WO2001090163A2

WO2001090163A2 - Use of the gabab receptor in assays to identify gamma hydroxybutyrate agonists, antagonists, and allosteric modulators of agonists

Info

Publication number: WO2001090163A2
Application number: PCT/CA2001/000770
Authority: WO
Inventors: Gordon Yiu Kon Ng
Original assignee: Merck Frosst Canada and Co
Current assignee: Merck Frosst Canada and Co
Priority date: 2000-05-25
Filing date: 2001-05-24
Publication date: 2001-11-29
Anticipated expiration: 2002-11-25
Also published as: WO2001090163A3

Abstract

An unexpected and novel mechanism of action for gamma hydroxybutyrate (GHB) has been discovered: agonist activity at the functional GABAB receptor. The discovery of this mechanism of action allows for the development of assays for the identification of modulators, such as agonists and antagonists, of GHB.

Description

TITLE OF THE INVENTION

USE OF THE GABAB RECEPTOR IN ASSAYS TO IDENTIFY GAMMA

HYDROXYBUTYRATE AGONISTS, ANTAGONISTS, AND ALLOSTERIC MODULATORS OF AGONISTS

FIELD OF THE INVENTION

This invention is directed to novel methods of using the GABAβ receptor to identify agonists, inverse agonists, antagonists, and allosteric modulators of gamma hydroxybutyrate (GHB).

BACKGROUND OF THE INVENTION

Gamma aminobutyric acid (GAB A); an endogenous neurotransmitter in the central and peripheral nervous systems, is responsible for mediating synaptic transmission through two ligand-gated channels, GAB AA and GAB Ac receptors, and a third receptor sub-type, GABAβ, which acts through G proteins to regulate potassium and calcium channels. Specifically, GABAβ receptor activation increases

K+ or decreases Ca++ conductance and also inhibits or potentiates stimulated adenylyl cyclase activity. The expression of GABAβ receptors is widely distributed in the mammalian brain (e.g., frontal cortex, cerebellar molecular layer, interpeduncular nucleus) and has been observed in many peripheral organs as well. Two GABAβ receptors, GABAβRla and GABAβRlb, have been identified that bind GAB Aβ receptor antagonists in transfected cells; Kaupmann et al, 1997 Nature 386:239-246. More recently, a third GABAβ receptor isoform, gblc, was reported to Genbank (Accession No. AJ012187). In application serial number (attorney docket No. MC022PV), filed herewith, another

GABAβ receptor, a novel isoform of the gblc receptor is disclosed. These receptors are extremely valuable in identifying modulators of the GAB Aβ receptor, such as GABAβ agonists and antagonists.

GABA, the ligand for GABAβ receptors, is related to L and D-amino acids, decarboxylated amino acids, and other small molecules like GHB (gamma- hydroxybutyrate, OHCH2CH2CH2COOH). Gamma hydroxybutyric acid, a central inhibitory neurotransmitter and a cerebral metabolite of gamma-aminobutyric acid, is present in high concentrations in the mammalian hypothalamus and basal ganglia, and is believed to act via specific pathways and receptors in brain. It has been effectively used as an intravenous anaesthetic agent, and as an oral sedative, and in the management of alcohol withdrawal syndrome. Beside its regulatory effects on dopaminergic transmission, gamma-hydroxybutyrate was thought for many years to interfere with gamma-aminobutyric acid (GAB A)ergic processes in the brain, but its molecular target remains unclear.

Although the relation of GHB to GAB A would seemingly suggest that GABAβ receptors may in fact be activated by GHB, there exists much debate over whether GHB is an agonist of the GAB A receptors, the general agreement being that if there is agonist activity at GABAβ receptors present at all it is exhibited at no less than millimolar concentrations rendering GHB a weak, partial agonist at best; Michel Maitre, 1997 Progress in Neurobiol. 51:337-361; Feigenbaum & Howard, 1996 Progress in Neurobiol. 50:1-7; Mosbacher et ah, 1999 The 8^{t l} Neuropharmacology Conference, Miami, Florida.

It would be highly desirable to identify a means by which GHB agonists, inverse agonists, antagonists and allosteric modulators of GHB agonists could be identified. Identification of such means would enable identification of alternate, perhaps more potent, compounds for the treatment of altered GHB physiology, conditions as serious as, for example, epilepsy, schizophrenia, sleep disorders, muscle wasting, growth retardation, obesity, and drug addiction.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery of an unexpected and novel mechanism of action for gamma hydroxybutyrate (GHB): agonist activity at the functional GAB Aβ receptor. The discovery of this mechanism of action allows for the development of assays for the identification of modulators, such as agonists and antagonists, of GHB.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows the nucleic acid (SEQ ID NO:l) and deduced amino acid (SEQ ID NO:2) sequences of human Gblc. The polypeptide sequence comprises a signal peptide (M -A^), a single Sushi Repeat (T26-R98)₅ two putative glycosylation sites (N2 N8 ), and exhibits weak amino acid identity (26% identity over 26 amino acid overlap) to selectins. The putative transmembrane domains, and their corresponding amino acid numbers, are as follows: TMl:Ser526-Phe550; TM2:Ser563-Gly589; TM3:Leu605-Trp625; TM4:Trp646-Ile665; TM5:Thr706- Ala726; TM6:His740-Thr761; and TM7:Gln767-Phe791.

FIGURE 2 shows the amino acid sequence of human gblc (Genbank Accession No. AJ012187; SEQ ID NO:8) aligned over the Gblc receptor polypeptide sequence of the instant invention.

FIGURE 3 shows the complete cDNA sequence of HG20 (SEQ ID NO:3).

