recognize that in some .0 embodiments, the solid supports used in the methods of the present invention may be derivatized with functional groups (e.g hydroxyls, amines, carboxyls, esters, and sulfhydryls) to provide reactive sites for the attachment of linkers or the direct attachment of the candidate agent or other assay component. Adhesion of an assay component to a solid support can be direct (ie. the component 5 directly contacts the solid surface) or indirect (i.e. an agent and/or component (e.g an antibody) is/are bound to a support, and the other assay component(s) binds to this agent or component rather than to the solid support). In some embodiments, the agent or component is covalently immobilized (e.g, utilizing single reactive thiol groups of cysteine for anchoring proteinaceous components (see e.g, Bioconjug Chem., 4:528-536 (1993)), or non-covalently, but specifically (e.g., '0 via immobilized antibodies or other specific binding proteins (see e.g, Adv. Mater., 3:388-391 (1991); Anal Chem., 67:83-87 (1995))), the biotin/streptavidin system (see e.g, Biophys. Biochem. es. Commun., 230:76-80 (1997)), or metal-chelating Langmuir-Blodgett films (see e.g, Langmuir 11:4048-4055 (1995); Angew. Chem. Int. Ed. Engl, 35:317-320 (1996); Pmc. Nat Acad. Sd. USA 93:4937-4941 (1996); and. Struct. Bio, 113:117-123 (1994)), and metal-chelating self-assembled 25 monolayers (see e.g,Anal Chem.,68:490-497 (1996)), for binding ofpolyhistidine fusion proteins. In some particularly preferred embodiments, standard direct or indirect ELISA, IFA, or RIA methods as generally known in the art are used to detect the binding of a candidate agent to a membrane protein of interest. In some embodiments, an increase in the level of surface expression of the membrane protein is detected in a sample, while in other embodiments, a -11- WO 2005/009441 PCTIUS2004/022751 decrease in the level of surface expression is detected. Thus, it is clear that the methods of the present invention are adaptable to the detection, identification, and characterization of multiple elements. Accordingly, in some particularly preferred embodiments of the methods of the present 5 invention, a sandwich ELISA (enzyme-linked immunosorbent assay) with a monoclonal or polyclonal antibody for capture ("a capture antibody") and a secondary antibody ("a reporter antibody") for detection of bound antibody-antigen complex (e.g., hERG bound to anti-hERG antibody or a hERG mutant bound to the corresponding antibody) may be used. In some preferred ELISA embodiments, alkaline phosphatase conjugates are used, while 10 in still other preferred embodiments, horseradish peroxidase conjugates are used. In addition, avidin/biotin systems may also be used, particularly for assay systems in which increased signal is desired. Suitable enzymes for use in preferred embodiments include, but are not limited to, peroxidases, luciferases, alkaline phosphatases, glucose oxidases, beta-galactosidases and mixtures of two or more thereof. 15 Thus, in one illustrative method of the present invention, 100 gl biotinylated antibody (e.g., directed against hERG or a mutant thereof) appropriately diluted in blocking buffer is added to each well of avidin-precoated ELISA plates (e.g., the neutravidin plates commercially available from Pierce). After 2 hr, the plate is washed with wash buffer (e.g, TBS/Tween 20 0.1%, with or without a blocking agent). Further nonspecific binding may be inhibited by 20 adding blocking buffer (e.g., by adding 300 V1 SuperBlock (Pierce) twice, as per the manufacturer's recommendations). Following incubation to allow binding of the biotinylated antibody to the surfaces of the wells, the plate is washed (e.g., 3 times) according to methods known in the art, to remove any unbound antibody present in the wells. Samples of cells expressing a membrane protein of interest may be diluted with an 25 appropriate buffer and added to the wells of the ELISA plate, as well as. standards and controls. The diluted standards, controls, and samples, maybe added to the wells of the ELISA plate (e.g, 100 sl/well). Standards, controls, and samples are generally tested in duplicate. The plate is incubated, for example, overnight, or for another appropriate length of time, typically on a rocking table at 5 RPM or other suitable agitation means in a humidor or the like. The plate is -12- WO 20051009441 PCT/US2004/022751 washed (e.g., 3 times or the like) with washing buffer as known in the art. Then, 100 i of appropriately diluted monoclonal or polyclonal reporter antibody (preferably preabsorbed with the antibody used to coat the wells of the plate), may be added and allowed to incubate at room temperature, preferably overnight (e.g, about 18-20 hours), or for another such incubition period 5 as may be appropriate. The plate may then be washed again, preferably as described above, and 100 d enzyme lab eled antibody, such as alkaline phosphatase-conjugated anti-rabbit Ig (commercially available from Pierce), appropriately diluted in a suitable blocking buffer (e.g., BSA Blocker in TBS) may be added, and allowed to incubate for a sufficient period (e.g 2 hours) with rocking or similar 0 agitation as described above. The plate may then preferably be washed again as described above. The enzyme substrate may be added to the wells and the reaction allowed to occur for an appropriate length of time, at the end of which the reaction is stopped using any appropriate method known in the art, and the optical densities of the solutions within the wells determined as known in the art. 5 Because background signal is often the limiting factor in amplified assays, in some embodiments of the present invention, measures may be undertaken to reduce background signal in these assays. In addition to the assay systems in which a solid support is utilized, the present invention provides methods in which the assay components remain suspended in solution. 