WO2007002359A2 - Methods for identifying delta subunit-containing gaba receptor modulatory agents - Google Patents

Abstract

The present invention provides methods of identifying agents that modulate δ subunit-containing GABA receptor activity. The agents so identified find use in a variety of research and treatment methods.

Description

METHODS FOR IDENTIFYING DELTA SUBUNIT-CONTAINING GABA RECEPTOR

MODULATORY AGENTS

CROSS-REFERENCE [0001] This application claims the benefit of U.S. Provisional Patent Application No.

60/693,844, filed June 24, 2005, and U.S. Provisional Patent Application No. 60/782,834, filed March 15, 2006, which applications are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The U.S. government may have certain rights in this invention, pursuant to grant nos.

NS35985, AA07680 and AAO 15460, awarded by the National Institutes of Health.

BACKGROUND

[0003] Receptors for the major inhibitory neurotransmitter, gamma-aminobutyric acid

(GABA), are divided into two main classes: (1) GABAA receptors, which are members of the ligand-gated ion channel superfamily; and (2) GABA_B receptors, which are members of the G- protein linked receptor superfamily. Since the first cDNAs encoding individual GABAA receptor subunits were cloned the number of known members of the mammalian family has grown to include at least six α subunits, three β subunits, three γ subunits, as well as a δ subunit, a ε subunit, a θ subunit, a π subunit, and at least two p subunits. Like other members of the ligand-gated ion channel family, the native GABAA receptor typically exists in pentameric form. The most common of GABAA receptors in the brain include two α subunits, two β subunits, and a γ subunit. The receptor binds two GABA molecules.

[0004] Other ligands (besides GABA) interact with the GABAA receptor to activate it

(agonists), to inhibit its activation (antagonists) or to increase or decrease its response to an agonist (positive and negative allosteric modulators). Such other ligands include benzodiazepines and non-benzodiazepine that bind to the benzodiazepine binding site (increase pore opening frequency; often the active ingredient of sleep pills and anxiety medications), barbiturates (increase pore opening duration; used as sedatives), and certain steroids, called neuroactive steroids.

[0005] Among antagonists are picrotoxin (which blocks the channel pore) and bicuculline

(which occupies the GABA site and prevents GABA from activating the receptor). The antagonist flumazenil is used medically to reverse the effects of the benzodiazepines. [0006] Although many proteins show changes in their function at very high alcohol concentrations (> 50 niM), the molecular basis for behavioral alcohol effects at low to moderately intoxicating doses experienced during social alcohol consumption remains elusive. Although most GABAAR subunit combinations can be activated by high (anesthetic) alcohol concentrations, so far only very specific GABAAR subunit combinations (containing the δ as well as the β3 subunit) exhibit dose-dependencies that mirror blood alcohol levels associated with mild to moderate intoxication in humans (ca. 3 - 30 mM, since the legal drinking limit is 17 mM or 0.08%). GABAARS containing the δ subunit are located either outside or in the perimeter of synapses, but not in the sub-synaptic membrane. Ethanol pharmacology shares many characteristics with allosteric activators of GABAARS (loosely referred to as GABAAR agonists), like benzodiazepines (BZs).

[0007] It has been thought that the "extrasynaptic" GABAAR δ subunit, that presumably takes the position of the γ2 subunit in functional pentameric GABAARS, renders receptors insensitive to benzodiazepines. In addition, most δ subunits are found associated with α4 and α6 subunits that differ from other GABAA receptor α subunits at a critical position (a histidine in αl,2,3,5, replaced by an arginine in α4,6) that makes α_4/6β_xγ₂ receptors insensitive to classical BZ agonists like diazepam or flunitrazepam. However, (wild type) arginine 100 in α4 and α6 receptors still allows high affinity binding of the imidazobenzodiazepines Ro 15-4513 and flumazenil (in αxβxγ2 GABA_ARS). In functional receptor assays, Ro 15-4513 is a weak partial inverse agonist (leads to a slight reduction in GABA_AR activity) on the most abundant GABAAR subtypes in the brain. Ro 15-4513 is a partial agonist (enhances GABA action, but less than diazepam even at saturating concentrations) on the cc4 and α6 receptors (with β and γ2 subunits), whereas flumazenil is essentially silent in functional assays. Interestingly, Ro 15- 4513, but not other inverse agonists (like the β-carbolines β-carboline-3-carboxyethyl ester (β- CCE) and methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM)), has been shown to have fairly dramatic alcohol antagonist actions, reported in a variety of mammals; in particular, alcohol effects at lower doses can be almost completely reversed by RoI 5-4513.

[0008] There is a need in the art for a method of identifying agents that specifically bind and modulate the activity of δ subunit-containing GABA receptors.

SUMMARY OF THE INVENTION

[0009] The present invention provides methods of identifying agents that modulate δ subunit- containing GABA receptor activity. The agents so identified find use in a variety of research and treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS [0010] Figures IA and IB depict the effect of the α6-R100Q mutation on ethanol sensitivity when expressed with β3 or β2 and δ subunits. [0011] Figure 2 depicts [³H]Ro 15-4513 saturation binding in native IP (δ-IP pellet) and δ- depleted (δ-IP supernatant) cerebellar GABA receptor fractions and to recombinant α4β3δ and α4β3γ2 GABA receptor expressed in the HEK 293T cell line.

[0012] Figures 3A and 3B depict results alcohol inhibition of [³H]Rol5-1413 binding.

[0013] Figure 4 depicts the results of a test for competitive antagonism between EtOH and

Rol5-4513. [0014] Figure 5 depicts the pharmacological characterization of the [³H]Ro 15-4513 binding site. [0015] Figure 6 depicts the structure-affinity and activity relationship of selected BZ site ligands. [0016] Figures 7A-C depict the effect of Ro 15-4513 on ethanol effects on recombinant α4β3δ receptor currents. [0017] Figures 8 A and 8B depict the effects of flumazenil, β-CCE, flunitrazepam and DMCM on Ro 15-4513 alcohol antagonism. [0018] Figure 9A and 9B depict the effect of β-CCE on the ethanol effect on α4β3δ GABA receptors. [0019] Figures 1 OA and 1 OB show that the β3N265M point mutation eliminates Ro 15-4513 insensitive ethanol enhancement at high alcohol concentrations.

DEFINITIONS [0020] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease or condition in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. [0021] The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, primates, including simians and humans.

[0022] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0023] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0025] It must be noted that as used herein and in the appended claims, the singular forms "a,"

"and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a test agent" includes a plurality of such test agents and reference to "the agonist" includes reference to one or more agonists and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0026] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

[0027] The present invention provides methods of identifying agents that modulate δ subunit- containing GABA receptor activity. The agents so identified find use in a variety of research and treatment methods.

[0028] The present invention is based in part on the observation that, contrary to what was previously believed, δ subunit-containing GABA receptors have a high affinity Ro 15-4513 binding site and that it is nature of this binding to this site that explains why Ro 15-4513 is an alcohol antagonist. In vitro screening methods

[0029] In some embodiments, a subject screening method is an in vitro method that generally involves: a) contacting a δ subunit-containing GABA receptor with: i) a test agent; and: ii) a ligand that binds a benzodiazepine ligand binding site on the receptor; and b) determining the effect, if any, of the test agent on binding of the ligand to the benzodiazepine ligand binding site.

[0030] A test agent of interest is a compound that modulates binding of the ligand ("BZ site ligand") to the benzodiazepine ligand binding site ("BZ binding site") on a δ subunit- containing GABA receptor. The BZ binding site is distinct from the GABA site; and from the etomidate, propofol anesthetic site.

[0031] In some embodiments, a test agent of interest is an agent that reduces binding of the BZ site ligand by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, compared to the binding of the BZ site ligand in the absence of the test agent.

[0032] A test agent of interest will in some embodiments be a test agent that inhibits binding of the BZ site ligand with an IC₅₀ of less than about 200 nM, e.g., less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM.

[0033] A test agent of interest will in some embodiments be a test agent that inhibits binding of the BZ site ligand with an IC₅₀ of less than about 200 μM, e.g., less than about 200 μM, less than about 150 μM, less than about 100 μM, less than about 50 μM, less than about 10 μM, less than about 1 μM, less than about 500 nM, less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM, or less than about 0.1 nM.

[0034] For example, in some embodiments, a test agent of interest is an agent that inhibits binding of the BZ site ligand with an IC₅₀ of from about 0.1 nM to about 0.5 nM, from about 0.5 nM to about 1 nM, from about 1 nM to about 5 nM, from about 5 nM to about 10 nM, from about 10 nM to about 25 nM, from about 25 nM to about 50 nM, from about 50 nM to about 100 nM, from about 100 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 500 nM, from about 500 nM to about 1 μM, from about 1 μM to about 10 μM, from about 10 μM to about 50 μM, from about 50 μM to about 100 μM, from about 100 μM to about 150 μM, or from about 150 μM to about 200 μM.