FIGURE 4 shows the complete amino acid sequence of HG20 (SEQ ID NO:4). FIGURE 5 A shows the major schematic topology/structural domains of gbla, gblb and gblc receptors, namely, the signal peptide, Sushi repeats, coiled- coil and PDZ domains. Figure 5B illustrates the amino acid alignment of the extracellular N-termini of human gbla, gblb, and Gblc isoforms. The proposed signal peptide cleavage site of gbl is marked with scissors, and arrows delimit the Sushi domains (SU). An arrow (Ψ) marks the start of the LIV-BP-like domain. Gaps (...) were introduced to maximize alignment. The novel Gblc isoform differs from gbla by an in-frame 62 amino acid deletion, and elimination of one protein-protein interacting Sushi Repeat (also known as short consensus repeat) leaving a single Sushi Repeat interacting module. The N-terminus common to the gbl isoforms exhibits weak amino acid identity to the bacterial periplasmic binding protein LIV-BP and is predicted to form two lobes that bind ligand according to a Venus flytrap model for receptor activation. The entire N-terminal domain of gbl, in the absence of the other portions of the receptor, is sufficient to specify agonist and antagonist binding.

FIGURE 6 shows a summary of BMAX (estimated maximal receptor density) and Kd for gblc and Gblc from 3H-CGP71872 saturation binding.

FIGURE 7 shows how GAB A mediated a dose-dependent inhibition of forskolin-stimulated cAMP (GABA EC50 value of 21±8 nM). Basal cAMP levels were approximately 1 pmol cAMP/2 X 105 cells and forskolin-induced cAMP levels typically greater than 20-fold. The functional and stably expressed Gblc-gb2 heterodimer subtype exhibited nanomolar potency for GABA similar to the reported affinity of GABA at neuronal GABAβ receptors.

FIGURE 8 shows GHB-mediated inhibition of cAMP production in gbl/gb2 HEK bulk stables (i.e., gbla/gb2, gblb/gb2 and Gblc/gb2 heteromers). FIGURE 9 shows the nucleic acid sequence (SEQ JO NO: 9) of human gbla.

FIGURE 10 shows the deduced amino acid sequence (SEQ ID NO: 10) of human gbla. The gb la-specific N-terminal sequence is comprised primarily of two protein-protein interacting Sushi repeat domains of approximately 60 amino acids, the first corresponding to T26-R98 and the second to K102- 160 first described by Kaupmann et al, 1998 Proc. Natl. Acad. Set USA 95:14991-14996.

FIGURE 11 shows the nucleic acid sequence (SEQ ID NO: 11) of human gblb. FIGURE 12 shows the deduced amino acid sequence (SEQ ID NO: 12) of human gblb. Gblb differs from gbla in that the first 164 amino acids of gbla are replaced by 47 different amino acids, thus gblb lacks N-terminus Sushi repeats.

DETAILED DESCRIPTION OF THE INVENTION As used throughout the specification and claims, the following definitions apply:

"Functional GABAβ receptor" herein refers to the receptor formed following co-expression of a GABAβ receptor polypeptide such as gbla, gblb, gblc or Gblc and a subunit known variously as GABAβR2 (White et al, 1998 Nature 396:679-682; and Jones et al, 1998 Nature 396:674-679); GBR2 (Kuner et al, 1999 Science 283:74-77); or gb2 (Ng et al, 1999 J. Biol Chem. 274:7607-7610, also known as HG20 (International Patent Application PCT/US99/02361, filed February 3, 1999). A functional GABAβ receptor, further, displays at least one functional response when exposed to GAB Aβ receptor agonists such as GABA or (-) baclofen. While functionality is attributed to heterodimer formation, it remains possible that GAB Aβ receptor monomers or homodimers are functional when in certain cellular environments. Examples of functional responses are: pigment aggregation in Xenopus melanophores, modulation of cAMP levels, coupling to inwardly rectifying potassium channels, mediation of late inhibitory postsynaptic potentials in neurons, increases in potassium conductance, and decreases in calcium conductance.

"HG20 or a functional equivalent thereof refers to HG20 (or gb2) and those polypeptides capable of interacting with a GABAβ receptor polypeptide to form a heterodimer active as a functional GABAβ receptor, e.g., GABAβR2 and GBR2. In accordance with the instant invention, Applicants have discovered that gamma hydroxybutyrate (GHB) acts as an agonist at functional GAB Aβ receptors resulting from the coexpression of HG20 and a GAB Aβ receptor polypeptide such as that of Gblc disclosed in copending application serial number (attorney docket No. MC023PV), filed herewith. This is the first report indicating that functional GABAβ receptors (e.g., gbl/gb2 heteromers such as gbla gb2, gblb/gb2, gblc/gb2, and Gblc/gb2) are negatively and differentially coupled to adenylyl cyclase and seemingly serve as a receptor for GHB. This was surprising as previous studies indicated that GHB was a weak, partial agonist at best at the GABAβ receptor; see Mosbacher et al, supra, confirming earlier studies indicating that GHB has little effect on either GABAA or GAB Aβ receptors at less than millimolar concentrations; see Feigenbaum & Howard, supra. Applicants, despite previous observations, identified a GABAβ receptor agonist capability for GHB on recombinant functional gbla, gblb and Gblc receptors in a cAMP accumulation assay; see Figure 8. Moreover, this activity was present at micromolar concentrations (~100μM, 500μM and ~100μM, respectively), a dosage not previously believed to exhibit GAB Aβ receptor activity. The agonism at the GAB Aβ receptors is competitively antagonised by the prototypical GAB Aβ antagonist CGP71872 but not NCS-382, a compound which partially inhibits GHB activity in vivo. Notably, GHB potence at the functional recombinant GABAβ receptors is not affected by the presence of valproic acid (an inhibitor of GHB- dehydrogenase which facilitates the breakdown of GHB to GABA), thus, disproving in part previous suggestions that the partial agonist activity noted at GABAβ receptors is due to GABA synthesis from GHB; see Maitre, supra. Accordingly, Applicants have proven that GABAβ receptors, and the Gblc receptor in particular, mediate at least part of the biological activities of GHB.