0 According to a first particularly preferred embodiment of the present invention, a method is provided for identifying an agent, such as a peptide, protein, antibody or chemical agent, that alters the level of surface expression of an integral membrane protein, such as hERG, in a mammalian cell. This method comprises: a) preparing a first medium containing mammalian cells that express a first mutant form of the membrane protein of interest , wherein 25 this first mutant form is expressed on the surface of the cells at a level less than a wild-type form of the protein; b) adding an effective amount of a candidate agent; c) incubating the cells in the presence of the candidate agent for a sufficient period of time; d) adding an effective amount of at least one antibody which binds to at least one extracellular epitope of the mutant; and e) -13- WO 20051009441 PCT/US20041022751 determining the level of binding of the antibody to the extracellular epitope of the protein following incubation of the cells with the candidate agent. Any change, such as an increase or decrease, in the level of binding in the presence of the candidate agent relative to control indicates that the candidate agent alters the level of surface 5 expression of the first mutant form of the membrane protein. According to a second particularly preferred embodiment of the present invention, the above method further comprises: f) preparing a second medium containing mammalian cells that express a second mutant form of the membrane protein of interest, wherein this second mutant form is different from the first mutant form and is also expressed on the surface of said D mammalian cells at a level lower than that of a wild-type form of the membrane protein; g) adding an effective amount of the candidate agent; h) incubating said cells in the presence of said candidate agent for a sufficient period of time; i) adding an effective amount of at least one antibody which binds to at least one extracellular epitope of the second mutant form; and j) determining the level of binding of the antibody to the extracellular epitope. Any change in the 5 level of binding following incubation with the candidate agentindicates that the candidate agent alters the level of surface expression of the second mutant form of the membrane protein of interest. According to preferred embodiments of the present invention, step (d) above comprises adding an effective amount of at least one primary antibody and an effective amount of at least D one secondary antibody. According to such embodiments, the primary antibody preferably binds to at least one extracellular epitope of the first mutant form of the membrane protein of interest. Even more preferably, according to such embodiments, the secondary antibody binds to the first antibody. According to still other preferred embodiments of the present invention, step (i) above 25 also comprises adding an effective amount of at least one primary antibody and an effective amount of at least one secondary antibody. According to such embodiments, the primary antibody preferably binds to at least one extracellular epitope of the second mutant form of the membrane protein of interest. Even more preferably, according to such embodiments, the secondary antibody binds to the first antibody. -14- WO 2005/009441 PCTIUS2004/022751 Preferably, the secondary antibody is coupled to an enzyme to facilitate detection and determination of the level of binding. Suitable enzymes for use in the methods of the present invention are known and available to those skilled in the art. Illustrative examples of suitable enzymes include, but are not limited to, peroxidases, luciferases, alkaline phosphatases, glucose 5 oxidases, beta-galactosidases and mixtures of two or more thereof. The determination of the level of surface expression of the integral membrane protein of interest may be performed using any of the methods and techniques known and available to those skilled in the art. Preferably, the level of binding is determined by fluorescence, . luminescence, radioactivity, absorbance or a combination of two or more of these. 0 According to certain particularly preferred embodiments of the present invention, the integral membrane protein is a membrane ion channel. Illustrative examples of suitable membrane ion channels include, but are not limited to, sodium channels, potassium channels, calcium channels or chloride channels. According to preferred embodiments of the present invention, the extracellular epitope 5 to which the antibody binds on the first mutant form of the membrane protein is preferably the same as a wild-type epitope, i.e. an extracellular epitope found on the naturally-occurring form(s) of the membrane protein of interest. Without wishing to be bound to any theory of operability or the like, such an arrangement may have the potential to reduce errors arising from differences in protein structure, for example by a change in one or more of the functional properties of the .0 protein. According to still other preferred embodiments ofthe present invention, the extracellular epitope to which the antibody binds on the second mutant form of the membrane protein is preferably the same as a wild-type epitope. According to particularly preferred embodiments of the