[0035] A test agent of interest will in some embodiments be a test agent that has a K_d value of less than about 200 μM, e.g., less than about 200 μM, less than about 100 μM, less than about 50 μM, less than about 10 μM, less than about 1 μM, less than about 500 nM, less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, or less than about 5 nM. Test agents that exhibit a K_d of less than about 200 μM are in some embodiments further evaluated in one or more additional assays, as described below.

[0036] A test agent of interest will in some embodiments be a test agent that has a K_d value of less than about 200 nM, e.g., less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, or less than about 5 nM. Test agents that exhibit a Kd of less than about 200 nM are in some embodiments further evaluated in one or more additional assays, as described below.

[0037] For example, a test agent of interest will in some embodiments have a Kd value of from about 0.1 nM to about 1.0 nM, from about 1.0 nM to about 5 nM, from about 5 nM to about 10 nM, from about 10 nM to about 20 nM, from about 20 nM to about 30 nM, from about 30 nM to about 40 nM, from about 40 nM to about 50 nM, from about 50 nM to about 75 nM, from about 75 nM to about 100 nM, from about 100 nM to about 125 nM, from about 125 nM to about 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 500 nM, from about 500 nM to about 750 nM, from about 750 nM to about 1 μM, from about 1 μM to about 10 μM, from about 10 μM to about 50 μM, from about 50 μM to about 75 μM, from about 75 μM to about 100 μM, from about 100 μM to about 150 μM, or from about 150 μM to about 200 μM.

[0038] A test agent of interest will in some embodiments specifically bind to a δ subunit- containing GABA receptor that comprises a β3 subunit, e.g., will not substantially bind to a δ subunit-containing GABA receptor that comprises any other type of β subunit. A test agent of interest will in some embodiments specifically bind to a δ subunit-containing GABA receptor that comprises a β2 subunit, e.g., will not substantially bind to a δ subunit-containing GABA receptor that comprises any other type of β subunit. A test agent of interest will in many embodiments not bind substantially to a GABA receptor that does not comprise a δ subunit, e.g., the test agent will not substantially bind to a GABA receptor that comprises a γ subunit.

[0039] The terms "candidate agent," "test agent," "agent," "substance," and "compound" are used interchangeably herein. Candidate agents encompass numerous chemical classes, typically synthetic, semi-synthetic, or naturally-occurring inorganic or organic molecules. Candidate agents include those found in large libraries of synthetic or natural compounds. For example, synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), ComGenex (South San Francisco, CA), and MicroSource (New Milford, CT). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from Pan Labs (Bothell, WA) or are readily producible.

[0040] Candidate agents may be small organic or inorganic compounds having a molecular weight of more than 50 daltons and less than about 2,500 daltons, e.g., test agents will generally be from about 50 daltons to about 100 daltons, from about 100 daltons to about 200 daltons, from about 200 daltons to about 300 daltons, from about 300 daltons to about 500 daltons, from about 500 daltons to about 1000 daltons, or from about 1000 daltons to about 2,500 daltons. Candidate agents may comprise functional groups necessary for structural interaction with proteins, e,g,, hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, and may contain at least two of the functional chemical groups. The candidate agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0041] Assays of the invention include controls, where suitable controls include a sample (e.g., a sample comprising a δ subunit-containing GABA receptor and a BZ site ligand) in the absence of the test agent. Generally a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

[0042] Where the screening assay is a binding assay, one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.

[0043] A variety of other reagents may be included in the screening assay. These include reagents such as salts; neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions; and the like. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The components of the assay mixture are added in any order that provides for the requisite binding or other activity. Incubations are performed at any suitable temperature, typically between 4°C and 4O⁰C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 hour and 1 hour will be sufficient.

[0044] The screening methods may be designed in a number of different ways, where a variety of assay configurations and protocols may be employed, as are known in the art. For example, one of the components may be bound to a solid support, and the remaining components contacted with the support bound component. The above components of the method may be combined at substantially the same time or at different times.

[0045] Where the assay is a binding assay, following the contact and incubation steps, the subject methods will generally, though not necessarily, further include a washing step to remove unbound components, where such a washing step is generally employed when required to remove label that would give rise to a background signal during detection, such as radioactive or fluorescently labeled non-specifϊcally bound components. Following the optional washing step, the presence of bound complexes will then be detected.

[0046] In some embodiments, the assay is an in vitro cell-free assay. A cell-free assay is generally conducted with a δ subunit-containing GABA receptor, which may or may not be associated with a membrane or membrane components. In other embodiments, the assay is an in vitro cell-based assay. Cell-based assays are conducted using cells that produce a δ subunit- containing GABA receptor. δ subunit-containing GABA receptors

[0047] A subject screening method involves contacting a δ subunit-containing GABA receptor with a BZ site ligand and a test agent. The δ subunit-containing GABA receptor is generally a pentameric receptor comprising two α subunits; two β subunits; and a δ subunit. In some embodiments, the β subunit is a β₃ subunit. In other embodiments, the β subunit is a β₂ subunit. In still other embodiments, the β subunit is a βi subunit. The α subunit can be any of (Xi, α₂, (X₃, α₄, α₅, or α₆. In some embodiments, the α subunit is an α₄ subunit.

[0048] In some embodiments, the δ subunit-containing GABA receptor is a naturally-occurring receptor and is isolated from a natural source of the δ subunit-containing GABA receptor. For example, in some embodiments, the δ subunit-containing GABA receptor is isolated from cerebellar tissue. Any animal source of the tissue is suitable for use, including, e.g., mammals such as bovines, equines, ovines, canines, felines, simians, and human tissue. In other embodiments, the δ subunit-containing GABA receptor is a recombinant δ subunit-containing GABA receptor. Recombinant sources of δ subunit-containing GABA receptor include δ subunit-containing GABA receptor produced in a mammalian host cell cultured in vitro, where the cell has been genetically modified with nucleic acid(s) comprising nucleotide sequences encoding δ subunit-containing GABA receptor subunits. Suitable mammalian host cells include primary cells, immortalized cell lines, and the like. Suitable immortalized cell lines include cell lines derived from human, mouse, rat, hamster, non-human primates, etc. Suitable cell lines include, but are not limited to, NIH 3T3 cells (e.g., ATCC CRL-1658), HEK293T cells (e.g., ATCC CRL-1573), CHO cells (e.g., ATCC CCL-61), HeLa cells (e.g., ATCC CCL- 2), and the like. Derivatives of such cell lines are also suitable for use. Many such cells are available from the American Type Culture Collection (ATCC).

[0049] In some embodiments, the δ subunit-containing GABA receptor is membrane associated. In some embodiments, δ subunit-containing GABA receptor is associated with one or more membrane components. In some embodiments, the δ subunit-containing GABA receptor is associated with lipids, e.g., in a liposome, a unilamellar vesicle, etc.

[0050] In other embodiments, the δ subunit-containing GABA receptor is not membrane associated and is not associated with one or more membrane components. In some of these embodiments, the δ subunit-containing GABA receptor is immobilized on an insoluble support. Suitable insoluble supports can take any of a number of forms, including, e.g, beads, sheets, wells of a multi-well plate, etc. Suitable insoluble supports can be of a variety of materials including, but not limited to, agarose, polystyrene, nitrocellulose, and the like. In some embodiments, the δ subunit-containing GABA receptor is immobilized directly to an insoluble support. In other embodiments, the δ subunit-containing GABA receptor is immobilized onto an insoluble support via a linker. Suitable linkers include, but are not limited to, oligopeptides; antibodies or antigen-binding fragments of antibodies; non-peptide organic linkers; and the like. In some embodiments, the δ subunit-containing GABA receptor is immobilized on an insoluble support via an antibody specific for the δ subunit. The antibody may be attached to the insoluble support in any of a variety of ways, e.g., via a protein A linkage to the insoluble support. Ligands

[0051] Ligands that bind a benzodiazepine (BZ) ligand binding site on the δ subunit- containing GABA receptor are referred to generically herein as "BZ site ligands." BZ site ligands include, but are not limited to, benzodiazepines that are capable of binding at the site, including but not limited to, RoI 5-4513, flumazenil, and the like; and β-carbolines, including but not limited to, β-carboline-β-carboxyethyl ester; and the like.

[0052] In some embodiments, the BZ site ligand is an agonist of the δ subunit-containing

GABA receptor. In other embodiments, the BZ site ligand is an antagonist of the δ subunit- containing GABA receptor. In other embodiments, the BZ site ligand is essentially functionally silent, but can serve as a ligand that reverses (antagonizes) the actions of agonists (e.g., ethanol) or antagonists active at this site.

[0053] In some embodiments, the BZ site ligand is detectably labeled. In some embodiments, the label is a directly detectable label, e.g., the label provides a signal that is directly detectable. In other embodiments, the label is indirectly detectable.