Gblc, the receptor polypeptide found to mediate in part the actions of GHB, is a novel cDNA which was obtained from adult human cerebellum mRNA. The novel receptor Gblc encodes a protein of 899 amino acids, differing from the previously published gblc submitted to Genbank (Accession No. AJ012187) by

Glaxo Wellcome in Genbank by a single amino acid substitution (a glutamine in place of a histidine at position 21). This difference is found in the extracellular N-terminus of the gblc receptor which is entirely responsible for ligand binding. As a result, the novel isoform exhibits higher affinity for antagonist and is more similar to native neuronal GABAβ receptors which exhibit, e.g., approximately 1 nM Kd for CGP71872.

The human polypeptide sequence of the Gblc polypeptide is provided in Figure 1. Particularly preferred embodiments of the instant invention employ Gblc receptor polypeptides comprising amino acids 18-899 of SEQ ID NO:2, the human Gblc lacking the putative signal sequence. A particular Gblc receptor sequence of use in the instant invention comprises a glutamine at position 21 in place of a histidine. Accordingly, those GABAβ receptor polypeptides of the gblc subgroup comprising a histidine to glutamine substitution at a position corresponding to position 21 of Gblc are expressly included within the present invention. Polypeptides of the gblc subgroup are readily distinguished structurally from those of the la and lb subgroups. Gblc subgroup polypeptides differ from gbla subgroup polypeptides by an in-frame 62 amino acid deletion, and elimination of one protein-protein interacting Sushi Repeat. Gblb subgroup polypeptides lack both N-terminus Sushi Repeats. Use of gbla and gblb GABAβ receptor polypeptides to identify GHB modulators constitute another aspect of the instant invention. Gbla and gblb receptors are known in the art; see Kaupmann et al, 1997 Nature 386:239-246, and are provided in Figures 9-12. Particularly preferred embodiments of the instant invention employ receptor polypeptides comprising the extracellular region of the polypeptides.

GAB Aβ receptor proteins contain various functional domains, including one or more domains which anchor the receptor in the cell membrane, and at least one ligand binding domain. As with many receptor proteins, it is possible to modify many of the amino acids, particularly those which are not found in the ligand binding domain, and still retain at least a percentage of the biological activity of the original receptor. Thus this invention specifically includes modified functionally equivalent GAB Aβ receptor polypeptides which have truncated, or mutated N- terminal portions. This invention also specifically includes modified functionally equivalent GAB Aβ receptor polypeptides which contain modifications and/or deletions in other domains, which are not accompanied by a loss of functional activity.

Additionally, it is possible to modify other functional domains such as those that interact with second messenger effector systems, by altering binding specificity and/or selectivity. Such functionally equivalent mutant receptors are also within the scope of this invention.

Particular embodiments of the instant invention employ those truncated forms of GAB Aβ receptor polypeptides which comprise the extracellular portion of the receptors, but lack the intracellular signaling portion of the receptor. Such truncated receptors are useful in various binding assays. Receptor chimeras (i.e., fusion proteins) which contain modifications and/or deletions not accompanied by a loss of functional activity are also included within the instant invention.

One of ordinary skill in the art is aware of the variety of expression vectors useful in effecting the expression of recombinant HG20 or a GABAβ receptor polypeptide of interest. Commercially available expression vectors which are suitable in this capacity include, but are not limited to, pMClneo (Stratagene), pSG5 (Stratagene), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1 (Invitrogen), EBO-pSV2-neo (ATCC 37593), pBPV-l(8-2) (ATCC 37110), pdBPV- MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), and the PT7TS oocyte expression vector (or similar expression vectors containing the globin 5' UTR and the globin 3' UTR). The choice of vector will depend upon the cell type used, the level of expression desired, and the like. Particularly in the assays of the instant invention, the present invention employs cells co-expressing HG20 and a GABAβ receptor polypeptide selected from the group consisting of gbla, gblb, gblc and Gblc, allowing for the formation of functional GABAβ receptors and HG20/G heterodimers. Such cells are generally produced by transfecting cells that do not normally express functional GAB Aβ receptors with either a single expression vector or independent vectors encoding HG20 and a Gbl isoform. The cells are then cultured under conditions such that heterodimers of HG20 and the Gbl isoform are formed where the heterodimers constitute functional GABAβ receptors. In this way, recombinant host cells expressing GAB Aβ receptors are produced. This does not exclude the use of cells endogenously expressing a functional GABAβ receptor, however.

Host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cells and cell lines which are suitable for recombinant expression of HG20 and a Gbl isoform which are commercially available, include but are not limited to, L cells L- M(TK^") (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), HEK293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92),

NTH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS- C-l (ATCC CCL 26), MRC-5 (ATCC CCL 171), Xenopus melanophores, and Xenopus oocytes. In the case oi Xenopus oocytes, co-expression of HG20 and a Gbl isoform is often effected by microinjecting RNA encoding HG20 and RNA encoding the Gbl isoform into the oocytes rather than by transfecting the oocytes with expression vectors encoding HG20 and the Gbl isoform. Microinjection of RNA into Xenopus oocytes in order to express proteins encoded by the RNA is well known in the art.

Another aspect of the instant invention are the assays afforded by the instant disclosure. These allow for the identification of modulators of GHB, e.g., agonists and antagonists (i.e., compounds which mimic/stimulate or which inhibit the function of gamma hydroxybutyrate on the Gblc receptor) potentially useful in the treatment and/or prevention of the various afflictions currently treated with GHB including, for example, epilepsy, schizophrenia, sleep disorders, muscle wasting, growth retardation, obesity, and drug addiction. Other types of modulators are inverse agonists and allosteric modulators of agonists.

One type of assay involves determining whether binding occurs between a candidate compound and a receptor polypeptide comprising the extracellular region of a GABAβ receptor polypeptide in the presence of GHB. Especially preferred embodiments employ receptor polypeptides comprising the extracellular region of gbla, gblb, gblc or Gblc receptors. Binding in such assays indicates the presence of a modulator of GHB. This assay expressly includes binding to soluble proteins lacking transmembrane and intracellular regions, cells or membrane preparations bearing a receptor polypeptide comprising the extracellular region, fusion proteins comprising the extracellular region of a GAB Aβ receptor and fragments of a GAB Aβ receptor polypeptide. The use of membranes rather than cells is well known in the art. Moreover, the above assay as well as all the assays disclosed relating to binding of ligand can be run with GAB Aβ receptor polypeptide monomers. Determination and measurement of binding can be done by means of a label directly or indirectly associated with the candidate compound.