present invention, the 25 extracellular epitope on the first and/or the second mutant forms of the membrane protein contains a tag. Suitable tags are known and available to those skilled in the art. A particularly preferred tag for use in the methods of the present invention is a hemagglutinin (HA) tag. The tag may be inserted in an extracellulat domain of the first and/or the second mutant forms of the membrane protein or may replace a portion of an extracellular domain thereof. -15- WO 2005/009441 PCT/TS2004/022751 According to the methods of the present invention, the first and second mutant forms of the membrane protein preferably both differ in at least one amino acid residue from the amino acid sequence(s) of the wild-type form(s) of the membrane protein. Moreover, the first and second mutant forms also preferably differ in at least one amino acid residue from the 5 amino acid sequence of each other. According to certain preferred embodiments, the first mutant form of the membrane protein may be a trafficking-deficient mutant and so is expressed on the surface of mammalian cells at a level lower than a corresponding wild-type form. According to still other preferred embodiments, the second mutant form may be a trafficking-deficient mutant and according to 10 yet still other preferred embodiments, both the first and the second mutant forms may be a trafficking-deficient mutants. According to certain particularly preferred embodiments of the present invention, the membrane protein of interest is a cardiac ion channel, most preferably a potassium ion channel. Such ion channels are known to those skilled in the art. An illustrative example of such a 15 potassium ion channel is hERG. Suitable first mutant forms of hERG for use in the methods of the present invention are known to those skilled in the art. According to preferred embodiments of the present invention, such mutant forms should function when expressed on the cell surface, but should be expressed on the surface at a lower level than any wild-type form(s), most preferably due to a trafficking 0 deficiency. Illustrative examples of suitable mutant forms include, but are not limited to, G6018 (which has been identified in a long QT syndrome family) and N470D. G601S is a trafficking deficient, hypomorphic channel which generates minimal, although kinetically unaltered, currents (see Furatani et al, Circulation 99:2290-2294 (1999)) and N470D is a hypomorphic missense mutation. Agents which increase surface expression of mutants such as G601S and 25 N470D also bind to a high affinity site in the hERG ion conduction pathway and are therefore potent hERG blockers. For purposes of illustration and not imitation, in a preferred embodiment of the present invention, a mutant form of anion channel, such as hERG-G601S, is engineered to express an extracellular tag, such as an HA tag, in the linker between transmembrane domains S1 and S2 -16- WO 2005/009441 PCT/US20041022751 (such a tag preferably should not alter the functional properties of the channel). Cells, such as HEK 293 cells, stably expressing this tagged mutant, e.g. hERG-G60IS-HA, are plated in a suitable container, such as a 9 6-well microtiter plate, and incubated with one or more candidate agents for a sufficient time, such as overnight. The cells are then preferably fixed, such as with 5 formaldehyde, but preferably not permeabilized and antibodies recognizing the HA tag are added. A secondary antibody, preferably conjugated to an enzyme, such as horseradish peroxidase, is used to bind the anti-HA antibody(ies) bound to the surfaces of the fixed cells. Cell surface signals may then be developed by any suitable method, such as a chemiluminescent reaction mix, and the level measured, for example, in a microtiter plate luminometer. Control 10 cells are usually incubated with water and/or any liquid vehicle used in conjunction with the candidate agent, such as DMSO. Agents which increase the surface expression of the first mutant form are potential blockers of hERG (or similar such ion channel). Suitable second mutant forms of hERG for use in the methods of the present invention are also known to those skilled in the art. According to preferred embodiments of the present 15 invention, such mutant forms should also be expressed on the surface at a lower level than any wild-type form(s), most preferably due to a trafficking deficiency, but should not function when expressed on the cell surface. Illustrative examples of suitable mutant forms of hERG include, but are not limited to, G601S/F656C and N470D/F656C. Agents which do not increase surface expression of mutants such as G601S/F656C and N470D/F656C are potent hERG 20 blockers. Again for purposes of illustration and not limitation, in a preferred embodiment of the present invention, a second mutant form of the ion channel, such as hERG-G601S/F656C, is engineered to express an extracellular tag, such as an HA tag, in the linker between transmembrane domains S1 and S2 (again, such a tag preferably should not alter the functional 25 properties of the channel). Cells, such as HEK 293 cells, stably expressing this tagged mutant, e.g. hERG-G601S/F656C-HA, are plated in a suitable container, such as a 9 6-well microtiter plate, and incubated for a sufficient time with a candidate agent, preferably a candidate agents which increased surface expression of the first mutant form. The cells are then preferably fixed and not permeabilized, and primary antibodies recognizing the HA tag are added. Secondary -17- WO 2005/009441 PCTUS2004/022751 antibodies, preferably conjugated to an enzyme are then used to bind the anti-HA antibody(ies) bound to the surfaces of the fixed cells. Cell surface signals may then be developed by any suitable method, such as a ch-emiluminescent reaction mix, and the level measured, for example, in a microtiter plate luminometer. Control cells are usually incubated with water and/or any 5 liquid vehicle used in conjunction with the candidate agent, such as DMSO. Those agents which do not increase the level of surface expression of the second mutant form are likely to be potent hERG blockers. Agents which increase the level of surface expression of the first mutant form and do not increase the level of surface expression of the second mutant form are highly likely to be potent hERG blockers. 