[0054] Directly detectable labels include, but are not limited to, radiolabels, e.g., radioisotopes e.g.³²?, ³⁵S, ³H; etc; fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5'- dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7- hexachlorofluorescein (HEX), 5-carboxyfiuorescein (5-FAM) orN,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA); and the like. Indirectly detectable labels include, e.g., biotin, which is readily detectable using avidin or other biotin-binding moiety, which avidin can be detectably labeled. Cell-based assays

[0055] In some embodiments, a subject method involves identifying a compound that inhibits binding of a BZ site ligand to a δ subunit-containing GABA receptor, as described above, where a test agent is identified that inhibits binding of a BZ site ligand to a δ subunit- containing GABA receptor; and further characterizing the identified test agent. Such a test agent is referred to as an "identified test agent." An identified test agent will in some embodiments be further characterized for its effect, if any, on induced currents across a cell membrane. In some embodiments, an identified agent will be contacted with an in vitro eukaryotic cell that produces a δ subunit-containing GABA receptor; and determining the effect, if any, on membrane current.

[0056] In some embodiments, the assay will generally involve contacting an in vitro eukaryotic cell that produces a δ subunit-containing GABA receptor with an identified test agent in the presence of ethanol (EtOH); and determining the effect, if any, of the test agent on EtOH-induced current enhancement. EtOH will generally be present at a concentration of from about 3 niM to about 300 mM, e.g., from about 3 mM to about 100 mM, from about 50 HiM to about 100 mM, from about 100 mM to about 150 mM, or from about 75 mM to about 125 mM.

[0057] In some embodiments of interest, an identified test agent is an agonist of the BZ binding site. In other embodiments of interest, an identified test agent is an antagonist of the BZ binding site. In vivo activity

[0058] In certain embodiments, a subject method comprises further analyzing in vivo activity of a test agent identified as described above. For example, a test agents identified by a subject in vitro screening method, where the identified test agent has an effect on binding of a ligand to a BZ ligand binding site of a δ subunit-containing GABA receptor, is assessed for a physiological effect, if any. Physiological effects include, but are not limited to, sleep induction; mood enhancement; alleviation of depression; sedation; anxiety reduction; reduction of convulsions; reduction of epileptic episodes; and the like.

[0059] In general, in vivo efficacy is determined by administering an identified test agent to a non-human animal. In some embodiments, the non-human animal is a model of a sleep disorder; and the physiological effect that is detected is sleep induction, hi other embodiments, the non-human animal is a model of epilepsy; and the physiological effect that is detected is reduction in the number and/or severity of epileptic episodes.

[0060] Suitable in vivo animal models include the elevated plus maze model for detecting anxiolytic activity; and the spontaneous locomotor activity model to determine sedative effects. A compound that shows a statistically significant effect in the animal model of anxiety and no statistically significant effect in the animal model of sedative effects is identified as having non-sedating anxiolytic properties.

[0061] In some embodiments, an in vivo evaluation of the ability of the compound to mediate antidepressant effects without causing sedation is carried out. This is done using animal models predictive of antidepressant activity and sedation. A compound that produces a statistically significant effect in an animal model predictive of antidepressant activity and no statistically significant effect in an animal model predictive of sedative effects is identified as having antidepressant properties. Suitable in vivo animal models include the Porsolt swim test for predicting antidepressant activity and the spontaneous locomotor activity model for determining sedative effects. IDENTIFIED AGENTS

[0062] The present invention provides agents identified using a subject method; and compositions, including pharmaceutical compositions, comprising the agents. In some embodiments, an identified test agent will be modified, e.g., to produce a derivative, an analog, or a pharmaceutically acceptable salt of the identified test agent. Thus, e.g., a modified test agent includes a pharmaceutically acceptable salt of an identified test agent; a derivative or analog of an identified test agent; and a pharmaceutically acceptable salt of such a derivative or analog.

[0063] A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20^th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al, eds., 7^th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3^rd ed. Amer. Pharmaceutical Assoc.

[0064] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public. UTILITY

[0065] A test agent identified using a subject screening method will find use in various applications, including research methods and treatment methods. For research applications, an identified test agent will in some embodiments be useful for analyzing the function of a particular GABA receptor subtype. For research applications, an identified test agent will in some embodiments be useful as a BZ site ligand, e.g., for use in a screening method (e.g., as described above), for identifying further agents that modulate the activity of a δ subunit- containing GABA receptor.

[0066] In some embodiments, an identified agent will be useful in various treatment methods, depending in part on the activity of the agent. An identified agent will be useful for, e.g., sleep induction; mood enhancement; reduction in the number and/or severity of epileptic episodes (e.g., seizures); reduction in the incidence and/or severity of convulsions; sedation; treatment of depression; treatment of anxiety; and the like.

[0067] Disorders and conditions that can be treated with an identified test agent (or a pharmaceutically acceptable salt of an identified test agent; or a derivative or analog of an identified test agent; or a pharmaceutically acceptable salt of such a derivative or analog) include, but are not limited to: 1) depression, e.g. depression, atypical depression, bipolar disorder, depressed phase of bipolar disorder; 2) anxiety, e.g. general anxiety disorder (GAD), agoraphobia, panic disorder +/- agoraphobia, social phobia, specific phobia, Post traumatic stress disorder, obsessive compulsive disorder (OCD), dysthymia, adjustment disorders with disturbance of mood and anxiety, separation anxiety disorder, anticipatory anxiety acute stress disorder, adjustment disorders, cyclothymia; 3) sleep disorders, e.g. sleep disorders including primary insomnia, circadian rhythm sleep disorder, dyssomnia NOS, parasomnias, including nightmare disorder, sleep terror disorder, sleep disorders secondary to depression and/or anxiety or other mental disorders, substance induced sleep disorder; 4) mood disorders; 5) epilepsy; and 6) convulsions.

EXAMPLES

[0068] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like.

Example 1 : Alcohol Induced Motor Impairment Caused by Increased Extrasynaptic GABAA_^

Receptor Activity

[0069] This example shows that alcohol impairs motor coordination by enhancing tonic inhibition mediated by a specific subtype of extrasynaptic GABAA receptor (GABAR), α6β3δ, expressed exclusively in cerebellar granule cells (GCs). In recombinant studies, a naturally occurring, single nucleotide polymorphism is characterized. This polymorphism leads to a single amino acid change (RlOOQ) in α6. This change leads to a GABAR that is dramatically more sensitive to alcohol. Behavioral and electrophysiological comparisons of α6-100RR and α6-100QQ animals strongly argue that alcohol impairs motor coordination by enhancing GC tonic inhibition. These finding identify extrasynaptic GABA receptors as critical targets underlying low dose alcohol intoxication and demonstrate that subtle changes in tonic inhibition in one class of neurons can alter behavior. Methods

[0070] Electrophysiology. Standard methods were used for isolation, injection and recordings from Xenopus oocytes and for preparation of cRNA. Wallner et al. Proc Natl Acad Sci USA 100, 15218-23 (2003). Oocytes were injected with 0.4 ng of α and β-subunit cRNA and 2-4 ng of δ-subunit cRNA. Currents were measured in two-electrode voltage clamp mode 3-8 days after injection inND96 (composition in niM: 96 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, 5 HEPES, pH 7.5). GABA and ethanol were added to this solution as indicated.

[0071] The Hill equation I/I_max = l/[l+(EC₅₀/[GABA]ⁿ], where EC₅₀ is the concentration of drug eliciting a half-maximal response, n is the Hill coefficient, I_max is the maximum current and I is the GABA-evoked current, was used to fit GABA dose-response curves (Figure 1 and Table 1). Values for EC₂₀, EC₅₀, and % ethanol enhancements were calculated for individual cells and averaged to generate the reported values.

[0072] For brain slice experiments, 300 μm parasagittal slices of cerebellum were prepared from 24-42 day-old Sprague-Dawley rats using standard techniques (Brickley et al. Nature 409, 88-92 (2001); Stell et al. Proc Nat! Acad Sci USA 100, 14439-44 (2003)) with the exceptions that slicing solution consisted of (in mM): 250 sucrose, 26 NaHCθ3, 10 glucose, 4 MgCl₂, 3 myo-inositol, 2.5 KCl, 2 Na-pyruvate, 1.25 NaH₂PO₄, 0.5 ascorbic acid, 0.1 CaCl₂, and 0.001 DL-APV. Slice storage and recording solutions were saturated with 95% O₂ - 5%

CO₂ and consisted of (in mM): 119 NaCl, 26 NaHCO₃, 11 glucose, 2.5 KCl, 2.5 CaCl₂, 1.3 MgCl₂, and 1 NaH₂PO₄; in addition, storage solution contained 0.001 DL-APV. AU procedures were in accordance with a protocol approved by the UCLA Chancellor's Animal Research Committee. For voltage clamp recordings, whole-cell pipettes contained (in mM): 140 CsCl, 10 HEPES, 1 EGTA, 4 Mg-ATP, 0.4 GTP, titrated to pH 7.3 with CsOH. Recording pipettes had a bath resistance of 6-10 MΩ. The following drugs were added to the extracellular solutions as indicated: 2 μM NBQX, 10 μM NO-711, 300 nM GABA and 0.5 μM tetrodotoxin (TTX).