Accordingly, the present invention includes a method for the identification of GHB agonists or antagonists which comprises exposing cells expressing HG20 or a functional equivalent thereof and a receptor polypeptide comprising the extracellular region of a GAB Aβ receptor polypeptide to a suspected agonist or antagonist in the presence of GHB. In the presence of a GHB agonist or antagonist, the amount of binding of GHB will be less than that seen upon contact of the receptor with GHB. In preferred embodiments, the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide are recombinant cells that have been transfected with a vector or vectors comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide.

Optionally, control experiments can be run utilizing control cells that are the same as the cells above except that the control cells do not express functional GABAβ receptors. The amount of binding of GHB to the control cells should be significantly less than the amount of binding of GHB to cells expressing functional GABAβ receptor. In this way, one can ensure that the binding of GHB in the above assays is actually due to binding of GHB to GABAβ receptors. Therefore, if the compound can block this binding, the compound is also likely to bind to GAB Aβ receptors.

Another type of assay constituting part of the instant invention is for the identification of compounds which modulate the function of GHB which comprises determining if a compound upon contact with a receptor polypeptide comprising the extracellular region of a GAB Aβ receptor results in a signal generated by activation or inhibition of the polypeptide, wherein contact is made in the presence of GHB. Preferred embodiments employ GABAβ receptor polypeptides of the gbla, gblb, gblc or Gblc subtypes. This can be done using detection systems appropriate to the cells or cell membranes bearing the polypeptide. A signal is an event indicating a functional response of the GABAβ receptor. Note, as above, this assay expressly includes use of soluble proteins lacking transmembrane and intracellular regions, cells or membrane preparations bearing a receptor polypeptide comprising the extracellular region, fusion proteins comprising the extracellular region of a GABAβ receptor and fragments of a GAB Aβ receptor polypeptide. One skilled in the art is familiar with a variety of methods of determining and measuring the functional responses of G-protein coupled receptors such as the GABAβ receptor; e.g., through the monitoring of changes in pigment distribution in melanophore cells, through the monitoring of changes in cAMP or calcium concentration or chemotaxis, through the monitoring of changes in membrane currents in Xenopus oocytes, through the monitoring of changes in calcium concentration measured using the aequorin assay, or through the monitoring of changes in inositol phosphate levels. Depending upon the cells in which heterodimers of HG20 and a Gbl isoform are expressed, and thus the G-proteins with which the functional GABAβ receptor is coupled, certain of such methods may be appropriate for measuring the functional responses of such functional GABAβ receptors. It is well with the competence of one skilled in the art to select the appropriate method of measuring the functional response for a given experimental system. Note that while the functionality of the various gbl isoform is attributed to heteromer formation, it remains possible that GABAβ receptor monomers or homodimers are functional when in certain cellular environments.

Preferred embodiments of the present invention are wherein HG20 or a functional equivalent thereof is expressed along with the receptor polypeptide such that heterodimers of the receptor polypeptide and HG20 are allowed to form. Accordingly, the present invention includes assays by which GHB receptor antagonists or inverse agonists may be identified by their ability to antagonize a functional response mediated by GHB in cells. One such method comprises contacting cells expressing HG20 or a functional equivalent thereof and a receptor polypeptide comprising the extracellular region of a GAB Aβ receptor polypeptide in the presence of GHB with a suspected antagonist or inverse agonist of GHB and determining whether a functional response follows. A GHB antagonist will antagonize GHB function whereas an inverse agonist will result in a function opposite to that of GHB (e.g., a decrease versus an increase in a specific function). A preferred embodiment of the present invention is wherein the response is measured and compared with the results obtained from contact with GHB. In this situation, the functional response of GHB would be less in the presence of a GHB antagonist or inverse agonist. Especially preferred embodiments comprise the further step of contacting a separate group of cells expressing the receptor polypeptide (the control group) with the suspected antagonist or inverse agonist; a lack of response in the control indicating the presence of an antagonist and a response in contradiction with (or in a direction opposite to) that of GHB indicating the presence of an inverse agonist.

In preferred embodiments, the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide comprising the extracellular region of a GAB Aβ receptor polypeptide are recombinant cells that were transfected with a vector or vectors comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide.

The present invention further includes assays by which GHB agonists or allosteric modulators of agonists may be identified by their ability to promote a functional response mediated by GHB in cells. One such method comprises contacting cells expressing HG20 or a functional equivalent thereof and a receptor polypeptide comprising the extracellular region of a GAB Aβ receptor polypeptide with a suspected agonist of GHB or an allosteric modulator of such in the presence of GHB and determining whether a functional response follows. Contact with a GHB agonist should stimulate GABAβ receptor function. By contrast, contact with an allosteric modulator of an agonist should enhance stimulation brought on by GHB. A preferred embodiment of the present invention is wherein the response is measured and compared with the results obtained from contact with GHB; the functional response being greater in the presence of a GHB agonist or allosteric modulator.

Especially preferred embodiments comprise the further step of contacting a separate group of cells expressing the receptor polypeptide (the control group) with the suspected agonist or allosteric modulator; a lack of response in the control indicating the presence of an allosteric modulator and a functional response indicating the presence of an agonist.

In preferred embodiments, the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide are recombinant cells that were transfected with a vector or vectors comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide. In particular embodiments, the functional response is selected from the group consisting of: modulation of the activity of an ion channel; changes in calcium concentration; changes in a signal from a reporter gene whose expression is controlled by a promoter that is induced by interaction of an agonist with the GABAβ receptor; and changes in membrane currents. In particular embodiments, the change in membrane current is measured in Xenopus oocytes. In other embodiments, the change in membrane current is the modulation of an inwardly rectifying potassium current.