0 According to more particularly preferred embodiments of the above methods, hERG surface expression is assayed by removing the microtiter plate(s) from the incubator(s) and removing the media bathing the cells. The wells are rinsed three times with PBS (100 pl) and then the cell fixed with paraformaldehyde (e.g 4% in PBS, pH 7.2, 100 ld), and then rinsed with PBS. Non-specific binding sites on the cell surface are preferably blocked, for example by 5 incubating the cells with 1% goat serum in PBS ("blocking buffer"). After removing the blocking buffer, the cells are incubated with the primary antibody, such as rat and-HA in blocking buffer. The primary antibody is then removed, the cells washed (e.g 3 times with blocking buffer). Secondary antibody, such as horseradish peroxidase-conjugated anti-rat goat antibody in blocking buffer, is added. The secondary antibody is then removed and the cells 0 preferably washed. According to such embodiments, chemiluminescent signals may be developed using any suitable technique, such as SuperSignal ELISA Femto Maximum Sensitivity Substrate (Pierce Chemical Co.). A suitable amount of reagent, e.g 50 Fl for each well of a microtiter plate, is added and a GloRunner luminometer (Turner Designs) used to obtain the data. 25 A fluorescent reaction may optionally be added to monitor the number of cells per well. II. Therapeutic Compositions and Methods The preferred embodiments of the present invention also methods of preventing or treating cardiac arrhythmia by administering to a mammal in need thereof, such as a human in -18- WO 20051009441 PCT/US2004/022751 need thereof, an effective amount of an agent identified using the assay described above. Preferably, the inventive methods of preventing or treating cardiac arrhythmia by administering to a mammal in need thereof, such as a human in need thereof, an effective amount of an agent which increases the level of surface expression of a first mutant form of hERG, such as G601S 5 or N470D. Even more preferably, such an agent does not increase the level of expression of a second mutant form of hERG, such as G60IS/F656C or N470D/F656C. The preferred embodiments of the present invention also include compositions for preventing or treating cardiac arrhythmia in a mammal, such as a human, comprising an effective amount of such an agent in combination with a pharmaceutically acceptable carrier. 10 An illustrative example of a particularly preferred agent suitable for use in the therapeutic methods and compositions of the present invention is vanoxerine (also known as GBR-12909). Vanoxerine, its manufacture and/or certain pharmaceutical uses thereof are described in U.S. Patents Nos. 4,202,896, 4,476,129 and 4,874,765, as well as European Patent EP 243,903 and PCT International Application WO 91/01732. 5 Pharmaceutically acceptable salts of vanoxerine may also be employed in the methods of the present invention. The pharmaceutically acceptable salts of vanoxerine which may be used in the inventive methods include, but are not limited to, salts of vanoxerine formed from non-toxic inorganic or organic acids. For example, pharmaceutically acceptable salts include, but are not limited to, the following: salts derived from inorganic acids, such as hydrochloric, 0 hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; salts derived from organic acids, such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, benzoic, salicylic, sulfanilic, 2 -acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like; and salts derived from amino acids, such as glutamic acid or aspartic acid. See U.S. Patent 25 6,187,802 and WO 91/01732. The pharmaceutically acceptable salts of vanoxerine useful in the methods of the present invention can be synthesized from vanoxerine by conventional chemical methods. Generally, the salts are prepared either by ion exchange chromatography or by reacting the free base with -19- WO 2005/009441 PCT/US2004/022751 stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. Pharmaceutically acceptable metabolites ofvanoxerine may be employed in the methods of the present invention, provided that they elicit the necessary pharmacological respsonse(s) 5 when administered to a mammal, such as a human, and are otherwise appropriate for use in the invention methods, e.g., exhibit an acceptable toxicology profile, are relatively stable under the conditions of use, etc. Illustrative examples of suitable metabolite which may be employed in the inventive methods include, but are not limited to, the following: 1-[2 (diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (which is also known as GBR 12935 and .0 is the principal metabolite of vanoxerine in humans) and pharmaceutically acceptable salts, analogs and derivatives thereof. Pharmaceutically acceptable derivatives of vanoxerine may also be employed in the methods of the present invention, provided that they elicit the necessary pharmacological responses when administered to a mammal, such as a human, and are otherwise appropriate for 5 use in the invention methods, e.g., exhibit an acceptable toxicology profile, are relatively stable under the conditions of use, etc. Illustrative examples of suitable derivatives which may be employed in the inventive methods include, but are not limited to, the following: GBR 13069 and GBR 12783, which are structurally similar to vanoxerine and GBR 12935, respectively, except that the 3-phenylpropyl moiety has been replaced by a 3-phenylpropen-2-yl moiety. 