[0073] An Axopatch 200 B amplifier (Axon Instruments, Inc., Foster City, CA) was used to make whole-cell recordings. Data were filtered at 5 kHz and acquired using pCLAMP 8.2 (Axon Instruments) at a sampling rate of 20 kHz. All GC recordings were performed in voltage-clamp mode at holding potentials of -70 mV and at room temperature. Analysis was conducted using customized routines written in IGOR Pro 4.0 (Wavemetrics). To determine sIPSC frequency and amplitude, data were analyzed in continuous data segments of 30 seconds. Tonic GABAR-mediated current was defined as the steady-state current blocked by 10 μM SR95531; its magnitude was calculated by plotting all-point histograms of relevant 30 second segments of data. Gaussian equations of the form:

[0074] were fit to these data, constraining fits to values 2 bins more negative than the peak.

This ensured that the tail of higher amplitude values (representing sIPSCs) did not influence the fit. For the statistical analysis, the effects of 10 mM ethanol on tonic current were compared to changes in tonic current observed over otherwise identical sham perfusion periods.

[0075] Genotyping. After isolation of genomic DNA from ear snips, the exon coding for α6-

100 position was amplified with primers designed to be located in introns flanking the region of interest (to avoid amplification of mRNA). The polymerase chain reaction (PCR) fragment was sequenced using standard fluorescent dye sequencing.

[0076] Behavior. Rats were housed with food and water ad libitum in a 12/12 light/dark cycle.

Homozygous male and female rats (α6-100RR and α6-100QQ, >P55) were used for the rotarod (MedAssociates Inc.) and sleep time (loss of righting reflex, LORR) studies. These animals were either obtained directly from a breeding colony at Charles River Laboratories or bred at UCLA. In the accelerating rotarod test the speed of rotation increases at a constant rate from 4 to 40 rpm over 5 minutes. All animals used in the rotarod tests were naϊve to ethanol and were used to test only one condition. Blood samples (20 - 50 μl) were taken from the tail and serum ethanol concentration was determined with an Analox enzymatic blood alcohol analyzer.

[0077] Statistics. Values are reported as mean ± S.E.M unless noted otherwise. To evaluate the effect of EtOH dose and time in the rotarod experiments, we used a GLM (general linear model) with repeated measures. The WiIk' s lambda multivariate test was used to test the effect of the ethanol dose/time interaction. These statistical analyses were conducted using SPSS for Windows version 12. AU other statistical comparisons were conducted using paired and unpaired Student's t-tests as appropriate. Results

A single amino acid change selectively enhances the ethanol sensitivity of GABARs composed of α6, β3 and δ subunits

[0078] The ethanol sensitivities of heteromeric GABARs containing α6-100R and α6-100Q were tested by expressing each variant in oocytes together with a β subunit and either δ or a γ2 subunit. Ethanol dose-response curves were evaluated for several combinations of GABAR subunits thought to exist in GCs (α6β2δ, α6β2γ2, α6β3γ2) as well as other combinations that may be present in these cells (Figure 1 and Table 1). It was found that introduction of the α6- RlOOQ polymorphism into GABARs composed of α6, β3, and γ2 gave rise to benzodiazepine sensitive receptors. However, the low ethanol sensitivity of α6(100R)β3γ2 GABARs was unchanged in α6(100Q)β3γ2 receptors (Table 1).

Table 1. Summary of ethanol and GABA sensitivity for GABARs of different subunit combinations.

Percent enhancement by ethanol (mM)

Receptors GABA EC₅o (n) n 10 30 100 300 α6(R100Q)β3δ 0.68 + 0.1 (5) 8 99.3 ± 15.0 180.1 + 28.2 275.3 + 32.4 389.2 + 65.0 α6β3δ 0.70 + 0.4 (6) 10 41.2 + 4.3 9 922..55 ++ 99..00 125.3 + 20.5 245.0 ± 33.6 α6(R100Q)β2δ 0.51 + 0.09 (5) 7 0 24.5 + 10.7 97.0 + 11.2 199.0 + 38.1 cc6β2δ 0.50 + 0.03 (5) 6 0 23.1 + 7.9 88.4 ± 15.6 175.0 ± 35.8 α6(R100Q)β1δ 0.62 + 0.04 8 0 24.1 + 4.0 50.3 ± 7.8 185.2 ± 9.4 α6β1δ 0.56 + 0.07 9 0 21.2 + 3.3 52.0 ± 5.6 167.9 ± 10.0 α6(R100Q)β2/3γ2_L 19 + 3.5 (6) 16 0 0 40.3 + 7.8 167.5 + 14.2 α6(R100Q)β2/3γ2s α6β2/3γ2_L 19 + 0.5 (6) 10 0 0 39.0 + 7.5 182.6 + 11.3 α6β2/3γ2_s α1 β3δ 0.56 + 0.05 (4) 8 32.5 + 5.4 88.2 + 7.0 117.5 + 10.6 295.3 + 19.7 α1β2/3γ2_L 6.8 + 0.8 (5), 9 0 0 34.4 + 9.9 147.3 +.12.9 α1 β2/3γ2_s 8.8 + 1.1 (6)

[0079] For each indicated subunit combination, GABA dose-response relationships were determined by fitting the Hill equation (see Methods). Current enhancement by ethanol was measured by co-application of ethanol and GABA (-EC₃₀). Reported values are mean (±S.D.) percent increases above responses to GABA alone, zero values represent measurements of no change in current and n values indicate the number of oocytes used to determine the average ethanol dose response curves. Because the identity of the β subunit in γ2-containing receptors did not lead to differences, results have been pooled.

[0080] In contrast, for the highly ethanol-sensitive α6β3δ GABARs, it was found that changing amino acid position 100 in α6 from R to Q leads to a further increase in ethanol sensitivity (Figure 1). Strikingly, α6β3δ receptors were the only GABARs that showed a significant change in ethanol sensitivity in response to the α6 RlOOQ polymorphism (Figure 1 and Table 1). Notably, α6β2δ, another species of GABAR which may contribute to tonic GABA currents in GCs, was unaffected.

Ethanol acts at extrasynaptic GABARs composed of cc6, β3 and δ subunits, enhancing GC tonic inhibition

[0081] Based on these results, it was hypothesized that introduction of the Rl 0OQ polymorphism into the α6 gene should enhance ethanol sensitivity of tonic GABA conductances in GCs. Surprisingly, it was discovered that the αόlOOQ allele is present in outbred, Sprague Dawley rats obtained from Charles River. Genotyping results from 35 animals showed 10 α6-100RR rats, 11 α6-100QQ rats, and 14 heterozygotes. Whole-cell recordings from GCs in cerebellar slices prepared from animals of the two homozygous genotypes were carried out. It was found that ethanol concentrations of 30 and 100 mM enhanced tonic GABA currents in GCs from rats of both genotypes. However, this enhancement was significantly larger in GCs from α6-100QQ rats. Ethanol also enhanced the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in GCs, and the mean increases in frequency were larger in recordings from the mutant rats. No ethanol-induced changes were found in the mean amplitudes of sIPSCs, nor in their decay rates. These findings demonstrate that tonic GABA current is sensitive to low concentrations of ethanol in wild type animals and that the presence of RlOOQ in α6 renders tonic GABA current even more sensitive to ethanol. Furthermore, the genotype-dependent effects on sIPSC frequency imply that the presynaptic effects on GABA release must result indirectly from changes in GC excitability since oc6 is not expressed in any other cerebellar cell type. Low concentrations of ethanol act postsynaptically to enhance tonic inhibition.

[0082] In recombinant studies, it was shown that α4/6(100R)β3δ GABARs are enhanced by

10 mM ethanol, a concentration below the legally permitted blood alcohol level for motor vehicle operation in many countries (typically 0.08% w/v or 17.4 mM). In order to examine postsynaptic effects of such low doses of ethanol on tonic GABA currents, experiments were performed in the presence of tetrodotoxin, NBQX, 300 nM added GABA and NO-711, a GABA transporter antagonist. Significant enhancement by 10 mM ethanol was observed for wild type GCs, while even larger increases were observed for mutant GCs. Together these results indicate that increases in tonic GABA current could be detected independent of changes in GABA release and in response to concentrations of ethanol associated with minimal intoxication. Ethanol more severely impairs motor coordination in the α6-100QQ rats.