Particular types of functional assays that can be used to identify modulators of GHB include transcription-based assays. Transcription-based assays involve the use of a reporter gene whose transcription is driven by an inducible promoter whose activity is regulated by a particular intracellular event such as, e.g., changes in intracellular calcium levels that are caused by the interaction of a receptor with a ligand. Transcription-based assays are reviewed in Rutter et al, 1998 Chemistry & Biology 5:R285-R290. The transcription-based assays of the present invention rely on the expression of reporter genes whose transcription is activated or repressed as a result of intracellular events that are caused by the interaction of an agonist with a heterodimer of HG20 and a Gbl isoform where the heterodimer forms a functional GABAβ receptor. In the above assays, compounds capable of effecting a greater signal from the reporter gene in the presence of GHB than GHB itself are noted as modulators of GHB (i.e., agonists or allosteric modulators of agonists) and warrant further study. Conversely, if a suspected antagonist is utilized along with GHB, the inquiry would be whether the cells treated with GHB emitted a greater signal than the cells treated with GHB and the suspected antagonist, in which case the compound would be an antagonist of GHB function. Suitable reporter genes for use in the above assays are green fluorescent protein (GEP), β-galactosidase, and luciferase.

A sensitive transcription based assay in accordance with the instant invention employs a plasmid encoding β-lactamase under the control of an inducible promoter. This plasmid is transfected into cells together with a plasmid encoding a receptor for which it is desired to identify modulators such as agonists or allosteric modulators of agonists. The inducible promoter on the β-lactamase is chosen so that it responds to at least one intracellular signal that is generated when an agonist or modulator of same binds to the receptor. Thus, following such binding of agonist or modulator to receptor, the level of β-lactamase in the transfected cells increases. This increase in β-lactamase is measured by treating the cells with a cell-permeable dye that is a substrate for cleavage by β-lactamase. The dye contains two fluorescent moieties. Li the intact dye, the two fluorescent moieties are close enough to one another that fluorescence resonance energy transfer (FRET) can take place between them. Following cleavage of the dye into two parts by β-lactamase, the two fluorescent moieties are located on different parts, and thus can drift apart. This increases the distance between the fluorescent moieties, thus decreasing the amount of FRET that can occur between them. It is this decrease in FRET that is measured in the assay.

Through the use of the above assay wherein the inducible promoter that drives β-lactamase is activated by an intracellular signal generated by the interaction of the GABAβ receptor and modulators thereof such as GHB, one of ordinary skill in the art could identify modulators of GHB function such as agonists, allosteric modulators of agonists, inverse agonists, and antagonists. To produce the GABAβ receptor, a plasmid encoding HG20 and a plasmid encoding a Gbl isoform are transfected into the cells. Cells endogenously expressing functional GABAβ receptor are also suitable in the instant assay. The cells are exposed to the cell- permeable dye and then exposed to compounds suspected of being modulators of GHB. Those compounds that cause a greater decrease in FRET in the presence of GHB are likely to be agonists of GHB.

Accordingly, the present invention includes a method for identifying modulators of GHB comprising: (a) providing cells expressing (i) HG20 or a functional equivalent thereof, (ii) a receptor polypeptide comprising the extracellular region of a GAB Aβ receptor polypeptide, and (iii) β-lactamase under the control of an inducible promoter that is activated by an intracellular signal generated by the interaction of GHB and the GAB Aβ receptor; (b) exposing the cells to a substrate of β-lactamase that is a cell-permeable dye that contains two fluorescent moieties where the two fluorescent moieties are on different parts of the dye and cleavage of the dye by β-lactamase allows the two fluorescent moieties to drift apart; and (c) measuring the amount of fluorescence resonance energy transfer (FRET) in the cells following contact with a suspected GHB modulator in the presence of GHB. In the instance wherein agonists are sought, the amount of FRET can be compared with that measured in the absence of the suspected agonist to obtain a value for the decrease in FRET caused by the suspected agonist.

A decrease in FRET resulting from GHB can be determined by measuring the amount of FRET in the presence and in the absence of GHB. By comparing the decrease in FRET caused by the suspected GHB agonist in the presence of GHB to the decrease in FRET caused by GHB, one can identify modulators, and agonists in particular, of GHB. An agonist in the presence of GHB would exhibit a greater decrease in FRET than GHB.

The above-described assay can be further modified to an assay for identifying antagonists of GHB. This method comprises (a) providing cells expressing (i) HG20 or a functional equivalent thereof, (ii) a receptor polypeptide comprising the extracellular region of a GAB Aβ receptor polypeptide, and (iii) β- lactamase under the control of an inducible promoter that is activated by an intracellular signal generated by the interaction of GHB and the GABAβ receptor; (b) exposing the cells to a substrate of β-lactamase that is a cell-permeable dye that contains two fluorescent moieties where the two fluorescent moieties are on different parts of the dye and cleavage of the dye by β-lactamase allows the two fluorescent moieties to drift apart; and (c) measuring the amount of fluorescence resonance energy transfer (FRET) in the cells following contact with a suspected GHB antagonist in the presence of GHB. This value can be compared with the value obtained from contact with GHB; a lesser amount of FRET in the cells contacted with GHB indicating the presence of a GHB antagonist.

In a particular embodiment of the above-described methods, the inducible promoter that is activated by at least one intracellular signal generated by interaction of an agonist with the GAB Aβ receptor is a promoter that is activated by changes in membrane currents, e.g., changes in potassium currents or activation of the map kinase signaling pathway. In other embodiments, the inducible promoter is activated by the transcription factor NFAT, or is activated by a signal transduced by a chimeric Gq protein, or a signal generated by protein kinase C activity, or by changes in intracellular calcium levels. The assays described above can be further modified to an additional assay for identifying GHB antagonists. Such modification would involve the use of β -lactamase under the control of a promoter that is repressed by at least one intracellular signal generated by interaction of GHB with the GAB Aβ receptor and would also involve running the assay in the presence of GHB. When the cells are exposed to compounds suspected of being GHB antagonists, β-lactamase will be induced, and FRET will decrease, if the compound tested is able to counteract the effect of GHB, i.e., if the compound tested is actually an antagonist. In a particular embodiment, the inducible promoter that is repressed by at least one intracellular signal generated by interaction of GHB with the GAB Aβ receptor is a promoter that is repressed by changes in potassium currents.