0 Other suitable derivatives include phenolic derivatives of vanoxerine, i.e. derivatives of vanoxerine in which the unsubstituted phenyl group ofvanoxerine is substituted by one or more hydroxy groups, as well as the methoxy congeners thereof. (See Rice et at, "Oxygenated analogues of 1-(2-(diphenylmethoxy)ethyl)- and 1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3 phenylpropyl) piperazines (GBR 12935 and GBR 12909) as Potential Extended-Action Cocaine 25 Abuse Therapeutic Agents,"J. Med Chem. 42(23):5029-5042 (2001); and Dutta etal, "Positional Importance of the Nitrogen Atom in Novel Piperidine Analogs of GBR 12909: Affinity and Selectivity for the Dopamine Transporter," Med Chem. Res. 3(4):209-222 (1993)). -20- WO 2005/009441 PCT/US2004/022751 Additional examples of suitable derivatives which may be employed in the methods of the present invention include 4-[2-bis(halophenyl)methoxy]-ethyl]-a-(substituted phenyl)-1 piperazine alkanol derivatives. See, e.g, U.S. Patent 4,476,129. When employed in the present methods, vanoxerine, or a pharmaceutically acceptable 5 salt, derivative or metabolite thereof, may be administered by any technique capable of introducing a pharmaceutically active agent to the desired site of action, including, but not limited to, buccal, sublingual, nasal, oral, topical, rectal and parenteral administration. Delivery of the compound may also be through the use of controlled release formulations in subcutaneous implants or transdermal patches. 10 For oral administration, a suitable composition containing -vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, may be prepared in the form of tablets, dragees, capsules, syrups and aqueous or oil suspensions. The inert ingredients used in the preparation of these compositions are known in the art. For example, tablets may be prepared by mixing the active compound with an inert diluent, such as lactose or calcium 15 phosphate, in the presence of a disintegrating agent, such as potato starch or microcrystalline cellulose, and a lubricating agent, such as magnesium stearate or talc, and then tableting the mixture by known methods. Tablets may also be formulated in a manner known in the art so as to give a sustained release of vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof. 20 Such tablets may, if desired, be provided with enteric coatings by known method, for example by the use of cellulose acetate phthalate. Suitable binding or granulating agents include, but are not limited to gelatine, sodium carboxymethycellulose, methylcellulose, polyvinylpyrrolidone or starch gum. Talc, colloidal silicic acid, stearin as well as calcium and magnesium stearate or the like can be used as anti-adhesive and gliding agents. 25 Tablets may also be prepared by wet granulation and subsequent compression. A mixture containing the vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, and at least one diluent, and optionally a part of the disintegrating agent, is granulated together with an aqueous, ethanolic or aqueous-ethanolic solution of the binding agents in an appropriate equipment, then the granulate is dried. Thereafter, other preservative, surface acting, -21- WO 2005/009441 PCT/US20041022751 dispersing, disintegrating, gliding and anti-adhesive additives can be mixed to the dried granulate and the mixture can be compressed to tablets or capsules. The tablets may also be prepared by the direct compression of the mixture containing the active ingredient together with the needed additives. If desired, the tablets may be transformed 5 to dragees by using protective, flavoring and dyeing agents such as sugar, cellulose derivatives (methyl- or ethylcellulose or sodium carboxymethylcellulose), polyvinylpyrrolidone, calcium phosphate, calcium carbonate, food dyes, aromatizing agents, iron oxide pigments and the like which ate commonly used in the pharmaceutical industry. For the preparation of capsules or caplets, a mixture ofvanoxerine, or a pharmaceutically 10 acceptable salt, derivative or metabolite thereof, and the desired additives may be filled into a capsule, such as a hard or soft gelatin capsule. The contents of a capsule and/or caplet may also be formulated using known methods to give sustained release of the active compound. Liquid oral dosage forms of vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, may be an elixir, suspension and/or syrup, where the compound is mixed L5 with a non-toxic suspending agent. Liquid oral dosage forms may also comprise one or more sweetening agent, flavoring agent, preservative and/or mixture thereof. For rectal administration, a suitable composition containing vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, may be prepared in the form of a suppository. In addition to the active ingredient, the suppository may contain a suppository 20 mass commonly used in pharmaceutical practice, such as Theobroma oil, glycerinated gelatin or a high molecular weight polyethylene glycol. For parenteral administration, a suitable