[0083] To explore whether increases in GC tonic inhibition in response to alcohol could account for behavioral phenotypes associated with intoxication the performance of α6-100RR and α6-100QQ animals on the rotarod, a cerebellum-dependent behavioral assay, was compared. The results indicated that, in a dose-dependent manner, low ethanol concentrations impaired α6-100RR rats, but led to significantly larger impairment of α6-100QQ rats. Blood ethanol concentrations obtained in these behavioral tests 25-30 minutes after administration of lg/kg ethanol (15.2 ± 2 mM in α6-100RR, n=4; 16.4 ± 2 mM in α6-100QQ, n=5; P=0.34) confirmed that plasma concentrations of ethanol were not affected by genotype and showed that the concentrations achieved were the equivalent of mildly intoxicating doses in humans.

[0084] The findings that this single amino acid difference is sufficient for increased ethanol sensitivity in vivo suggest that it makes an important contribution to the ANT phenotype. Moreover, as the RlOOQ polymorphism enhances ethanol sensitivity only in GABARs composed of α6, β3 and δ subunits, extrasynaptic receptors of this molecular makeup in GCs are likely to be responsible for the more severe intoxication exhibited by the α6-100QQ rats. Since these animals differ in the sensitivity of GC extrasynaptic but not synaptic current to ethanol, these data show that selective enhancement of tonic inhibition can account for ethanol- induced motor impairment.

Example 2: Ethanol potently and competitively inhibits binding of the alcohol antagonist

Ro 15-4513 to cu/gβ₃δ GABAA receptors

Methods

[0085] Radiolabeled [³H]Rol5-4513 (ethyl 8-azido-5,6-dihydro-5-methyl-6-oxo-4H- imidazo(l,5-α)benzodiazepine-3~carboxylate (33.3 Ci/mmol) was purchased from Perkin Elmer Life Sciences) and is supplied dissolved in ethanol. To change the solvent from ethanol to dimethylsulfoxide (DMSO), the [³H]Rol5-4513 ethanol solution was dried in a vacuum concentrator and [³H]Ro 15-4513 re-dissolved in DMSO. Unlike ethanol, DMSO at final concentrations (less than 1%) did not change [³H]Ro 15-4513 binding to cerebellar immunoprecipitated δ-receptors.

[0086] Rol5-4513, flumazenil (ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-

4H-imidazo[l,5-α][l,4]benzodiazepine-3-carboxylate), diazepam and flunitrazepam were from Hoffman-LaRoche (Nutley, NJ), DMCM (methyl-6,7-dimethoxy-4-ethyl-β-carboline-3- carboxylate) was a gift from Ferrosan (Copenhagen, Denmark), FG7142 (N-methyl-β- carboline-3-carboxamide) and β-CCE (β-carboline-3-carboxy ethyl ester) were provided by Schering (Berlin, Germany). EtOH, GABA, and bicuculline were purchased from Sigma. Compounds were dissolved in DMSO as a 10 mM stock solution and used at the indicated concentrations. Protein concentration was determined with the BCA protein assay kit (Pierce) with bovine serum albumin as standard. Membrane preparation

[0087] Bovine cerebellum was obtained from a local supplier and stored frozen at -7O⁰C.

Tissue was thawed and homogenized by sonication in 10 volumes of homogenization buffer (50 mM Tris/HCl, pH 8.0, 50 mM KCl, 1 mM EDTA, 0.32 M sucrose, 0.5 mM DTT₅ 0.01% bacitracin supplemented with either protease inhibitors (2 mM benzamidine, 0.1 mM benzethonium chloride, 0.3 mM PMSF) or a protease inhibitor cocktail (Complete Mini, Roche)), and centrifuged (550 x g) for 10 min at 4°C to pellet nuclei and cells. The supernatant fraction was collected by three sonication-centrifugation cycles (in homogenization buffer without sucrose). Membrane pellets were collected at 35,000 x g for 1 hr at 4°C and used for ligand binding, or stored frozen at -20⁰C. Recombinant cell expression

[0088] HEK-293T cells were transfected with rat cDNAs under the control of a CMV promoter (α: β:δ or γ2 in a 1 : 1 :2 ratio) as previously described (Meera et al. (1997) Proc Natl Acad Set USA 94:14066-14071) and cells were harvested 60 - 100 hr after transfection. Membranes from these HEK-293T pellets were homogenized by sonication in 10 volumes of assay buffer (in mM, 100 KCl₅ 10 KH₂PO₄/K₂HPO₄, pH 7.5 at 4°C) with a protease inhibitor cocktail (Complete Mini, Roche), and subjected to three centrifugation-resuspension cycles before being used for ligand binding assays. [³H]Rol5-4513 ligand binding assay

[0089] Membranes (or immunoprecipitated receptors bound to Protein-G agarose beads) were resuspended in assay buffer (50 mM Tris/HCl, pH 8.0, 1 M KCl, 1 mM EDTA, 0.5 mM DTT, 2 mM benzamidine, 0.01% bacitracin, 0.3 mM PMSF, 10 μg/ml trypsin inhibitor) by sonication. Resuspended membranes were incubated (in a volume of 0.5 ml) for 60 min on ice in the presence of [ H]RoI 5-4513 (33.3 Ci/mmol, Perkin Elmer Life Sciences) and various concentrations of competing ligands. Membranes (10-40 μg of protein/filter) were collected by rapid filtration on Whatman GF/B filters. After three washing steps with 10 ml of assay buffer, the filter-retained radioactivity was counted in a Beckman LS3800 liquid scintillation counter. Nonspecific binding was determined in the presence of 10 μM Ro 15-4513 and diazepam- insensitive (DZ-IS) binding in the presence of 10 μM diazepam. Data for binding curves were fitted using a nonlinear least-squares method using the equation, B(c) = B_m?βL/(l + (KJc)"), for binding curves, and B(c) = B_msκ/(l + (C//C₅₀)") where c is the concentration of ligand, B is binding, i?_max is maximal binding, K_d is the dissociation constant, and n is the Hill coefficient, IC₅₀ is half-maximal inhibitory concentration. These calculations were performed using Graph- Pad Prism software. All error bars in the figures are standard deviations (SD). Immunoprecipitation

[0090] Immunoprecipitations were performed from membrane preparations solubilized in assay buffer (see above), supplemented with 8 mM of the non-ionic detergent nonaoxyethylene-dodecyl ether (C12E9), using rabbit GABAAR π subunit-specific antibody (30) and the Protein G IP50 immunoprecipitation kit (Sigma). Briefly: 100 μl (~1 μg/μl) of protein extracts were incubated in 600 μl immunoprecipitation-buffer plus 60 μl 0.5 M NaCl with antibody (at appropriate dilutions) overnight at 4°C. Following the addition of 30 μl protein G agarose, the tubes were incubated for another two hours with mixing. The beads were washed five times with cold IP-buffer and binding assays were performed on antibody- bound receptors (Pδltl et al. (2003) JNeurochem 87:1444-1455). Results

[0091] The alcohol antagonist RoI 5-4513 binds to δ subunit-containing GABA_ARS

[0092] Based on the observations that δ subunit-containing receptors are uniquely sensitive to ethanol, and that a BZ-site single nucleotide polymorphism (α6-R100Q) increases alcohol sensitivity in α6β3δ receptors, whether native and recombinant δ subunit-containing receptors could bind the behavioral alcohol antagonist Rol5-4513, commercially available as a tritiated radioligand, was investigated.

[0093] To study native receptors, δ subunit-containing GABAA receptors were immuno- purified from cow cerebellum. The most likely subunit composition of these immuno-purified receptors is αόβδ. Based on the high ethanol sensitivity of cerebellar tonic currents and the increase in alcohol sensitivity observed with the α6R100Q allele, it seems likely that the majority of these receptors contain the β3 subunit. In addition to native receptors, recombinant α4β3δ receptors expressed in eukaryotic (HEK293 T) cells were also used.

[0094] Saturation binding experiments showed that both receptor preparations exhibited high affinity [³H]Ro 15-4513 binding (K_d = 7.5+0.1 nM on recombinant α4β3δ receptors) that is in the same range as that found for recombinant α4β3γ2 receptors (K_d = 2.9 + 0.7 nM, Fig. 2). The K_d for [³H]Rol5-4513 binding for native δ-immuno-precipitated pellet (δ-IP pellet) was 7.0 + 0.4 nM, and 5.7 + 0.6 nM in the immuno-depleted supernatant (δ-IP supernatant) containing native γ2-containing receptors.