In particular embodiments of the above-described methods, β- lactamase is TEM-1 β-lactamase from Escherichia coli.

In other embodiments, the substrate of β-lactamase is CCF2/AM (Zlokarnik et al, 1998 Science 279:84-88).

In particular embodiments of the above-described methods, the cells express a promiscuous G-protein, e.g., Gocl5 or G l6. In other embodiments, the cells have been transfected with an expression vector that directs the expression of a G-protein subunit or subunits.

The assays described above could be modified to identify inverse agonists of GHB. In such assays, one would expect a decrease in β-lactamase activity where agonists such as GHB would produce an increase. The assays described above could also be modified to identify allosteric modulators of GHB. In such assays, one would not expect a response from the modulator in the absence of GHB.

In particular embodiments of the above-described methods, HG20 is a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:4; positions 9-941 of SEQ ID NO:4; positions 35-941 of SEQ ID NO:4; positions 36-941 of SEQ ID NO:4; positions 38-941 of SEQ ID NO:4; positions 39-941 of SEQ ID NO:4; positions 42-941 of SEQ ID NO:4; positions 44- 941 of SEQ ID NO:4; positions 46-941 of SEQ ID NO:4; positions 52-941 of SEQ ID NO:4; positions 57-941 of SEQ ID NO:4; the amino acid sequence encoded by the DNA sequence deposited in GenBank Accession No. AF056085; the amino acid sequence encoded by the DNA sequence deposited in GenBank Accession No. AJ012188; and the amino acid sequence encoded by the DNA sequence deposited in GenBank Accession No. ASF074482.

It is important to note that in all of the above assays, HG20 (or gb2) can be substituted with a functional equivalent such as GABAβR2 or GBR2.

In particular embodiments of the above-described methods, HG20 is a chimeric HG20 protein. By chimeric HG20 protein is meant a contiguous polypeptide sequence of HG20 fused in frame to a polypeptide sequence of a non- HG20 protein. For example, the N-terminal domain and seven transmembrane spanning domains of HG20 fused at the C-terminus in frame to a G protein is a chimeric HG20 protein. Another example of a chimeric HG20 protein is a polypeptide comprising the FLAG epitope fused in frame at the amino terminus of amino acids 52-941 of SEQ ID NO:4. Especially preferred forms of chimeric HG20 proteins are those in which a non-HG20 polypeptide replaces a portion of the N- terminus of HG20. Chimeric GAB Aβ receptor proteins or fragments thereof may also be used in the present invention. In particular embodiments, the chimeric receptor proteins comprise the extracellular region of a GAB Aβ receptor polypeptide with or without the signal sequence. In particularly preferred embodiments, a polypeptide comprising the extracellular region of a GABAβ receptor polypeptide is fused to another non-GABAβ protein or portion thereof.

In preferred embodiments, the expression vector encoding HG20 comprises a nucleotide sequence selected from the group consisting of: positions 293-

3,115 of SEQ ID NO:3; positions 317-3,115 of SEQ JX) NO:3; positions 395-3,115 of SEQ ID NO:3; positions 398-3,115 of SEQ ID NO:3; positions 404-3,115 of SEQ ID

NO:3; positions 407-3,115 of SEQ ID NO:3; positions 416-3,115 of SEQ JO NO:3; positions 422-3,115 of SEQ ID NO:3; positions 428-3,115 of SEQ ID NO:3; positions 446-3,115 of SEQ ID NO:3; and positions 461-3,115 of SEQ ID NO:3. Some of the above-described methods can be modified to take advantage of other ways of assaying for GHB agonist activity at the GABAβ receptor. GHB agonists/antagonists/inverse agonists may affect the internalization or trafficking of functional GAB Aβ receptors. For example, in the case of the β2- adrenergic receptor, agonist exposure results in receptor internalization. Therefore, receptor trafficking between intracellular pools and the cytoplasmic membrane may be considered an assay of agonist activity. It may be that GAB Aβ receptor trafficking is modulated by GHB agonists in a similar manner. It would then be possible to identify agonist activity by monitoring GAB Aβ receptor trafficking. Such trafficking can be monitored by whole cell immunohistochemistry and confocal microscopy or by surface and intracellular receptor labeling and flow cytometry. Furthermore, because the functional GABAβ receptor may be a heterodimer, then GHB agonists/antagonists/inverse agonists may be expected to alter the ratio of heterodimer to monomer. Hence the disruption or appearance of a heterodimer may be considered an additional screening assay. In this case, the monitoring of receptor dimerization or disappearance may be done by the appearance or disruption of FRET. Each of the monomers are labeled with a fluorophore such that close proximity would allow FRET to occur. Upon GHB agonist binding, one might see disruption of FRET, indicating disruption of dimers or increase in FRET indicating more dimerization in the course of agonist activation. Assays which make up further aspects of this invention include binding assays such as competition for 125i_GABAβ receptor agonist binding, coupling assays including agonist-mediated inhibition of forskolin-stimulated adenylate cyclase in cells expressing the Gblc receptor polypeptide, measurement of agonist-stimulated calcium release in cells expressing Gblc receptors and promiscuous G proteins such as G15/G16/Gqi5/Gqo5 such as aequorin assays and calcium-based dye assays, stimulation of inward rectifying potassium channels (GJRK channels, measured by voltage changes) in cells expressing Gblc receptors, and measurement of pH changes upon agonist stimulation of cells expressing Gblc receptors as measured with a microphysiometer. In certain cellular environments, stimulation of Gblc receptors may also lead to inhibition of calcium channels. The above assays can be carried out with GHB in the presence of compounds suspected of modulating GHB function and compared to results obtained following contact with GHB. In this manner, GHB modulators can be identified using the above-taught principles. While the above-described methods are explicitly directed to testing whether "a" compound is an agonist or antagonist of GHB, it will be clear to one skilled in the art that such methods can be adapted to test collections of compounds, e.g., combinatorial libraries, collections of natural produces, etc., to determine whether any members of such collections are activators or inhibitors of GHB. Accordingly, the use of collections of compounds, or individual members of such collections, as the compound in the above-described methods is within the scope of the present invention.