composition of vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, may be prepared in the form of an injectable solution or suspension. For the preparation of injectable solutions or 25 suspensions, the active ingredients can be dissolved in aqueous or non-aqueous isotonic sterile injection solutions or suspensions, such as glycol ethers, or optionally in the presence of solubilizing agents such as polyoxyethylene sorbitan monolaurate, monooleate or monostearate. These solutions or suspension may be prepared from sterile powders or granules having one or more carriers or diluents mentioned for use in the formulations for oral administration. -22- WO 20051009441 PCTIUS2004/022751 Parenteral administration may be through intravenous, intradermal, intramuscular or subcutaneous injections. A composition containing vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, may also be administered nasally, for example by sprays, aerosols, 5 nebulised solutions and/or powders. Metered dose systems known to those in the art may also be used. Pharmaceutical compositions of vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, may be administered to the buccal cavity (for example, sublingually) in known pharmaceutical forms for such administration, such as slow dissolving 10 tablets, chewing gums, troches, lozenges, pastilles, gels, pastes, mouthwashes, rinses and/or powders. Compositions containing vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, for topical administration may comprise a matrix in which the pharmacologically active compound is dispersed such that it is held in contact with the skin in 15 order to administer the compound transdermally. A suitable transdermal composition may be prepared by mixing vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, with a topical vehicle, such as a mineral oil, petrolatum and/or a wax, for example paraffin wax or beeswax, together with a potential transdermal accelerant such as dimethyl sulphoxide or propylene glycol. Alternatively, vanoxerine, or a pharmaceutically acceptable salt, 20 derivative or metabolite thereof, may be dispersed in a pharmaceutically acceptable cream or ointment base. The amount of vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, contained in a topical formulation should be such that a therapeutically effective amount delivered during the period of time for which the topical formulation is intended to be on the skin. 25 Vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, may also be administered by continuous infusion either from an external source, for example by intravenous infusion or from a source of the compound placed within the body. Internal sources include implanted reservoirs containing the vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, to be infused which is continuously released for example by -23- WO 2005/009441 PCT/U1S20041022751 osmosis and implants which may be (a) liquid such as a suspension or solution in a pharmaceutically acceptable oil of the compound to be infused for example in the form of a very sparingly water-soluble derivative such as a dodecanoate salt or (b) solid in the form of an implanted support, for example of a synthetic resin or waxy material, for the compound to be 5 infused. The support may be a single body containing all the compound or a series of several bodies each containing part of the compound to be delivered. The amount of vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, present in an internal source should be such that a therapeutically effective amount is delivered over a long period of time. In addition, an injectable solution of vanoxerine, or a pharmaceutically acceptable salt, 10 derivative or metabolite thereof, can contain various additives such as preservatives, such as benzyl alcohol, methyl or propyl 4-hydroxybenzoate, benzalkonium chloride, phenylmercury borate and the like; as well as antioxidants, such as ascorbic acid, tocopherol, sodium pyrosulfate and optionally complex forming agents, such as an ethylenediamine tetraacetate salt for binding the metal traces, as well as buffers for adjusting the pH value and optionally a local 15 anaesthetizing agent, e.g. lidocaine. The injectable solution containing vanoxerine, or a pharmaceutically acceptable salt, derivative or metabolite thereof, is filtered before filling into the ampule and sterilized after filling. Other agents which could be used in such therapeutic methods of the present invention include any of the known blockers of hERG, such as astermizole, terfenadine, E-4031, cisapride, 20 chloroquine and the like. EXAMPLES Example 1 - Rescue of hERG-G601S surface expression by astemizole HEK-293 cells stably expressing hERG-G601 S-HA were plated in a BIOCOAT poly-D 25 lysine cellware 96-well black plate with a clear bottom (BD Discovery Labware). Cells were plated (8 x 10' cells/well) in complete medium containing DMEM/F12 with 10% FBS plus penicillin-streptomycin and geneticin (G418; 0.5mg/ml) and incubated for8 hours at37*C/5%
    CO
    2 prior to addition of drugs. Stock solutions(200 p.M, 1 mM and 5 mM) of the drugs (astemizole, norastemizole, fexofenadine) were prepared. Just prior to addition, working -24- WO 2005/009441 PCT/US2004/022751 dilutions (200 nM, 1pM and 5pM) were prepared in DMEM/F12 with 10% FBS. Vehicle consisted of 0.1% DMSO in DMEM/F12 with 10% FBS. The media bathing the cells was removed and replaced with drug containing or vehicle control media (100 pl/well). For each drug there were three test wells for each concentration and three control wells. The plates were 5 incubated overnight (approx. 16 hours) at 37*C/5% CO 2 prior to the start of the surface expression assay. Surface expression assays were performed on the bench top at room temperature. The cells were washed three times with 100 pl PBS, followed by fixation with freshly prepared, ice cold 4% paraformaldehyde in PBS (pH 7.2, 100d, 20 min.) The fixative was removed and the [0 cells rinsed with 100 l PBS. Nonspecific binding sites on the cells were blocked by incubation with 1% goat serum in pBS (blocking buffer, 100 Id for 30 min). Blocking buffer was removed and the cells incubated for three hours with rat anti-IA (1:500 dilution, 100 1, Roche) diluted in blocking buffer. After removing the primary antibody, the cells were washed three times with 1% goat serum in PBS (100 pl and 10 min/wash). HRP-conjugated goat anti-rat antibody 15 (1:2000; Jackson Labs) was diluted win blocking buffer and incubated with the cells for 1 hour (100 A/well). Following incubation, cells were washed with 1lOOpI of 1% goat serum in PBS (10 min) and then three times with 100 pl PBS (10 rin/wash). Chemiluminescent signals were developed with the SuperSignal ELISA Femto Maximum Sensitivity Substrate (Pierce Chemical Co.). PBS was removed from the wells and replaced with 0 100 I detection reagent per well. Signals were immediately captured using a GloRunner luminometer (Turner Designs). The data obtained showed a direct correlation between the ability of a drug to rescue surface expression of hERG-G601S-I-A and the potency with which it blocks hERG. Thus, astemizole increased surface expression much more than norastemizole, which is amuch weaker 25 blocker. Similarly, fexofenadine, which does not block hERG at all, did not increase surface expression of hERG-G601S-HA. Example 2 - Removal of the drug binding site in hERG removes rescue by astemizole -25- WO 2005/009441 PCT/US2004/022751 HEK-293 cells in 35 mm dishes were transiently transfected with cDNAs encoding hERG-G601S-HA or hERG-G601S/F656C-HA. Twenty-four hours post-transfection, astemizole (1 pM) was added to some plates and vehicle control (DMSO) added to others and the plates incubated overnight. 5 Following incubation, the cells were fixed with para formaldehyde and processed for surface expression of the HA tag as described above (with the exception of proportionally larger reagent volumes). Chemiluminescent signals were developed in a TD20/20 luminometer (Turner Biosysterns). The data obtained showed that astemizole did not rescue expression of the double 0 mutant hERG-G601S/F656C, but did rescue expression of hERG-G601S, showing that it is a potent blocker of hERG. Having now fully described this invention, it will be understood to those of ordinary skill in the art that the methods of the present invention can be carried out with a wide and equivalent range of conditions, formulations, and other parameters without departing from the 5 scope of the invention or any embodiments thereof. All patents and publications cited herein are hereby fully incorporated by reference in their entirety. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that such publication is prior art or that the present invention is not entitled to antedate such publication by virtue of prior invention. -26-
    权利要求:
    Claims (25)
    [1] 1. A method of identifying an agent that alters the level of surface expression of an integral membrane protein in a mammalian cell, said method comprising: a) preparing a first medium containing mammalian cells that express a first mutant form of said integral membrane protein , wherein said first mutant form is expressed on the surface of said cells at a level less than a wild type form of said protein; b) adding to said first medium containing' mammalian cells a test amount of a candidate agent; c) incubating said cells in the presence of said candidate agent for at least 16 hours; d) adding to said first medium containing mammalian cells an effective amount of at least one antibody which binds to at least one extracellular epitope of said mutant form of said integral membrane protein; and e) determining the level of binding of said at least one antibody to said extracellular epitope following incubation with said candidate agent, wherein a change in said level of binding relative to control indicates that said candidate agent alters the level of surface expression of said mutant form of said integral membrane protein.
    [2] 2. The method according to claim 1, further comprising the steps of: f) preparing a second medium containing mammalian cells that express a second mutant form of said integral membrane protein , said second mutant form is different from said first mutant form and is expressed on the surface of said mammalian cells at a level lower than that of a wild-type form of said integral membrane protein; 27 g) adding to said second medium containing mammalian cells an effective amount of said candidate agent; h) incubating said cells in the presence of said candidate agent for at least 16 hours; i) adding to said second medium. containing mammalian cells an effective amount of at least one antibody which binds to at least one exteacellnl2r epitope of said second mutant form of said integral membrane protein; and j) determining the level of binding of said at least one antibody to said extracellular epitope of said second mutant form of said integral membrane protein following incubation with said candidate agent, k) wherein a change in said level of binding relative to control indicates that said candidate agent alters the level of surface expression of said second mutant form of said integral membrane protein.
    [3] 3. The method according to claim 1 or 2, wherein step (d) comprises adding an effective amount of at least one primary antibody and an effective amount of at least one secondary antibody, wherein said primary antibody binds to at least one extracellular epitope of said first mutant form of said integral membrane protein and said secondary antibody binds to said first antibody.
    [4] 4. The method according to claim 1 or 2, wherein said level of binding is measured by fluorescence, luminescence, radioactivity, absorbance or a combination of two or more thereof
    [5] 5. The method according to clan'n 1 or. 2, wherein said integral membrane protein is a membrane ion channel. 28
    [6] 6. The method according to claim 5, wherein said membrane ion channel is a sodium channel, a potassium channel, a calcium channel or a chloride channel.
    [7] 7. The method according to claim 1 or 2, wherein said at least one extracellular epitope comprises a wild-type epitope.
    [8] 8. The method according to claim 1 or 2, wherein said at least one extracellular epitope contains a tag.
    [9] 9. The method according to claim 8, wherein said extracellular tag replaces at least a portion of an extracellular domain of said integral membrane protein.
    [10] 10. The method according to claim 8, wherein said extracellular tag is inserted in an extracellular domain of said integral membrane protein.
    [11] 11. The method according to claim 8, wherein said extracellular tag comprises a hemagglutinin (HA) tag.
    [12] 12. The method according to claim 3, wherein said primary antibody and/or said secondary antibody is coupled to an enzyme.
    [13] 13. The method according to claim 12, wherein said enzyme is selected from the group consisting of peroxidases, luciferases, alkaline phosphatases, glucose oxidases, beta-galactosidases and mixtures of two or more thereof
    [14] 14. The method according to claim 1 or 2, wherein said first mutant form comprises an amino acid sequence which differs in at least one amino acid residue from the amino acid sequence of a wild-type form of said integral membrane protein.
    [15] 15. The method according to claim 2, wherein said second mutant form comprises an amino acid sequence which differs in at least one amino acid residue from the amino acid sequence of a wild-type form of said integral membrane protein. 29
    [16] 16. The method according to claim 1 or 2, wherein said first mutant form is a trafficking-deficient mutant.
    [17] 17. The method according to claim 2, wherein said second mutant form is a trafficking-deficient mutant.
    [18] 18. The method according to claim 1 or 2, wherein said integral membrane protein is a potassium ion channel.
    [19] 19. The method according to claim 18, wherein said potassium ion channel is hERG.
    [20] 20. The method according to claim 19, wherein said first mutant form of hERG is G601S.
    [21] 21. The method according to claim 19, wherein said first mutant form of hERG is N470D.
    [22] 22. The method according to claim 19, wherein said second mutant form of hERG is G601S/F656C.
    [23] 23. The method according to claim 19, wherein said second mutant form of hERG is N470D/F656C.
    [24] 24. The method according to claim 2, wherein step i) comprises adding an effective amount of at least one primary antibody and an effective amount of at least one secondary antibody, wherein said primary antibody binds to at least one extracellular epitope of said second mutant form and said secondary antibody binds to said first antibody.
    [25] 25. A method as claimed in claim 1 substantially as herein described with reference to any example thereof. 30
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    AU2013200842B2|2017-02-02|
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    2014-08-07| PC1| Assignment before grant (sect. 113)|Owner name: CASE WESTERN RESERVE UNIVERSITY; THE METRO HEALTH Free format text: FORMER APPLICANT(S): CHANTEST CORPORATION |
    2017-06-01| FGA| Letters patent sealed or granted (standard patent)|
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    申请号 | 申请日 | 专利标题
    US10/619,184|US20050054010A1|2003-07-15|2003-07-15|High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins|
    US10/619,184||2003-07-15||
    US10/635,010|US7211407B2|2003-07-15|2003-08-06|High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins|
    US10/635,010||2003-08-06||
    PCT/US2004/022751|WO2005009441A1|2003-07-15|2004-07-15|High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins|
    AU2004259199A|AU2004259199A1|2003-07-15|2004-07-15|High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins|
    AU2011200195A|AU2011200195B2|2003-07-15|2011-01-19|High Throughput Assay Systems And Methods For Identifying Agents That Alter Surface Expressions Of Intergral Membrane Proteins|
    AU2013200842A|AU2013200842B2|2003-07-15|2013-02-15|High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins|AU2013200842A| AU2013200842B2|2003-07-15|2013-02-15|High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins|
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