[0095] Fig.2. Ethanol-sensitive α4/6β3δ GABA_AR have a high affinity Rol5-4513 binding site. [³H]Ro 15-4513 saturation binding in native immuno-purified (δ-IP pellet) and δ-depleted (δ-IP supernatant) cerebellar GABAR fractions, and to recombinant α4β3δ and α4β3γ2 GABAR expressed in the HEK293T cell line. [³H]Rol5-4513 binding to δ subunit-containing GABAARS is inhibited by ethanol

[0096] Inspired by the finding that these alcohol-sensitive receptors bind the alcohol antagonist

Ro 15-4513 with high affinity, it was investigated if [³H]Ro 15-4513 binding to δ subunit- containing GABAAR can be inhibited by ethanol. Figure 3a illustrates that [³H]Ro 15-4513 binding to native cerebellar δ subunit immuno-purified receptors (δ-IP pellet), as well as to recombinant α4β3δ receptors, was dose-dependently inhibited by 3 - 300 mM EtOH, IC₅O ~ 12 rnM. In marked contrast, [³H]RoI 5-4513 binding to α4β3γ2 and the δ subunit immuno- depleted supernatant (with γ2 subunit-containing receptors) was essentially insensitive to ethanol, even at very high concentrations. However, in marked contrast to, and despite its close structural similarity with Rol5-4513, [³H]flumazenil (see structure in Fig. 6) binding to recombinant α4β3δ receptors was not inhibited by up to 300 mM ethanol. Alcohol inhibits a fraction (~6 %) of cerebellar [³H]Rol5-4513 binding

[0097] [ H]Rol5-4513 binding to GABA_ARS in the cerebellum has been considered to include sites sensitive to classical BZ agonists (DZ-S binding) on αlβγ2 isoforms, and diazepam- insensitive (DZ-IS) binding sites observed in the presence of 10 - 100 μM diazepam. The latter are thought to be largely composed of α6βγ2 subunits. Based on our finding that δ subunit- containing GABAAR bind [³H]Ro 15-4513 with high affinity and that this binding is sensitive to displacement by ethanol, it was reasoned that a fraction of the DZ-IS binding sites in brain might be due to δ subunit-containing receptors. About 20% of the [³H]Ro 15-4513 binding to cerebellar membranes was not blocked by 10 μM diazepam (Fig. 3b inset). Approximately 30% of this diazepam-insensitive [³H]Rol5-4513 binding was dose-dependently inhibited by ethanol (IC₅₀ ~ 7 mM with a maximum inhibition at 100 mM ethanol (Fig. 3b). Given that DZ- IS binding of [³H]Ro 15-4513 in the cerebellum is about 20% of total binding, ethanol- displaceable Ro 15-4513 binding is about 6% of total cerebellar Ro 15-4513 binding. This is consistent with the δ antibody-precipitated fraction, enriched in αόβδ receptors and in the same range as the fraction of αόβxδ receptors (~11%) determined by biochemical methods in rat cerebellum. Ethanol did not inhibit the [³H]Ro 15-4513 binding to classical diazepam-sensitive (DZ-S) sites under the same conditions. Rol5-4513 is a competitive alcohol antagonist

[0098] The complete and dose-dependent displacement of [³H]RoI 5-4513 by ethanol on recombinant α4β3δ receptors (Fig. 3A) suggests the possibility that this is due to a competitive antagonism between ethanol and Ro 15 -4513. To further evaluate and distinguish competitive (direct) from allosteric (indirect) interaction, [³H]Ro 15-4513 saturation binding experiments were performed in the continuous presence of non-saturating ethanol concentrations. Receptor occupancy of ligands (in this case [³H]Rol5-4513) decreases in a predictable way in the presence of a presumed competitive antagonist (EtOH). The inclusion of 10, 30 or 50 mM ethanol in [³H]Rol5-4513 saturation binding experiments led to a dose-dependent parallel shift of the [³H]Rol5-4513 binding curve to the right (Fig. 4). The simultaneous least-square fit using a combined Hill/Schild-Gaddum equation (using a K_d for Ro 15-4513 of 7.5 nM) resulted in a Hill coefficient of 1.1, and a K_d for ethanol of 8.1 mM (solid line curves in Fig. 4). The fact that the prediction for competitive antagonism overlaps with our experimental data provides further evidence that alcohol and Ro 15-4513 likely occupy overlapping binding sites. [0099] The decrease in the receptor occupancy in the presence of competitive antagonists, as described by the Schild-Gaddum equation, is due to a reduction in the apparent association rate of ligands (binding sites occupied by the competitive ligand are not available for binding), without changes in the dissociation rate (i.e., the residence time of ligands in their binding sites). The dissociation rate of [³H]Rol5-4513 from recombinant α4β3δ receptors was determined, by measuring unbinding after the addition of excess cold (1 μM) Ro 15-4513 or a high concentration of ethanol (200 mM) to a receptor preparation equilibrated with 5 nM [³H]RoI 5-4513. Excess (1 μM) Rol5-4513 as well as 200 mM ethanol led to essentially identical [³H]RoI 5-4513 ligand dissociation rates, showing that ethanol does not decrease the residence time of [³H]Ro 15-4513 in its receptor (Fig.4, inset). This suggests that ethanol, even at 200 mM, does not allosterically alter the [³H]Ro 15-4513 binding pocket. Rol5-4513 binding to α4β3δ GABA_ARs is inhibited by ligands that prevent Rol5-4513's behavioral alcohol antagonism

[00100] To further characterize the [³H]Ro 15-4513 binding site on the δ subunit receptors competition binding experiments were performed on recombinant (α4β3δ) and native (cow cerebellum) immuno-puriiϊed δ subunit-containing receptors with BZ site ligands that have been previously shown to reverse RoI 5-4513's behavioral alcohol antagonism (flumazenil, β- CCE and FG7142) and the imidazobenzodiazepines (RoI 5-4513/flumazenil structural analogs) RY024 and RY080. RY024 has been described as a behavioral alcohol antagonist. Like "cold" Rol5-4513, flumazenil, RY024 and RY080, and the β-carbolines β-CCE and FG7142 displaced [³H]Rol5-4513 from its binding site on α4β3δ receptors (Fig. 5). This shows that ligands that are able to block ethanol enhancement of α4/6β3δ GABAAR currents, such as Ro 15-4513, or that are able to reverse the alcohol antagonist activity of Ro 15-4513 (flumazenil and β-CCE), are also able to displace [³H]Rol5-4513 binding. However, most other BZ site ligands, including all classical BZ agonists tested (diazepam, fmrazepam, flunitrazepam and midazolam), the β-carboline DMCM, and the BZ site ligands Zolpidem and zopiclone, that are known to bind with high affinity to the classical BZ sites in γ2 subunit- containing receptors, do not displace [³H]Ro 15-4513 from this binding site on δ receptors at reasonable concentrations (see Fig. 6). This is consistent with findings that these compounds do not prevent Rol5-4513's alcohol antagonist activity. The GABAAR channel antagonist picrotoxinin (100 μM) did not inhibit [³H]Rol5-4513 binding.

[00101] Flumazenil, Rol5-4513₅ and RY080 all differ only at the moiety at the C7 position (see

Fig. 6; fluorine in flumazenil, an azido-group in Ro 15-4513, and an acetylene-group in RY080). The less potent compound RY024 is identical to RY80 except that it contains the carboxy-t-butyl ester instead of the carboxy-ethyl ester moiety. In addition, the only difference between FG7142 and β-CCE is that the lower affinity compound FG7142 carries a carboxy- methyl amide instead of a carboxy-ethyl ester. It is therefore likely that the carboxy-ethyl ester moiety (see the structures shown in Fig. 6), present in all compounds with affinities K_d < 10 nM, is important for high affinity binding to the alcohol/BZ site in α4/6β3δ receptors. The lack of affinity of classical BZ agonists and DMCM could be due to the absence of the carboxy- ethyl ester group at the appropriate location. In addition, chemical moieties in these inactive compounds, e.g., the pendant phenyl group in classical BZs (see Fig. 6) may lead to steric hindrance.

[00102] Ki values were determined based on the ability to displace [³H]Ro 15-4513 (5 nM) from recombinant α4β3δ receptors harvested from transiently transfected HEK293 cells. The concentrations resulting in half-maximal inhibition of specific [³H]RoI 5-4513 binding were converted to K; values by using the Cheng-Prusoff relationship and the K_d value for [³H]Ro 15- 4513 of 7.5 nM. K; values above 1000 nM are grouped into two categories: less than 10% inhibition (»1000) and between 10% and 50% inhibition at 1000 nM (>1000).

[00103] Fig. 3. The alcohol receptor binding assay: Ethanol-displaceable high affinity

[³H]Ro 15-4513 binding to native and recombinant δ subunit-containing GABAAR: (a) [³H]Ro 15-4513 binding is inhibited by low concentrations of ethanol. Receptors were equilibrated with 10 nM [³H]Ro 15-4513 and varying EtOH concentrations. We tested native immuno-purified (δ-IP pellet) and δ-depleted (δ-IP supernatant) cerebellar GABAR fractions, and recombinant α4β3δ and α4β3γ2 GABAARS expressed in the HEK-293T cell line, (b) About one third of the diazepam-insensitive [³H]Ro 15-4513 binding to cow cerebellar GABAARS is antagonized by low ethanol concentrations. Inset: Total, 10 μM diazepam- sensitive (DZ-S) and -insensitive (DZ-IS) 30 nM [³H]Rol5-4513 binding to cow cerebellar membranes.

[00104] Fig. 4. Test for competitive antagonism between ethanol and Rol5-4513. Ro 15-

4513 saturation binding was performed in the presence of 10, 30 and 50 mM ethanol. Individual curves were fitted with the Hill equation (shown as dashed line curves). Solid line curves are derived from a simultaneous least square fit of all the data-set using the equation [[³H]Ro 15-4513] = (f/l-f)^1/h * K_d* (1 + [EtOH]/K_{d E}t_0H). [³H]Ro 15-4513] is the concentration of [³H]Ro 15-4513 to reach a fractional occupancy (f), h is the Hill coefficient, Kd is the dissociation constant for [³H]RoI 5-4513, and the term (1+ [EtOH]/Kd EIOH) is derived from the

Schild-Gaddum equation and describes the decreased receptor occupancy by Ro 15-4513 in the presence of the proposed competitive antagonist ethanol, where [EtOH] is the ethanol concentration and KdEtOH is the dissociation constant for EtOH. The best fit of curves was obtained with a Hill coefficient of 1.1 and a K_{d EIO}H of 8.0 mM and a K_d for RoI 5-4513 of 7.5 nM. Inset: Dissociation of [³H]Rol5-4513 from α4β3δ receptors equilibrated with 5 nM [³H]Rol5-4513. The unbinding rate of [³H]Rol5-4513 was measured by adding excess (1 μM) cold Rol5-4513 followed by rapid filtration after approximately 0.5, 1, 2, 5 and 10 min, and counting the amount of bound hot ligand. To test the effect of ethanol on the dissociation rate, 200 mM ethanol was added instead of cold Ro 15-4513 to prevent rebinding of the radioligand after dissociation. The experiment shown is representative for a total of three experiments performed. [00105] Fig. 5. Pharmacological characterization of the [³H]Rol5-4513 binding site.

Displacement of 10 nM [³H]Rol5-4513 by the BZ antagonist flumazenil (Rol5-1788), the Rol5-4513 congeners RY080 and RY024, and the BZ site ligands FG7142, β-CCE on recombinant α4β3δ receptors expressed in HEK cells. Very similar results were obtained with native immuno-purified δ subunit-containing cow cerebellar GABAAS.

Example 3: Low dose alcohol actions on α4β3δ GABAA receptors are reversed by the behavioral alcohol antagonist R015-4513 Materials and Methods Electrophysiology

[00106] Clones used were as described previously and were confirmed by sequencing to ensure that they are free of errors and agree with the consensus sequences for rat α4, α6, β3 and δ subunit proteins. Wallner et al. (2003) Proc. Natl. Acad. Sci. USA 100:15218-15223. Mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene). cRNA was transcribed after plasmid linearization using the mMessage mMachine kit (Ambion). Transcripts were purified by LiCl precipitation and RNA concentration was determined on a gel and by photometry. Oocytes were co-injected with a mixture of α, β and δ (or γ2) subunits in a 1 : 1 : 5 (or 1:1:10) subunit molar ratio . Currents were measured at room temperature (22-24⁰C) in the two-electrode voltage clamp configuration at -80 mV holding potential with an Axoclamp 2A Axon Instruments amplifier. Two electrode voltage clamp on Xenopus oocytes was performed in ND96 salt solution (composition, 96 mM NaCl, 2 mM KCl₅ 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, pH 7.2). Because of the slow onset in the expression of highly alcohol-sensitive δ subunit-containing receptors, oocytes were measured 7

- 14 days after injection. Currents were measured either in a tonic current mimicking mode as steady-state current (Fig. 7, 8), or by brief (<10 sec) co-applications of 300 nM GABA with drugs (ethanol, BZ-site ligands), to evoke peak currents, followed by a recovery time of at least 1 min. Reagents

[00107] Hoffman-LaRoche (Nutley, NJ) kindly provided Rol5-4513, Rol5-1788 (ethyl 8- fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[l,5-α][l,4]benzodiazepine-3-carboxylate), diazepam and flunitrazepam. DMCM (methyl-6,7-dimethoxy-4-ethyl-β-carboline-3- carboxylate) was a gift from Ferrosan (Copenhagen, Denmark) and FG7142 (N-methyl-β- carboline-3-carboxamide) as well as β-CCE (β-carboline-3-carboxy ethyl ester) were provided by Schering (Berlin, Germany). Ethanol, GABA, and bicuculline were purchased from Sigma. Compounds were dissolved in DMSO as a 10 mM stock solution and used at the indicated concentrations. DMSO at final concentrations used did not lead to changes in GABA receptor currents. Results Low dose ethanol enhancement on α4/6β3δ GABAARS is antagonized by Ro 15-4513.

[00108] The effect of the benzodiazepine alcohol antagonist Ro 15-4513 on the alcohol-induced current enhancement in α4/6β3δ receptors expressed in oocytes was investigated. Figure 7 shows that 300 nM Rol5-4513 completely reversed the ethanol enhancement of α4β3δ GABAARS for ethanol concentrations up to 30 mM. This "anti-alcohol effect" of Ro 15-4513 is surprisingly specific, because at concentrations where it abolished the alcohol-induced current increase (up to 300 nM), Rol5-4513 did not lead to a reduction in the "basal" GABA-induced current on these receptors; i.e., it is not an inverse agonist in this assay at concentrations that inhibit the ethanol-augmentation of GABA currents (Fig 7c). The dose dependence of this effect revealed that the concentration of Rol5-4513 required to inhibit 50% of the 30 mM ethanol enhancement (IC₅o) was about 10 nM (Fig. 7b). The alcohol antagonist action of low dose RoI 5-4513 suggests that, against common knowledge, α4/6β3δ GABAAR have a high affinity Ro 15-4513 binding site, with a K_d of ~ 10 nM.

[00109] At higher alcohol concentrations (> 100 mM) a fraction of the alcohol-induced enhancement was not blocked by 300 nM Rol5-4513 (Fig. 7a). This high dose ethanol enhancement was not surmountable by increasing the Ro 15-4513 concentrations. Therefore, α4β3δ GABAARS have a distinct, RoI 5-4513 -insensitive component of alcohol enhancement. To demonstrate the dose dependence, increasing concentrations of Ro 15-4513 were applied to currents evoked by 300 nM GABA plus 10, 30 or 50 mM ethanol. Rol5-4513 led to a dose- dependent block that was complete for the 10 and 30 mM dose (with 300 nM Rol5-4513) (Fig. 7b, 7c). Again, at the 50 niM ethanol dose, a small fraction of the alcohol enhancement was not blocked by Ro 15-4513 (Fig. 7c). The complete and specific antagonism of low dose alcohol effects on these receptors by Rol5-4513 suggests the intriguing possibility that Rol5-4513 might work by competitively displacing EtOH from its binding site.

[00110] Fig.7. Rol5-4513 antagonizes ethanol effects on recombinant α4β3δ receptor currents, (a, b) To mimic tonic GABA current, 300 nM GABA was perfused on to Xenopus oocytes expressing rat α4β3δ receptors that where held at -80 mV and currents measured with a two-electrode voltage clamp system. The indicated doses of ethanol and drugs were applied, (a) 300 nM RoI 5-4513 completely antagonizes ethanol enhancement up to an ethanol concentration of 30 niM (b) Cumulative Rol5-4513 dose response curve shows a dose- dependent inhibition of ethanol effects. GABA evoked currents are blocked by 30 μM bicuculline. (c) GABA peak currents with and without ethanol and the indicated concentrations of Rol5-4513. Rol5-4513 leads to a dose dependent inhibition of (10, 30 & 50 niM) ethanol enhancement on α4β3δ receptors. At concentrations up to 300 nM Rol5-4513 did not block the basal current on α4β3δ receptors (evoked by 300 nM GABA). Figures 7a and 7b show representative recordings of 6 and 5 experiments respectively. Antagonizing Rol5-4513's alcohol antagonism by flumazenil and β-CCE

[00111] Certain benzodiazepine site ligands, like the general benzodiazepine antagonist flumazenil (Rol5-1788) and the structurally unrelated BZ-site ligands β-CCE and FG7142, were shown to prevent the alcohol antagonistic effects of Ro 15-4513 in behavioral assays. It was reasoned that this could be due to displacement of Ro 15-4513 from its binding site by these compounds, which do not show alcohol antagonism by themselves. Four selected BZ site ligands were tested for their ability to reverse or mimic Ro 15-4513 antagonism of ethanol effects. These compounds were applied to α4β3δ receptors (expressed in oocytes) in the presence of 300 nM (~EC₂₀) GABA (to mimic tonic GABA current), 30 mM ethanol (to increase the GABA current), and 100 nM Rol5-4513 (to reverse the enhancement by 30 mM ethanol). Flumazenil (Rol5-1788) and β-carboline-ethyl ester (β-CCE) at 300 nM reversed the RoI 5-4513-induced alcohol antagonism. However, the classical benzodiazepine agonist flunitrazepam, as well as DMCM (a β-carboline with pronounced inverse agonist efficacy on γ2 subunit-containing receptors), each at 1 μM, did not reverse the effects of Rol5-4513 (Fig. 8a), indicating that the Ro 15-4513/BZ binding site on δ subunit-containing receptors is unique and binds only certain BZs and BZ site ligands with high affinity. None of the four compounds tested blocked ethanol enhancement on their own. These data are consistent with previous findings that Ro 15-4513 's alcohol antagonism can be antagonized by certain BZ-site ligands in ³⁶Cl^" flux assays in synaptoneurosomes and provide an in vitro correlate to the behavioral data that show that flumazenil and β-CCE can reverse the alcohol antagonist effects of RoI 5-4513. A comparison of the structures of Ro 15-4513 and flumazenil shows that these two molecules are identical, except for one moiety, which is an azido group in Ro 15 -4513 and a fluorine in flumazenil (Fig. 8b). β-carboline-ethyl ester (β-CCE) is a positive GABA modulator on α4β3δ receptors

[00112] It was observed that β-CCE led to an "overshoot" when it was used to antagonize the alcohol antagonist effects of Ro 15-4513 on GABA/ethanol-induced currents (see Fig. 8), suggesting that β-CCE might potentiate alcohol effects on α4/6β3δ GABAAR. β-CCE alone and in combination with 3 mM ethanol was tested for their functional effects on α4β3δ receptors. Figure 9a shows that β-CCE not only enhances alcohol actions, but also increases the activity of α4β3δ GABAARS in the absence of alcohol. In the same way as alcohol effects on α4β3δ GABAAR, the β-CCE-induced enhancement of GABA currents is reversed by Rol5- 4513 (Fig. 3b). A likely explanation for these findings is that β-CCE as well as Ro 15-4513 occupy an overlapping and mutually exclusive binding site, whereas β-CCE and ethanol might be able to bind next to each other in a side-by-side binding pocket, both microdomains blocked by Ro 15-4513 (see Fig. 8b). Loss of Rol5-4513-insensitive ethanol actions in α4β3N265Mδ GABAAR

[00113] The Rol5-4513-insensitive component of ethanol enhancement is observed at high alcohol concentrations (> 30 mM), where most recombinant and native GABAAR show ethanol enhancement that is likely due to alcohol sites determined by mutations in the second and third transmembrane region of GABAARS. It was shown (Fig. 10) that in α4β3N265Mδ receptors, where the β3 wild-type subunit is replaced with the mutated β3N265M subunit, retain the Rol5-4513-sensitive alcohol enhancement. However, the β3N265M mutation completely abolished the Ro 15 -4513 -insensitive ethanol enhancement observed at 100 and 300 mM ethanol (Fig. 10a), and even at 1 M ethanol. GABAAR composed of α4β3N265Mδ and α4β3δ subunits show identical ethanol enhancement at alcohol concentrations up to 30 mM and differ only at the 100 and 300 mM dose (Fig 10b). As a consequence of this loss of Rol5- 4513 insensitive high concentration alcohol effects, recombinant α4β3N265Mδ receptors now have a saturable ethanol response curve with a half-maximal response of 16 mM (at EC₂₀ [300 nM] GABA₅ Fig. 8b), a concentration close to the legal blood alcohol limit (17 mM) for driving in most US states. [00114] Fig. 8. Rol5-4513 alcohol antagonism is antagonized by flumazenil and β-CCE, but not flunitrazepam and DMCM. (a) Currents were evoked by 300 nM GABA, potentiated by 30 niM ethanol, and this potentiation was reversed by 100 nM RoI 5-4513. In constant presence of 300 nM GABA₅ 30 mM ethanol and 100 nM Rol5-4513, the BZ site ligands Rol5-1788 (300 nM), β-CCE (300 nM), flunitrazepam, and DMCM (each 1 μM) were sequentially applied to test if they reverse Rol5-4513's ethanol antagonistic effects. At the end of the recording, 30 μM bicuculline was used to block the GABA-induced current. Shown is a representative recording of a total of 3 experiments, (b) Chemical structures of the imidazobenzodiazepines Rol5-4513 and Rol5-1788 shows that they differ only at one single position in the molecule. The clinically used benzodiazepine antagonist flumazenil (Ro 15-4513) carries a fluorine at the C7 position of the benzodiazepine ring, whereas Rol5-4513 carries the larger azido group.

[00115] Fig. 9. β-CCE enhances ethanol effects and is an agonist on α4β3δ receptors, (a)

The β-carboline β-CCE at the indicated concentrations was applied alone or together with ethanol (always in the presence of 300 nM GABA) to oocytes expressing α4β3δ receptors and peak GABA/Cr currents were measured, (b) Dose dependent reversal of 300 nM β-CCE enhancement of α4β3δ currents by Rol5-4513.

[00116] Fig. 10. A point mutation eliminates Rol5-4513-insensitive alcohol effects at high alcohol concentrations, (a) A single point mutation (β3N265M in membrane-spanning segment TM2 of the β3 subunit) abolishes the Ro 15-4513 resistant alcohol enhancement observed at high ethanol concentrations in α4β3N265Mδ receptors, (b) Alcohol dose response curve, determined by brief co-applications of ethanol and GABA EC₂₀ (300 nM for α4β3δ and α4β3N265Mδ, 30 μM GABA for α4β3γ2 GABA_AR). Currents through α4β3N265Mδ GABAAR show a saturable alcohol enhancement and an EC₅₀ of around 16 mM. The complete reversal of even very high dose alcohol effects by 300 nM Rol5-4513 in these experiments is surprising, as this is not expected from an ideal competitive relationship of ligands with apparent dissociation constants (10 nM for Ro 15-4513 and 16 mM for ethanol). The reasons why Ro 15-4513 is so potent in antagonizing high dose ethanol actions on functional receptors remain to be clarified.

[00117] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMSWhat is claimed is:

1. An in vitro method for identifying an agent that modulates δ subunit- containing gamma amino butyric acid (GABA) receptor activity, the method comprising: a) contacting a δ subunit-containing GABA receptor with: i) a test agent; and: ii) a ligand that binds a benzodiazepine ligand binding site on the receptor; and b) determining the effect, if any, of the test agent on binding of the ligand to the benzodiazepine ligand binding site.

2. The method of claim 1, wherein the ligand is a compound is a benzodiazepine or a β-carboline.

3. The method of claim 1 , wherein the ligand is Ro 15-4513 or a derivative or analog thereof.

4. The method of claim 1, wherein the ligand comprises a detectable label.

5. The method of claim 4, wherein the label is a radiolabel.

6. The method of claim 1 , wherein the GABA receptor comprises a β₃ subunit.

7. The method of claim 1, wherein the GABA receptor comprises a β₂ subunit.

8. The method of claim 1, wherein the GABA receptor is membrane associated.

9. The method of claim 8, wherein the GABA receptor is a recombinant GABA receptor.

10. The method of claim 1 , wherein the GABA receptor is immobilized onto an insoluble support.

11. The method of claim 10, wherein the insoluble support is spherical.

12. The method of claim 10, wherein the GABA receptor is immobilized onto the insoluble support via at least one antibody specific for a subunit of the receptor.

13. The method of claim 1 , wherein a test agent that modulates binding of the ligand to the receptor is identified, and wherein the method further comprises determining the EC₅₀ of the test agent.

14. The method of claim 1, wherein a test agent that modulates binding of the ligand to the receptor is identified, and wherein the method further comprises determining whether the test agent has agonist or antagonist activity.

15. The method of claim 14, wherein the method of determining whether the test agent has antagonist activity comprises: a) contacting the test agent with a cell that expresses the GABA receptor in the presence of ethanol; and b) determining the effect, if any, of the test agent on ethanol-induced current enhancement, wherein the ethanol is present at a concentration of from about 3 mM to about 100 mM, wherein a test agent that reduces the ethanol-induced current is an ethanol antagonist of the receptor.

16. The method of claim 14, wherein the method of determining whether the test agent has agonist activity comprises: a) contacting the test agent with a cell that expresses the GABA receptor; and b) determining the effect, if any, of the test agent on a current across the cell membrane, wherein a test agent that increases the current is an antagonist of the receptor.

17. The method of claim 15 or claim 16, wherein the cell is an oocyte.

18. A method of identifying a test agent that is an agonist of a δ subunit- containing gamma amino butyric acid (GABA) receptor in vivo, the method comprising: a) administering a test agent identified by the method of claim 1 to a non-human animal model expressing the receptor, wherein the identified test agent modulates binding of the ligand to the receptor and has a K_d of less than 100 nM; and b) determining a physiological effect, if any, of the test agent on the animal model, wherein the at least one physiological effect is selected from sleep induction, mood enhancement, reduction of depression, sedation, anxiety reduction, reduction of convulsions, and reduction of epileptic episode.