The following non-limiting Examples are presented to better illustrate the invention.

EXAMPLE 1 Construction of GABAβ Receptor Expression Constructs

The ORFs of human gbla, gblb, and Gblc isoforms were obtained from human cerebellum cDNA (Clontech) by nested PCR cloning using a High- fidelity HF-PCR cloning kit (Clontech).

For Gblc, the first step of amplification used primers: hgblcF-ECORl 5'-GAT ATC GAA TTC GCC ACC ATG TTG CTG CTG CTG CTA CTG GCG CCA CTC-3' (SEQ ID NO:5) and gblRR 5'-gc cct tec cct etc cct ttc cct ccc-3' (SEQ ID NO:6). PCR conditions were: precycle denaturation of 94°C for 15s, followed by denaturation at 94°C for 15s and an annealing/extension step at 68°C for 4 min for 30 cycles. A final extension step was done at 72°C for 3 min. The second amplification used primers: hgblcF-ECORl and hgblECORl-R 5'-GCC GAG GAA TTC TCA CTT ATA AAG CAA ATG CAC TCG ACT CCC (SEQ ID NO:7). PCR conditions were as described above. PCR products were subcloned into PCR-TA cloning vector (Invitrogen), p T7 vector for expression in Xenopus oocytes, pcDNA3.1/Zeo(+) (Invitrogen) for transient high-level expression in COS-7 cells, or pIRES-bleomycin bicistronic vector (Clontech) for stable expression in HEK-293 cells. The ORF of human gbla was obtained from human cerebellum cDNA

(Clontech) by nested PCR cloning using primers based on gbla (Genbank Accession No. AJ225028). Primers for the first round PCR were as follows: gblFF: 5 -TGA GGC CCG GGG AGA GCC GGG GAG-3 (SEQ ID NO: 13) and gblRR: 5'-GC CCT TCC CCT CTC CCT TTC CCT CCC-3' (SEQ ID NO: 14), primers for nested PCR: hgblaECORI-F: 5'-GCC GAG GAATTC GCC ACC ATGTTG CTGCTGCTG

TTA CTG GCG CCA CTC-3' (SEQ ID NO: 15) and hgblECORI-R: GCC GAG GAA TTC TCA CTT ATA AAG CAA ATG CAC TCG ACT CCC-3' (SEQ ID NO: 16). The ORF of human gblb (Genbank Accession No. AJ225029) was also obtained from human cerebellum cDNA (Clontech) by nested PCR cloning. Primers for the first round PCR were as follows: hgblb-FF: 5'-CCC CCT GAG CTC CTA ACG CTC-3' (SEQ ID NO: 17) and gblRR: 5'-GC CCT TCC CCT CTC CCT TTC CCT CCC-3' (SEQ ID NO: 18), primers for nested PCR: hgblbF-Notl: 5'-GAT GAT ATC GCG GCC GCA TGG GGC CCG GGG CCC CTT TTG CCC-3' (SEQ ID NO: 19) and hgblECORI-R: GCC GAG GAA TTC TCA CTT ATA AAG CAA ATG CAC TCG ACT CCC-3' (SEQ ID NO:20).

PCR products were subcloned into pT7 vector for expression in Xenopus oocytes, pcDNA3.1/Zeo(+) (Invitrogen) for transient high-level expression in COS-7 cells, or pIRES-bleomycin bicistronic vector (Clontech) for stable expression in HEK-293 cells. All constructs were verified by sequencing on both strands and expression by in vitro translated in the presence of [35s]methionine in the TNT® Coupled Reticulocyte Lysate system (Promega).

EXAMPLE 2 Transient Expression for Radioligand binding

COS-7 (ATCC) cells were grown to -70% confluence. 6μg of Glaxo lc or Merck lc plasmid DNA (Qiagen) were transfected into 1.2 million COS-7 cells using 36μl of lipofectamine as recommended by manufacturer (Gibco BRL).

EXAMPLE 3 Receptor Binding

Cells were harvested 72hr-post transfection. Crude membrane preparations were prepared as described in Belley et al, 1999 Bioorganic &

Medicinal Chemistry 7:2697-2704. 1H-CGP71872 (a GABAβ receptor antagonist) was custom synthesized by NEN. Saturation binding experiments were conducted using 25μg of membrane and increasing concentration of radiolabeled 3H-CGP71872 ranging from ~0.05nM to 75nM. Non-specific binding was obtained in the presence of lμM cold CGP71872. Each condition was made in duplicate. Incubation was carried out in 200μl of binding buffer (50mM Tris, pH7.4, 2.5mM CaCl₂, IX protease inhibitor cocktail - Complete Tablet™ (Boehringer Mannheim)) with constant shaking at room temperature for 2hr. Brandel harvester was used to trap bound ligand onto Whatman GF/B filters. Data analysis was performed using GraphPad Prism (San Diego).

EXAMPLE 4 Transient Expression and Generation of Gbl-Gb2 Stable HEK-293 Cell Lines

Receptor DNAs (6 μg total DNA/1.2 X 10⁶ cells) were transfected into

COS-7 or HEK-293 cells using 36 μl Lipofectamine reagent (Gibco BRL) according to the manufacturer's instructions. Stable gb2-expressing HEK-293 clones were selected by growth in puromycin (5μg/ml) containing media and dilution cloning. RNA was prepared from 95 clones using Trizol reagent (GibcoBRL) and 10 μg total RNA spotted by vacuum using a dot-blot apparatus onto BrightStar-Plus nylon membranes (Ambion). The blot was hybridized with a ³²P-labeled DNA fragment encoding the full-length gb2 receptor (10⁶ cpm/ml) in Zip-Hyb solution (Ambion) for 10 h at 50 °C, and washed at 55 °C for 90 min in high-stringency wash buffer. Two high gb2 receptor RNA-expressing clones (gb2.46, and gb2.10) were analysed for cell surface gb2 receptor expression by indirect staining using gb2 antisera 1630.1-1630.2 and goat anti-rabbit antibodies coupled with Alexa-488 on a Becton Dickinson FACS Vantage SE flow-cytometer configured to detect FITC fluorescence. Gbl isoform DNAs were transfected into the gb2.10 clone as described, and bulk gbla-, gblb-, and Gblc-gb2 stables were selected by growth in puromycin (5μg/ml) and phleomycin (50 μg/ml) containing media. Control stable pIRES -puromycin and prRES-bleomycin vector expressing HEK-293 cell lines were generated also by growth in antibiotic selection media.

EXAMPLE 5 cAMP Functional Assays

cAMP determinations were made using a scintillation proximity assay (SPA) kit (Amersham, ONT). Briefly, HEK-293 cells were washed, detached, and 77,000-100,000 cells/well resuspended in Hank's Balanced Salt Solution containing 25 mM HEPES pH 7.4, 100 μM 4-(3-butoxy-4-methoxybenzyl)-2-imadazolidinone (Ro 20-1724, BIOMOL, PA) and incubated for 20 min at 37 °C. 2 μM forskolin and ligands (10^"9-10^"3 M) were added and incubated for 30 min at 37 °C. Cells were lysed by boiling and c AMP levels were determined by SPA according to the manufacturer's instructions. Data were analyzed by nonlinear least-squares regression using the computer-fitting program GraphPad Prism version 2.01 (San Diego).

EXAMPLE 6 In Vitro Receptor Expression

In vitro coupled transcription/translation reactions were performed in the presence of [³⁵S]methionine in the TNT® Coupled Reticulocyte Lysate system (Promega, Madison, Wl) using pcDNA3.1 plasmids containing the gbla, gblb, and Gblc DNAs. Translation products were analysed by electrophoresis on 8-16% Tris- Glycine SDS gradient gels (Novex pre-cast gel system, San Diego, CA) under denaturing and reducing conditions. Gels were fixed, dried and exposed to Kodak X- AR film at -70 °C for 4 to 24 h.

Claims

WHAT IS CLAIMED:

1. A method for the identification of modulators of gamma- hydroxybutyrate (GHB) which comprises: (a) contacting a receptor polypeptide comprising the extracellular region of a GAB Aβ receptor polypeptide with a candidate compound, and

(b) determining if binding occurs in the presence of GHB; binding indicating the presence of a GHB modulator.

2. The method of claim 1 wherein the GABAβ receptor polypeptide is selected from the group consisting of gbla, gblb, gblc and Gblc receptor polypeptides.

3. The method of claim 2 wherein the GABAβ receptor polypeptide is Gblc.

4. The method of claim 3 wherein the receptor polypeptide comprises amino acid 18-525 of SEQ ID NO:2.

5. The method of claim 1 wherein the binding of the candidate compound in the presence of GHB is compared to that of a control, the control comprising contacting the receptor polypeptide with GHB.

6. The method of claim 1 wherein the modulators are agonists or antagonists.

7. The method of claim 5 which comprises exposing cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide to a suspected agonist or antagonist in the presence of GHB; the amount of binding of GHB being less in the presence of an agonist or antagonist of GHB.

8. The method of claim 7 wherein the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide are recombinant cells that were transfected with a vector or vectors comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide.

9. A method for the identification of compounds which modulate the function of gamma hydroxybutyrate (GHB) which comprises determining whether a candidate compound upon contact with a receptor polypeptide comprising the extracellular region of a GAB Aβ receptor polypeptide results in a signal generated by activation or inhibition of the polypeptide in the presence of GHB.

10. The method of claim 9 wherein the GABAβ receptor polypeptide is selected from the group consisting of gbla, gblb, gblc and Gblc receptor polypeptides.

11. The method of claim 10 wherein the GAB Aβ receptor polypeptide is Gblc.

12. The method of claim 11 wherein the receptor polypeptide comprises amino acid 18-525 of SEQ ID NO:2.

13. The method of claim 9 wherein HG20 or a functional equivalent thereof is expressed along with the receptor polypeptide such that heterodimers of the receptor polypeptide and HG20 are allowed to form.

14. A method in accordance with claim 13 for the identification of GHB receptor antagonists or inverse agonists which comprises:

(a) contacting cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide in the presence of GHB with a suspected antagonist or inverse agonist of GHB, and

(b) determining whether a functional response follows.

15. The method of claim 14 wherein the response is measured and compared to that of a control wherein the receptor polypeptide is contacted with GHB; the functional response of GHB being less in the presence of a GHB antagonist or inverse agonist.

16. The method of claim 14 wherein the response is compared to that of a control, the control comprising contacting a separate group of cells with the antagonist or inverse agonist; a lack of response in the control indicating the presence of an antagonist and a response in contradiction with that of GHB indicating the presence of an inverse agonist.

17. The method of claim 14 wherein the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide are recombinant cells that were transfected with a vector or vectors comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide.

18. A method in accordance with claim 13 for the identification of GHB agonists or allosteric modulators of agonists which comprises: (a) contacting cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide in the presence of GHB with a suspected agonist of GHB or an allosteric modulator of such, and

(b) determining whether a functional response follows.

19. The method of claim 18 wherein the response is measured and compared to that of a control, the control comprising contacting the receptor polypeptide with GHB; the functional response being greater in the presence of a GHB agonist or allosteric modulator.

20. The method of claim 18 wherein the response is measured and compared to that of a control, the control comprising contacting a separate group of cells with the agonist or allosteric modulator; a lack of response in the control indicating the presence of an allosteric modulator and a functional response indicating the presence of an agonist.

21. The method of claim 18 wherein the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide are recombinant cells that were transfected with a vector comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide.