CA2160038A1 - Methods of screening compounds for their pharmacological relevance based on downregulation of recombinant receptors - Google Patents

Abstract

This invention relates to the use of reagents in a novel manner to screen compounds in vitro for their ability to interact with and modulate the functional properties of receptors coupled to guanyl nucleotide-binding regulatory proteins (G-proteins).Methods are described herein for determining the ability of compounds to act as antagonists by observing their ability to downregulate recombinant G-protein coupled receptors in membrane preparations. The methods described permit the downregulation effect to be observed within a time frame that is dramatically accelerated compared to other known methods of testing the same effect, thereby providing novel and improved efficient means of screening compounds for their probable pharmaceutical efficacy. This invention also relates to the use of such reagents and methods in kits.

Description

216~038 BACKGROUND OF THE INVENTION
One manner in which cells . with one another (either within an organismor between the environment and the organism) or receive i--ru- from the ellvilu is by signaling via ~ole;~ ,...ole~,ules, known as receptors, which are frequently embedded in the outer surface of cell mf~ r~lr~ A chemical substance (either released from one cell within the organism or entering the organism from the environment) will interact with the receptor located on the cell (or within the cell) of the organism by binding to a specific area on the }eceptor, thereby producing a eu..rul " ~ change in the structure of the receptor. Depending upon where the 0 receptor is located within a cell, and with which other molecules and ~ ul~ol~uules it is in contact, a specific biochemical change will be generated in the immediate vicinity by the change in receptor eu..rullll~Liull, which is able to bring about greater biochemical changes within the cell through a cascade or domino effect of subsequent chemical signaling. Examples of chemical messengers that are produced to act on receptors in this way to effect cell-cell, are Ll such as, but not limited to, u~ I, hormones and pl~ u~loll~s.
There are many different types of receptors, which fall within families of related proteins depending on features including their localization within the cell, their basic structural, ~ , and the mechanism by which they transmit signals upon binding a chemical messenger. Three well-~ .~t~,.i~d classes of cell-surface receptor proteins are channel-linked, catalytic and G protein-linked receptors.
Channel-linked receptors form channels in a lipid membrane that will undergo a ~o..ru I change upon chemical-messenger binding, to open or close, either allowing specific ions to pass, or preventing them from passing through the membrane.
The change in the flow of ions through the membrane can initiate significant changes in the environment of a cell such as the ion fluxes that underlie signal l,~,.. "i~
within a nerve cell. Catalytic receptors function as enzymes when activated by achemical messenger. Usually, the chemical messenger binds to a portion of the receptor that is exposed on the external surface of the membrane causing a portion if 21~q~
the enzyme to catalyze a reaction on the inner surface of the cell. G protein-linked receptors are proteins which function as one part of a multi-component complex whereby binding of transmitter to the receptor component initiates a chain reaction through an associated G protein that is transmitted to an effector component to effect change in the molecular environment.
The class of receptors known as G protein-linked receptors are typically clla~ i~d by a hepta-helical u-~ ;~lio.~, in which the receptor protein traverses the membrane seven times. Members of this class of receptors also share a common signaling mechanism which involves intracellular transducer elements known as G proteins in the 0 Ll ' of the signal across the membrane. When a chemical messenger binds to a specific site on the nrtr~rPll~ r surface of the receptor, the r `Illa~iOll of the receptor changes so that it can interact with and activate a G protein. The activated G
protein causes a molecule of guanosine ~ r (GDP), that is bound to the surface of the G protein, to be replaced by a molecule of guanosine Ll ir ' , ' (GTP), 5 triggering another ~o.. rO~ iul,~l change in the G protein. When GTP is bound to its surface, the G protein regulates the the activity of an effector. These effectors include enzymes such as adenylyl cyclase and phospholipase C, they include certain transport proteins and they also include channels that are specific for calcium ions (Ca2+), potassium ions (K+), or sodium ions (Na+).

2 0 In general, activation of G protein-coupled receptors by ~i will induce one or another of the following effector responses: activation of adenylyl cyclase, inhibition of adenylyl cyclase or stimulation of Aul~u ~Jh~ C activity. When the effector adenylyl cyclase is either activated or inhibited it produces changes in the l of the molecule cyclic adenosine ...o, rl ,' (cAMP). Another effector, 1 ' ~ ip~c,, C, causes one molecule of p~lu~ idylillubi~ul-lJ;~ , ' (PIP2) to becleaved into one molecule each of inositol LlilJ' ,' ' (IP3) and diacylglycerol (DAG);
IP3 then causes calcium ions (Ca2+) to be released into the cytoplasm. Alterations in cellular levels of cAMP and Ca2+ are two of the most important inir~ln,~ll ' messages that in turn act to alter the behaviour of other target proteins in the cell.

` 2160038 Receptors may be classified according to the type of signaling pathway they activate within cells. This fl~ occurs at the level of the G proteins, which detect and direct signals from diverse receptors to the d~)~lULII- ' effector-response pathway.
Three main groups of G proteins are: Gs-like, which mediate adenylyl cyclase activation; Gi-like, which mediate inhibition of adenylyl cyclase; and Gq-like, which mediate activation of phosphoplipase C. Since one receptor is able to activate many G
proteins, the signal i ' pathway may amplify the signal greatly.
A cell may contain multiple G proteins, each of which may interact with many different receptors and regulate several different effectors. When several receptors 0 utilize a common G protein 'transducer' this signdl-~.d,.OIu~,liol~ pathway allows for sigrdl integration because many individual PYtr~lrPIllll~r signals cdn be integrated to yield a cumulative in~r~f Pll~ r signal, such as elevated I dliU.I., of calcium ion.
Thus, the G protein-linked receptor/effector systems generate complex networks of molecular 111~ that allow for versatile regulation of intercellular and cell function.
A wide variety of chemical ~ involved in regulating key functions in the body act through G protein-coupled receptors. These include, among others, Il~U~ such as dopamine, d~ ,llVlillc and serotonin, hormones of the endocrine system such as ~ , glucagon and adL-,llvcul~iw~-u~. , and lipid mediators such as y~ and leukotrienes. Over one hundred different G
protein-coupled receptors have been identified in humans, and many more are expected to be discovered. All of these receptors are believed to utilize one of the three principal G protein-effector signaling pathways (stimulation or inhibition of adenylyl cyclase or activation of p' , ' 'i, C).
In view of the diverse functions of G protein-coupled receptors in the body, it is not surprising that many existing therapeutic drugs act by directly modifying the function of G protein-coupled receptors. In most cases, these drugs exert their effects on receptor function by binding to the same site on the receptor component as the natural ` ~ 21600~8 chemical messenger. Such drugs may be classified into two types: I) agonists, which mimic the action of natural transmitter by provoking activation of G protein-effector signaling pathways when they bind to the transmitter site; and 2) uu...~Lilivc , which block the binding of the transmitter by occupying the transmitter binding site but do not themselves activate G protein-effector pathways. A useful analogy is that of a lock and key, whereby agonists are different keys which are able to open the same receptor lock, whereas antagonists will block the key-hole but will not open the lock. In a more general view, compounds which can bind to a specific region of the receptor are called ligands; agonists and antagonists are ligands which bind to the transmitter recognition site on the receptor.
Analysis of the effects of ligands on the ability of G protein-coupled receptors to activate signaling pathways has suggested that the receptors exist in two forms or ,u..~ ~, an 'inactive' ~ul~ru which is silent and an 'active' ~:u..rul~ Liull which triggers G protein activation and effector signaling (Gilman, A.G., 1987, Annu.
Rev, Biochem. 56: 615-649; Levitski, A., 1988, Science 241:800-806). Generally, ligands that can cause the receptor to assume the active cullro.l..~Liul. turn on signaling and are thus agonists. These compounds, at maximally effective , can elicit a full or partial response, and are termed "full" and "partial" agonists,respectively. Ligands that block or otherwise interfere with the interaction of agonists with the receptor, and thereby prevent agonists from activating the receptor, are known as "competitive antagonists". These . l~uul-d~ are generally thought to act by binding to the transmitter site, but to possess no intrinsic activity themselves (i.e. they do not turn on the signaling function of the receptor.) Studies have shown that .;UIll~,tiLiliv~ antagonists can be further categorized into two classes, 'neutral antagonists' which block agonist binding but have no effect on signaling, and 'inverse agonists' (also known as negative antagonists) which can inhibit the 'h~hæluulld' or basal level of signaling displayed by receptors in the absence of agonists.
This evidence has led to a model in which active and inactive receptors co-exist in the cell in equilibrium, with agonists pushing the e4uilil,. to the active form, inverse s ` 216~8 aOonists pushing it to the inactive form, and neutral antagonists blocking the chemical messenger site while not favoring either I r ~lldliOI~.
Agonist-induced receptor duw--l~, ' is a well-~ rd F in which continued exposure to high ~ollcc~ d~iulls of agonist results in a reduction of the number of receptors. The process is believed to involve in~Prn~ n of agonist-receptor complexes with subsequent proteolysis in Iysosomes and is reviewed in Caron, M.G. & R.J. Leflkowitz, 1993, Recent. Prog. Horm. Res. 48: 277-290. Recent evidence has implicated an initial agonist-induced ICid~ oclll~ of receptors at the cell surface in receptor downregulation (von Zastrow, M. & B.K. Kobilka, 1994, J. Biol.
0 Chem. 269: 18448-18452).
Certain selective antagonists of the serotonin subtype 2A and 2C receptors (5-llyJIu~y~ly,ui 2A and 2C, or 5HT~A and 5HT~C) which rlPm( therapeutic efficacy have also been shown to produce a decrease in receptor levels in various brain regions when d.l~ d chronically to rats. This loss of radioligand binding by receptors upon prolonged antagonist treatment has been termed 'atypical du..~ ouldIi()l~', reflecting the general view that agonists normally duwll~
whereas antagonists up-regulate receptor numbers. It has been suggested that this atypical action of antagonists represents an adaptive response which could underlie the therapeutic efficacy of s~ antagonists as dll~id~~ and dll~ilJ yCl~U~
agents (Meltzer, H.Y. & J.F. Nash, 1991, Parmacol. Rev., 43: 588-600; Coward, D.M., 1993, "The Ph(u.. ~ .Oy of Clozapine-like Atypical Ar~ y~h~lics, Antipsychotic Drugs and their Side-Effects" Academic Press Inc., San Diego, U.S.A., 28-44.).
The development of a method of testing ~ull r- ' for their abilities to induce receptor dUWIII~ ~ ' '' as antagonists has great utility for many industries whose goal is to develop chemical substances that interact with G protein-coupled receptors. Since G protein-coupled receptors are ubiquitous and widely used in nature to transmitcellular signals, this invention has utility for different industries, such as, but not 216~038 limited to: the pl.c,.", . I industry, the pest-control industry, the industry, the food industry and the fragrance industry.
The L~ r-~ 'l sector is LJdl~ ulalIy interested in the potential therapeutic applications of antagonist-induced receptor downregulation in treatments for p~ gi~ associated with depression and psychosis. Moreover, there are several genetic diseases shown to be associated with mutations in G protein-coupled receptors resulting in constitutive receptor signaling. Drugs which possess O related receptor downregulating activity could have particular therapeutic relevance for such , conditions.
Current methods of screening compounds for their ability to interact with G protein-linked receptors are well-known in the art but are less than adequate. For example, one type of assay, termed a ligand binding assay, measures the abilitity of a compound to bind to the transmitter recognition site on the receptor. In its most widely-applied form, the assay involves incubating an aqueous suspension of the receptor (generally a cell membrane preparation containing the receptor) with a radioactive derivative of a ligand known to bind to the transmitter site. By measuring the amount of radioactive ligand bound in the presence of the test compound, it is possible to detect ligands for the same site by the inhibition of .ddioli~ ' binding to the receptor. This type of assay, and variations based on alternative labels (e.g. fluorescent ligands) or technical improvements to facilitate automation (e.g. II ~il-n proximity assay) are frequently the first screening test carried out to identify compounds which interact with receptors and thus merit further, as potential therapeutic candidates.
Other types of assays, which are generally used to investigate those . ~ .v..l~ which are found to be active in the ligand binding assay, permit the di~ I of agonist from antagonist ligands. The distinction between these two drug classes is obscured and unreliable in the ligand binding assay, and systems which can measure receptor activation of G protein-effector signalling activities are required for such ~ ;ri. ~
to be made with ronfirlpn~p Such assays are generally termed bioassays, and were 21600~8 traditionally carried out with animal tissues in organ baths where the activity of the receptor could be measured as a complex response of the tissue (e.g. muscle ronfr:lrfinn) In these p~ " , agonists are identified by their ability to activate the tissue respoDse, while antagonists do not activate the response themselves but competitively block the activation by agonists. Variations on this screening procedure use biochemical assays, such as the production of the cAMP or IP3, to measure the effects of compounds on receptor coupling to effector response pathways. They are well known in the art.
In addition to di;~ i..6 between agonists and ~t~nnisfs~ these bioassay systems 0 also permit more detailed analysis of the properties of the drug candidate, such as potency (the Wll~ ld~iUII at which the ligand exerts its effect on receptor signaling), as well as efficacy (the maximal effect of the drug on receptor activity). The Ill~s~ll~llltlll of ligand efficacy permits further . I- ~;r~ ",. of agonist ligands into full agonists and partial agonists, the latter group producing only a partial response even at maximal In these assays, antagonists have ~ero efficacy (i.e. they produce no activation of the response pathway). These parameters permit selection of the most promising drug candidates, based on their ranking in the different assays, for further .l~h,~ - and analyses in more complex models (e.g. animal models of disease).
With the enormous progress in the cloning, sequencing and expression of genes which encode G protein-coupled receptors, (and drug target proteins in general), there has been a major shift from the use of animal tissues to using I~CU.llb' ' receptors.
Re~u..ll,i receptors are produced by expression of the cloned gene in cultured cells.
In those cases where the receptor cDNA has been isolated and cloned such I~;~ulllbil~dilL receptors are now the principal source of receptors for drug screening in ligand binding assays and in bioassays. The use of l~u..ll. receptors has many advantages over tissue sources, including the ability to use human receptors expressed from human genes, the facility with which large amounts of the protein can be produced, and the fact that a single receptor subtype can be tested and compared with ` ~ 2160038 closely related subtypes (subtypes are closely related but distinct receptors which use the same natural ~Idl.~lllil~l).
Bioassay systems for l~,UIllb' ' G protein-coupled receptors that are known in the art are based on the ability of the expressed receptor to activate 1uE~ signaling pathways in the host cell. The first assays measured the activity of effectors (adenylyl cyclase and pl~v,~l,vli~.as~ C) using known b; ' I assays originally used for tissue-based assays. These generally employ li~n cell lines which have been made to express the cloned receptor DNA using techniques (tldll~re~iull, ~iUII) which are well known to and routinely practised by technicians trained 0 in the art. One example uses fluorescent dyes sensitive to the . of . specific ions, primarily calcium, to measure changes in the intracellular ion r associated with activation of receptors coupled to Gq-phopsholipase C
signaling. An increasing number of new systems involve genetic Cll~ of the host cell to facilitate, ~ ,..; of the effector response to receptor activation. In one example, the gene for an enzyme that is readily assayed, such as beta-tl~Citl~C~', iS inserted into the host cell genome under the control of a promoter element sensitive to cAMP levels. Receptors which activate adenylyl cyclase and thereby increase cAMP levels in these cells will activate expression of the beta-rf~citl~c~ ~reporter~ gene. ~ of the enzyme activity in a simple assay thus provides a measure of receptor activity.
In general terms, all of these bioassay systems are designed for, and well-adapted to, the task of di~ il.g these ligands which activate the receptor (i.e. agonists) from those which do not activate it (i.e. antagonists).
Du.. ~gllLI~iull of receptor numbers is known to be induced by certain agonists and Ub~ iUII:~ in whole animals have suggested that this pl~ ulll~llv~l may also occur with specific antagonists. However, the antagonist-related process has not been generated in cultured cell systems with Itl,UIllll' ' receptors. Hence, there is a need for the d~v~lv~/ of an assay or screening system which permits the testing of ` ~ 216~03g compounds for their ability to act as antagonists and to stimulate receptor downregulation .
Testing for du .. ,u~,ul~ has previously been successful only in whole animal models. In such systems chronic treatment with agonists or antagonists is required over a period of days or weeks before measurable effects are observed. Not only is this extended time period i~u~ iblc with the practical requirements of screeningprocedures, but using animal models generates variability in the results obtained, creating a need for even greater effects before the results are statistically significant.
In addition to sample variation, animal models necessitate labour-intensive and expensive protocols, besides raising questions of ~ u-L.~ib;lity and being challenged by the ethical motivation to search for alternatives.
Currently there is no method of screening r~-~rolln~ in cultured cells or membrane liol., for antagonist stimulated downregulation, and the whole animal or tissue-based methods require time periods far in excess of those needed by the present invention. Accordingly, it is an object to provide a method for screening candidate drugs i71 vivo for antagonist activity within a time frame that is dramatically accelerated compared to other known methods of testing the same effect, thereby providing novel and improved efficient means of screening compounds for their probable pll,.,llld~u~ d efficacy.
The bdch~luu.ld illru is provided for the purpose of making known il.f~ iu.
believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the bc~h~
irlr~ constitutes prior art against the present invention. Moreover, ~ ~I
referred to throughout the application are hereby ill~.Ul~UI ' ' by reference in their entireties in this application. This present invention also relates to the use of the method and reagents in the form of a kit.

` 2160038 SUMMARY OF TE~E INVENTION
These objects are a.,uu.,.~ h~,d by the use of novel methods for screening compounds in vitro for their ability to interact with and modulate the functional properties of receptors coupled to guanyl nucleotide-binding regulatory proteins (G proteins). The present invention, then, provides a novel method for testing and ranking ~;u-- r-for their abilities act as antagonists and to induce receptor downregulation, yielding significant advantages of time and scale over traditional techniques which are unsuitable for general screening procedures. In particular, a method is provided for testing and ranking rn-Tlro~ln~ for their abilities to du.. , ~, ' G protein coupled lû receptors when these receptors are chosen and expressed as ~-,u---l,i-.. ~ gene products in a particular cultured cell-line. The methods require that the receptors be expressed at ~UII~ iUi~ that are adequate to provide ample signal detection to rank ~,011 r- ' on a statistically significant basis, but are i~ ' ' to induce cell toxicity which causes variable results (U.S. Patent Application # 08 / 336,248). Moreover, the method invented generates conditions under which a cultured cell system expressing such l~col-lbii~ll~ receptors will respond to treatment with receptorantagonists with irreversible downregulation of the receptor to generate an in vitro assay to screen antagonists for ability to downregulate membrane-bound G-proteinlinked receptors which is quicker, less-expensive, more sensitive and reliable than the whole animal systems that are currently necessary for this purpose.
TARl.li~ ANI) nGllRES =~
Table 1 shows some antagonist binding affinities measured in Ill~::lllb~ from Sf9 cells expressing 5-HT~C receptors and IP responses, measured in whole cells.

` ~ ~160~8 Table 2 shows the binding affinity of [3H~ hl~ after apparent loss of 5-HT2c receptor sites expressed in Sf9 cells lu.~ with drugs.

Table 3 presents results ~...~ L-dli.lg apparent loss of binding sites induced by antagonists with 5-HT2C receptors expressed in Sf9 cells.

Figu~re l depicts the results of ~ the functional coupling of the rat 5-HT2C receptor to ( l~ pl~ C in Sf9 cells.

Figu~re 2 shows effects of sc.ui ~i~ agonists of IP release in 5-HT2c receptor-expressing Sf9 cells.

Figure 3 ~IPm( the apparent loss of [3H ~il¢ binding after antagonist 0 pre-treatment in whle cells and l.. ~,.. lbldl~CS.

Figu~re 4 depicts effects of SC:IU~U(I~ ;U antagonists on the apparent loss of t3H]n~Pc(llPr~;inp binding sites in 5-HT2c receptor-containing Sf9 ~ after antagonist u- ~

Figure 5 shows time 11r~ of [3H]I.l~ il.e binding in I ~ from Sf9 cells expressing the 5-HT2C receptor.

` ~ 216Q038 DETAILED DESCRIPTION OF TIIE INVENTION

The following common ablJ~ iaLiull~ are used throughout the ~ir ând in the ClâimS:
"IP" refers to inositol phosphâte.
"5-HT" refers to 5~ dlv}~yLly~L~
"D.O.I." refers to 2,5-dimethoxy~- ' . ' llyvlvb "PBS" refers to phosphâte buffered sâline.

The term "downregulâtion" means the process whereby the number of receptors located at the cell surface, both able and available to ind ligands is decreased in a controlled manner.

The term "reagent" means a substance used to bring about a chemical reaction in another substdnce. For example, ~ lllb~ i G protein coupled receptor molecules ' in whole cells or membrane Ln~ Id~iOII~, to permit the testing of ligands for their ability to participate in receptor binding reactions with or without any associated physiological effects.

In one rmhf)~limPI~ the present invention provides a method for screening the activities of antagonists to dUWIII~ ~ ' biologicdl receptors.

Another ~.I.v~ of the present invention involves its use in a test kit, said kit comprising solutiorls and devices for carrying out an assay for the ordering of ` ~ 21600~8 antagonist to d~wlul " ' ' G protein-linked receptors, for example, the 5HT~C
receptor.

In a more preferred ~ ",l,~,li".. f the present invention provides for a method for screening the activities of antagonists to downregulate G protein-eoupled receptors.
5 In yet a more perferred ~,.. lb~dilll~llL, the present invention involves a method for testing chemical WllllJUUlldlS for their abilities to du.. , O ' G protein-eoupled reeeptors involving: (a) expressing DNA eneoding a G protein-eoupled reeeptor in a eell expression system in sueh a manner as to generate a I~J~UIIU~ le reagent that allows for lli~,lilllill~lliU.. of chemical compounds based on relative ability to du~ , ' said G protein-eoupled reeeptor; (b) measuring a quantifiable parameter using bioehemical or other assay procedures that indieate the du.. ~UC;O~ ' of said reeeptor in said system eomprising whole cells or membrane fragments containing G
protein, an d~)lJlUI)l' ' effeetor, and cloned G-protein linked receptor;
(c) contacting a test-compound with the system under conditions permitting interaction of the test-compound with said reeeptor; and (d) measuring the change, if any, of the 4Udll~irldl,lc parameter which reflects the ability of the test compound to du the G protein-coupled receptor.

In the most preferred ~l"l.c..li,.,r,ll, the present invention provides a method for ordering the ability of antagonists to downregulate the 5-HT2C receptor expressed in a l 'c~vi-u,/Sfg system.

The present invention provides novel methods for testing and ranking eomrmln~i~ for their abilitics act as antagonists and to induce reeeptor du.. , " ' ' , yielding significant advantages of time and scale over traditional techniques which are unsuitable for general screening procedures. In particular, a method is provided for testing and ranking: A ~ for their abilities to dUWI~ ' ' G protein-coupled receptors when these receptors are chosen and expressed as I~COIIIbi~ gene produets in a particular cultured cell-line. The methods require that the receptors be expressed at rr,nr~rlr~linn~ that are adequate to provide ample signal detection to rank connrolln~i~ on a statistically significant basis, but are ~ ' to induce cell toxicity which causes variable results (U.S. Patent Application # 08 / 336,248). Moreover, the method invented generates conditions under which a cultured cell system expressing such l~cu.,.l, receptors will respond to treatment with receptor antagonists with il.~ il,lc duw~ll~ ukl~io.~ of the receptor to generate an in vilro assay to screen antagonists for ability to du.. I~ ' membrane-bound G-protein linked receptors which is quicker, less-expensive, more sensitive and reliable than the whole animal systems that are currently necessary for this purpose.

The system can be presented in a ~ lly packaged form as a . , - .: or a mixture wherein compatibility of the reagents will allow for a test ~ iUII (most typically as a test kit) a packaged cu..lbill~.~ioll of one or more containers, devices, or the like, holding the necessary reagents and usually including written illi~tll ' describing the p~.rul.ll.~ of the assays. Reagent systems of the present invention involve all possible configurations and: . . for p~l[ullllill~ the various assay : 2160~38 .
formats described herein. A test kit form of the reagent system may further include ancillary chemicals.
The present invention hæ been developed from the d~ oll~lldliu~ that treatment of intact Sf9 cells or ".. l",.i~. c containing the baculovirus-expressed 5HT~C receptor with ~ ~ v , followed by removal of the drug by extensive wæhing, resulted in a loss of binding capacity for both the antagonist [3H]mesulergine and the agonist [3H]SHT. Several ubo~ liu~lD made during the development of the invention strongly suggest that this action of antagonists in Sf9 cells is related to the ability of these drugs to decreæe the level of 5HT~C receptors in various brain regions following chronic dl]lllill;o;ld~iOII in vivo (Pranzatelli, M.R. et al., 1992, Eur. J. Phar~nacol. 244:
1-5; Smith, R.L., et al., 1990, J. Pharmacol. Exp. 17ler., 254: 484-488; Sanders-Bush, E. & M. Breeding, 1988, J. Pharmacol. Exp. Ther., 247: 169-173). A striking correlation exists between the relative abilities of antagonists to promote such 'atypical downregulation' of SHT2C receptors in vivo, and the potencies of the same antagonists in decreasing [3H]mesulergine binding to the baculovirus-expressed SHT~C receptor.
The ligands exhibiting the highest potencies in reducing sites in Sf9 cell membranes are the same ones that produce the greatest decrease in 5HT~C receptor levels following in vivo treatments: metergoline, ritanserin and mesulergine (Pranzatelli, M.R., et al., 1992, Eur. J. Pharmacol., 244: 1-5). Similarly, antagonists that produced a lesser but demonstrable effect on receptor levels in vivo, such æ mianserin, ketæerin and clozapine, showed intermediate potencies on the baculovirus-expressed receptor, while the antagonist spiperone yields no effect in either system (K~ rpsl~a~-i, M.T., et al., 1992, J. r~ Y~7. Ther. 264: 1262-1267; Pranzatelli, M.R., et al., 1992, E~r.
J. Phar~nacol., 244: 1-5).
Antagonist trcatment of the baculovirus / Sf9 inseet cells or m~mhr~nP~ containing a I~Culllbil~ m~~m~ n receptor, followed by washout of the residual drug, results in a decrcased number of binding sites for the agonist or agonist analogs without any ehange in affinity for these reagents. The decrease in binding is irreversible, is not due to the presence of residual antagonist, and is not observed following treatment with agonists in lieu of antagonist. The effect is dose dependent, with a rank order of potency distinct from that for inverse agonist activity (U.S. Patent Application # 08 /
0 336,248), indicating that the two effects reflect distinct actions of antagonists on the receptors. The relative abilities of antagonists to produce loss of binding shows a good correlation with their reported abilities to 'du.. , O ' ' ' 5HT2C receptors in vivo following chronic trcatment, suggesting that these actions reflect the same underlying process.
Thus while this downregulation of G protein-coupled receptors simulates the ehronie effects of the antagonists in treatment of isolated tissues, whole animals and human subjects, it is not observed in culturcs of m:lmm~ n cells, which would be expected to resemble the li~n organism more closely. Not only do the methods described provide an u~ulLulli~y to screen compounds for effects which imitate their pharn~ lo~ir~l efficaey in vivo, but the observation of these effccts is dramatically ~ 2160038 accelerated by using the methods described, further enhancing their ~rr~ ., for screening programs.
Other common features of the antagonist effect on 5HT2C receptor levels in vivo and in Sf9 cells are the dose~ PI-d~ and irreversibility of the action. Clearly seen in the Sf9 cell studies forming the basis of this invention, dose ~IrL~ has been reported for the effect of clozapine on 5HT2C receptor numbers in choroid plexus following chronic treatment of rats (Kllopra~ i, M.T., et Ql., 1992, J. Phr ~ F~r. Ther.
264: 1262-1267). Recovery of sites following cessation of treatment in both systems requires synthesis of new receptors, as shown with intact Sf9 cells and in membrane LJI~aliul-;~ made from them, by the lack of recovery of lost binding sites for at least 24 hours after removal of the antagonist, a finding consistent with ill~,V.,l~ibl~, IOSS of receptor binding capacity.
The antagonists shown to to promote loss of binding for the baculovirus-expressed SHT~C receptor in the studies from which this invention os derived include drugs in current ~ uli~ use such as the ~ s~ u~i~ clozapine and the ;t-l~iJ~ .;~
mianserine. The methods described in this invention are appropriate for d~
whether other G protein-coupled receptors, such as the 5HT2A receptor, are able to show similar antagonist profiles when expressed in insect cells.
The present invention will now be illustrated, but is not intended to be limited, by the 2 o following examples.

` ~ 21600~8 E2~AMlPLES
Example 1 P ~.,.,. ' of Dru~ Screenin~ Rea~ent A. Rea~ents Buffer chemicals and protease inhibitors were purchased from Sigma, and cell culture media from Gibco/BRL. Unlabeled ligands were supplied by Research Bi l. h~
Illt~ iu~l, with the exception of RU24969, which was a gift from Roussel Uclaf.
[3H]l, ' ~,il.~, (78-82 Ci/mM) was purchased from Amersham and [3H]myo-inositol (10-20 Ci/mM) was purchased from NEN-Dupont. The ion exchange resin AG 1 X-8 0 was supplied by Bio-Rad.
The l~u..~ baculovirus used for expression of the rat SHT2C receptor was provided by the B~ lo~y Research Institute of Montreal. The virus was constructed using a synthetic DNA fragment encoding the rat 5HT~C receptor, based on the published sequence of the cloned cDNA from choroid plexus (Julius, D., et. al., Science (~V ' v D.C.) 241:558-564, 1988). The synthetic cDNA was prepared and its sequence verified by Allelix Biu~ le The cDNA was inserted into the IpDC-126 baculovirus transfer vector and a IC~l.,U--lb . Ul~ b~ulvviuu~ was produced and purifled as previously described (O'Reilly, D.R., et. al., Baculo~irus Ecpression Vectors: A Laboratory Manual. W.H. Freeman and Company, New York, 1992).

` ~ 2160038 B. Cell culture and receptor expression.
Sf9 cells were cultured in 50 ml batches in 250 ml shaker flasks at 27C in Sf-900 II
serum free medium containing 50 g/ml of gentamicin sulfate. Cells were grown to a density of 3X106 cells/ml and infected with the 5HT~C l~ulllb;.lalll baculovirus, or with wild type AcNPV baculovirus, at a multiplicity of infection (m.o.i.) of 2. Viral stocks for infections were grown in Grace's insect medium containing 5% fetal bovine serum (Hyclone), and were added to cultures at a dilution of d~ 'y 1:20 upon infection. The infected cells were maintained in culture for various periods and used for analysis of ligand binding and Il~ lL of IP production as described below.

C. T~ ' bindin~ assays.
For the estimation of total receptor numbers in intact Sf9 cells, the cells were pelleted by low-speed ~...firu~;a~ioll (3 min at 800 RPM in Sorvall H6000A rotor), followed by 1~ , in PBS and l~ iru~a~iol~, and ll , ' ' in either PBS or binding buffer (50 mM Tris/HCI, pH 7.4, 15 mM MgCI~, 2 mM EDTA, 0.1% ascorbic acid, 5 ng/ml leupeptine, 10 mg/ml aprotinin, 20 rng/ml 1:1. ' , 50 mg/ml TPCK and 50 rng/ml trypsin inhibitor). Cell viability after washing was estimated at 60-70% by trypan blue exclusion. Aliquots of 10,000 cells were incubated for I hr at 27C in a final volume of 540 nl containing 20 nM of [3H]mesulergine. T ' " were terminated by vacuum filtration over GF/C filters and washing with binding buffer at 4C. Bound radioactivity was measured on filters i,.. ~ ' with MeltiLexTY melt-on scintillant using a Wallac MicroBeta counter. Non-specific binding was estimated in parallel ' containing 10 mM mianserin or metergoline.

" ~ 21600~8 For analysis of ligand binding to membrane ~ aldLiOII~, cultures were harvested at 48 hr po~t :..re~io.., Iysed and a membrane pellet was prepared as previously described (Labrecque, J.M., et al., FEBS Lett. 304:157-162, 1992) and stored at -80C. Protein were determined by the nitrocellulose amido black method (Schaffner, W. and C. Weissmann, Anal. Biochem. 56:502-51, 1973). ~ lblalæs were thawed on ice and ~ in binding buffer by 11m.,~". ..i,~li..ll in a Potter ' The ..~ (5 mg) were incubated with 13Hl~ ul~ for 1 hr at 27C in a final volume of 540 rnl, and the assays terminated as described above for intact cells.
Saturation binding assays yielded a Kd for [3H' ' O of 2 nM, and l .J~ .r~
binding assays with unlabeled drugs were carried out with 3 nM [3H` - ' Oi--e (Table 1).

D. Analysis of binding kinetics.
Time course association ~ with [3H' ' Oi..e were carried out by measuring [3H] ' Oi.le bound at different times following addition of radioligand as described above (see also figure legends). Dissociation r~rli,.~ were performed by incubating ' for 60 min at 27C with [3H]III~UI~IO;IR at a C~ Pntr~ti of 20 nM or 500 nM. The labeled mPmhr~nP~ were then centrifuged (2 min at 12000 rpm in a microfuge) and .. ~ .r~ Pd in a 1000 fold e~cess of either binding buffer alone or buffer containing unlabeled drugs. The diluted mPmhr:~nPc were then incubated at 27C and aliquots were collected in triplicate with a 30cc syringe and vacuum filtered over 24 mm GF/C filters using a millipore 1225 sampling manifold.
Bound iadiuàl,Livily was measured on the fllters by srinti~ti~n counting in 1 ml of ` ` ` 2160038 .` ~ , ` . . ~ . . .
., ~ . . . .

3 ;: - m ~, 3 a r _ _, --, o ~ 0 O ~ .D N ~ _ _, O O O O O O O O O _ ' o _ r~a o o o --o -- ~
~ O o r~ o o ~ o ~ _ _, -- _ ~ Vl C.~l Q 0 '~
- <a 0 o ~ ~n a ~ _ ! . ~ ~
It It It It It It 1~ It It ~ . ~ ~ ~
oocoooooo O .- . f 0 r~ ~ ~, 0 0 0 , , ,, _ o o o o o It It It It It It ~t It ~ a 0000000 0 : ~, Vl, .
O ~J
O' ~ O 0 0 ~. ~ C.~--0 ~ 0 00 0 O0 ~ ~--~ ~
' t ' ' It I t It 1~ It It It -- _ o ~ O 0 0 0 ~ C~
,, ,i , ` ~ 2160~38 Hi-load LKB srinfill cocktail. Control ' were processed in parallel toassess stability.
E. Inrcifr,l phl ' vr~ rti~m Growing cell cultures (lx106 cells/ml) were prelabeled for 24 hr with I mCi/ml [3H]myo-inositol prior to infection, and the labeled cells were then transferred to 50 ml shaker flasks for infection. Assays (n==2 or 3 as indicated) were started by addition of labeled cells (2.5x105 to lOx103 cells/well) to a 96-well deepwell plate (Beckman) containing the drugs to be tested, followed by immediate mixing. The cells were then incubated for 20 min at 27C in a shaking incubator, and the ill~,Ub~lLiOII~ stopped by 0 addition of perchloric acid. Total [3H]-labeled IP (total inositol polyphosphate) were measured by crinfill~linn counting following isolation by anion exchange ;IlI`UllldLU~ / on AG I X-8 resin, as previously described (Fargin, A., et. al., J.
Biol. Cne7n. 264:14848-14852, 1989).
F. ExprPccinn of filnrfi-ln~l 5~T~ rece~tor in Sf9 i rPlls A bd~uluvi~.D encoding the rat SHT2C receptor was used to express the , ~ L
receptor in cultures of Sf9 insect cells. Receptor levels, as measured in whole cells by the binding of [3H].,I~,,ul~ i.¢ (10 nM), increased with time after infection to reach d~ y I million sites per cell at 72 hr post-infection (Figure 1~. The ability ofthe expressed 5HT2C receptor to modulate r,~ phospholipase C activity in intact Sf9 cells was assessed by measuring the production of total IP ûver 20 min as described above. The levels of inlr~rPlll~lslr IP at different times post-infection and under various pharm~rûlrl~ir~l treatments are presented in Figure 1. The basal level of " ~ 2160038 IP production increased over the time course of the infection, and at 38 hr post-infection were roughly double that at 20 hr. Basal IP production dramatically decreased at 72 hr, reaching levels ~:U~ to those observed for cells infected with wild-type baculovirus. This loss of activity at 72 hr, the point of highest receptor expression, paralleled a drop in cell viability (to under 10%), consistent with a generalized loss of cell function at late stages of the viral infection.
As seen in Figure 1, the natural agonist 5HT was able to stimulate IP production in whole cells expressing the SHT2C receptor; the response to 5HT, however, varied over the course of the infection. At 20 hr and 38 hr post-infection, SHT stimulated IP
0 production by 30% and 13%, ~ ivtly, over basal activity measured at the same time point. The response to 5HT was completely inhibited by the antagonist mianserin (10 mM). The agonist had no effect on basal IP release at 12 hr post-infection, when there was little or no receptor expression (Figure 1), nor in cells infected with wild-type virus. The response to 5HT was lost at 72 hr, in parallel with the drop in basal activity.
The ~ r of the stimulation of IP production by the ~
agonists 5HT, D.O.I. and RU24959 was studied in whole cells at 20 hr post-infection as seen in Figure 2. The rank order of potency for the agonists was 5HT = D.O.I. >
RU24959 (see legend Figure 2). While D.O.I. and 5HT stimulated IP production to equal extents (~-, 'y 30% stimlllo~ n), RU24959 acted as a partial agonist of the response (12% ~timllloti-~n) Treatment of the cells with pertussis toxin (up to 100 ` ~ 21600~8 ng/ml) for 24 hour preceding ~ ,a~U~ of IP production did not âlter the y effect of SHT.
The antagonist mianserin, in addition to blocking the ' y effect of 5HT, also produced ân inhibition of basal IP production in the absence of added âgonist. The extent of this inhibition varied, from 20% ât 20 hr post-infection to roughly 40%
inhibition at 38 hr. Mianserin hâd no effect on basal levels of IP production at 12 hr or 72 hr post-infection. These results show that the baculovirus-expressed SHT2creceptor exhibits ~ , agonist~ d-r ' activity which can be inhibited by the antagonist mianserin. Mianserin acted as an inverse agonist at the SHT2C receptor, 0 in agreement with a previous report (Barker, E.L., et. al., J. Biol. Chem. 269:11887-11890, 1994).
G. Analysis of Data The binding of [3H]Il.~.ul.,.~i,lc (satuMtiOn ~ ) and the inhibition of [3H, ' ~i"`' binding by unlabeled ~;IU~ iC ligands were analyzed in terms of a single class of binding sites using the computer program LIGAND (Munson, P.J., and D. Rodbard, Anal. Biochem., 107:220-239, 1980). Data from three i-,~
binding ~ I i were fitted individually and the affinities presented for [3H]~ ul~ c (Kd) and other ligands (Ki) represent as the average values (~t SEM)from 3 sets of data. Dose-response data for IP and: ~ induced decreases in [3H]mesulergine binding capacity were scaled taking values measured in the absence of added ligand as 100%. The scaled data were analyzed according to a 4 parameter logistic equation analogous to the Hill equation (ALLFIT; A. DeLean, Department of `` ~ 2160Q38 Pharmacology, Université de Monkéal or INPLOT; GraphPad Software, SanDiego CA). For the decrease in [3H]mesulergine binding with each ligand, 3-4 sets of data were fitted " ~~ '~ using ALL~IT with the slope factor set equal to 1. Further details are described in the legends ~. . o~ .yi-,g each of the Tables and figures.
Maximal inverse agonist activities measured fo~ each drug tested were compared statistically by a two tailed t test (a = 0.05).
Example 2 Use of R- ' to Screen D~ ` ' of Rat 5HT2C Rece~tor E.~1,1.i.,.~,1Lb were carried out by treating intact Sf9 cells expressing the 5HT,C receptor 0 (at 38 hr post-infection) for 12 hr with high ~;(, . (100 mM) of various drugs added to the culture medium. The drugs were then washed out as described above and the effect of pre-treatment on receptor levels was determined by measuring the binding of [3H]-l.~ul.,~ (I0 nM) in the standard filtration assay. As shown in Pigure 3 (shaded bars), antagonist treatment resulted in decreases in [3H] ' ~,i-.e binding, with the extent of the decrease varying from over 90% for lll~;L~l~uli.. ~, and ritanserin to little or no decrease for spiperone. The agonists SHT and D.O.I produced no significant change in the level of binding. When these ~ ,.lL~ were carried out using membrane preparations (Figure 3, solid bars), essentially the same profile of drug effects on the level of rL3H~ binding was observed as in whole cells.
For certain ~IlLa~:,v ', particularly mesulergine and clo~apine, the magnitude of the ~` 2160038 decrease in binding sites was somewhat greater in membranes than in whole cells, but the rank order for loss of sites was the same in cells and in l.._~llb~dl~

The loss of [3H]mesulergine binding sites induced by antagonists was ,l~ud~ ,d in greater detail in membrane ~lc;,udldLiu...,. As shown in Figure 4, pre-treatment with varying .u~ . of the different ~r~r^,~ ' ', followed by extensive washing to remove bound antagonist, produced dose-dependent decreases in [3H]~ ulu~
binding. The potencies of the antagonists in reducing the binding of [3H, ' y,il.~
varied over several orders of magnitude, with l..~t~ Uli.._ being the most potent and spiperone having no effect at ~ n~ ~ .,I.,.~in"~ up to I mM. Treatment of l~ lbldll~
with 10 nM metergoline resulted in equivalent decreases (approximately 90%) in the binding of [3H]mesulergine (lO nM) and [3H]-5HT (350 nM)" indicating that the binding of antagonists and agonists were equally effected by antagonist pre-treatment.

To illustrate that the loss of sites was not due to residual antagonist remaining bound to the receptor after the wash, ' were treated with either mianserin (1 mM), ritanserin (10 nM) or l--~;Lt;l~,uli--e (10 nM), washed, and saturation binding was performed with [3H]I..~sul~.~ i.le. As seen in Table 2, the antagonists produced marked decreases in the Bmax values, but did not alter the apparent affinity of [3H]mesulergine. Although ritanserin treatment seemed to slightly increase the Kd, the analysis of variance indicated that this change was not statistically significant (a =
0.001, p = 0.012). This result shows that the reduction in the number of [3H' '~ ~illt binding sites for mianserin, ~ L~l~ oli--~ or ritanserin is not due to residual antagonist reversibly bound to the receptor .

` ~` 2160038 7Ai~LE 2 Bllldil)g a~ ity o~ mesutorr~iine ~rter appareltt loss o~ 5-HT~C
receptor slto9 expro3sod In S~9 cell~.
Mesulo~glno binding a~ y (Kd) alld tocop~or dr!nsl~y (Bm, ~ wore moosured In membrsnos (~rom cells 48 h~ a~er Inlectlon) tl-at had been ~.~'r~ lb~led lor 60 mll1 wi~l~ drugs arld t~1en extenslvoly was~1ed be~oro tho saturallon blndll1g al1al-ysis as described in Malerlals arld Me~llods i iesul~s rrom anlagontst Irealmenl o~
mer~-brarles wero delorrtlllled (lom Illlee i..J.~,~,.J~,,lt experln~enls witll n~enll values and slandald errors calcllla~od llom separate esllrnateg lilled will~ o program LlCAi ~D.
Mesulergins blndln9 palamele~s Trealmellls K,' Bm-l~
nu rtrl1r~'mo ',~ or corlt~ol Colltrol 21 ~ 1 2 22.0 ' 2.0 100 ' 9 Miansel~ M) 3 8 + 2 2 14 5 ' 2.0 66 ' 9 MJte~g~ ,a (îO nM) 2.0 ' 0 6 2 9 ' 1.1 12 ~ 6 Ritanserln (10 nM) 7.~ ' 3.0 5.5 ~ 3.0 25 _ 12 ' Analysls ol vLIlonco wg.~ porlormed on Kd ~sllmcto~ ~or eont~ol ~nd dr~lg-trealcd n~on~brrlne~ (n 0 001, p e 0.012) '~ A/-alysls o~ VAr~ar~C0 wns porlr7rr~od orl r~' n., ~t5~1mnto~ wltll thn rrlnclr~r~ n~
l~yprttl~e5is (n - o ool p ~ O oOl) Ol~o lniled Dllrlr~r~ lrst P~O I~on"o~
n = 0.05) was used lo comparo d-ug l-onlrnorl- Bn~ valuos wl~h eon~rol ` ~` 21~0~
The association-dissociation of [3H]mesulergine, an antagonist which itself causes a decrease in binding sites was analyzed, to illustrate tllat the system was not an artifact due to residual antagonist (see Figure 4). The time course of association of 20 nM
[3H]"l~.~k,.~i"e, measured in, ..,l"",.. ~ by filtration at different time points after r~liolig~n~l addition, exhibited first-order kinetics to reach a steady state within 60 min at 27~C (Figure S top curve). In parallel ~ bl were incubated for 60 min with [3H]mesulergine at 500 nM, a . ' sufficient to cause a >50%
reduction in binding sites (c.f. Fig 4). In the latter ~ of the bound [3H]mesulergine was then initiated by ~ r~ g the ' ~ and 0 l~ i. g them in a large volume of buffer (Ix105-fold the original incubation volume), and residual bound ligand was measured at various times using a filtration assay (see Legend to Figure 5). As shown in Figure 5, ~" ' ~y 95% of the bound [3H]mesulergine had dissociated from the membranes following a 2 hr incubation at 27C (tl~l = 40 min). When these membranes were then tested for their ability to bind 10 nM [3H]III~ IIC;IL , the total binding was reduced by ~" I~, 60% compared to control Ill~;lllb~ ,o (i.e. no mesulergine pre-treatment) processed in parallel, consistent with the effect of 500 nM unlabeled IIU~ (ca Figure 4).
These results clearly IICII1011~ll ' that the ability of ~ ule.L (and ~Jlc~ ll~ly the other antagonists) to reduce the number of binding sites is not due to residual ligand occupying the receptor, since the lost binding sites were not recovered even after o~ ;llll of over 95% of the bound lll~ul~

To illustrate that the loss of sites produced by antagonist treatment was a reversible process, intact cells or ll-~ ulc~ were treated with metergoline (10 nM), washed ` ~ 216003~
according to the standard protocol and incubated for up to 24 hr in the absence of added drug. Analysis of [3H]mesulergine binding following such treatments showed no recovery of binding sites, while control samples (i.e. no ~ tu~ pre-treatment) processed in parallel exhibited only a slight decrease in [3H]I.._;~Uh;lL, binding. The loss of sites for the baculovirus-expressed SHT~C receptor following antagonist treatment thus appears to be irreversible under the conditions examined here.
Finally, the dose-response curves for loss of binding sites after agonist ~
(Figure 4) were analyzed (Table 2) to generate EC~o (i.e., ~ l . producing a 50% decrease in binding) values for the series of: ~ The results presented in 0 Table 3 provided the following rank order of potency for loss of sites~ ~ Oulille >
ritanserin >
y jl,r > clozapine > mianserin > ,~ d~ le > ketanserin > >
.Oide. Although the antagonists showed a wide range of potencies in reducing the level of binding sites, the results in Figure 4 suggest that the maximal reduction was the same (d~ Iy 90% loss of sites) for all of the O , although the effects of ~J .I.)Ad~l ill~ and methylsergide appeared to be incomplete at the highest .,,,.~r.,~l,l;.,.,~ used.

` ~ 2180~38 Tti8LE 3 Appare~1t loss o~ bl~ldlllg sltcs Induced by ill1tngol1ist~ wlll~
5-~T~c receptor~ expressed 1l1 S~9 cclls llr sidual blndir~g silos were o~ r~alr!d in ~llerl\b~ r~s (l~o~ 8 ~1~ irlrr~clr~d cclls) Ih~l had boQn prelrea~ed ~o- 60 min wi~h drugs al dillo~ ll c~ ,s arld Illcn e)l~ensively was~lod belo~ o birldi-lg all21ysis DS ~Ic5c~1bcri ill M~lr-~ials and Methods. Nrsnspocillc binding was de~c-~-linrd wi~h 10 l~r~ l~lio~lsc~in /~ D9 onisl polencies ~EC~o) (~llcan ~: slandard er~o~1 lor i~lducillg Dn a~ e~l loss ol siles were lakon Irom rlg 7.
App~l~nl loss ol ~ n~esulc~oil~ billriillo siles Anlagonisls -PECso il~ SCICCIIYjlY lailo~
M~ .s~" ,a 1û.25 + 0.12 79 + 7 û.036 Mell~yse~gldo >3 ~1 x 10"
ilitanselin ô.q2 ~ 0.16 49 ~ 15 0.32 Mianselln 5.60 + 0.15 0 72.6 Mesulergine 7.9O + 0.09 38 + 7 0.23 Kelal1serln 4.q5 + 0.07 2 + 5 1.76 Clo~apino 6.05 + 0.16 10 + 7 5.1 Spiperono Nl)' 0 >1 >~ 10~
Spiroxatril10 4.64 + 0.03 35 + 5 <1 x 10 5 ' t~e ioss ol l'l~]mesuiorglne blnding witl~ a~lngo~isl occu;lDr~cy a~ lû :s K~
Y~as t~ lorn ~i9. 7.
~ Seleclivlly lalio = (EC~o ~ol loss or blndirlgl~Cs, ~or invcrso agor~isrll) (T2blo 1). Values ol cl seleclive lor loss ol binding: valucs ol ~l sr~lcclivo lor inver~e a9o,~is,n, ' ~D nol delermlned.

Claims (3)

1. A method for testing chemical compounds for their abilities to downregulate G
protein-coupled receptors involving:

(a) expressing DNA encoding a G protein-coupled receptor in a cell expression system in such a manner as to generate a reproducible reagent that allows for discrimination of chemical compounds based on relative ability to downregulate said G protein-coupled receptor;

(b) measuring a quantifiable parameter using biochemical or other assay procedures that indicate the downregulation of said receptor in said system comprising whole cells or membrane fragments containing G protein, an appropriate effector, and cloned G-protein linked receptor;

(c) contacting a test-compound with the system under conditions permitting interaction of the test-compound with said receptor; and (d) measuring the change, if any, of the quantifiable parameter which reflects the ability of the test compound to downregulate the G protein-coupled receptor.

2. A method as claimed in Claim 1 wherein said receptors is 5HT2C

3. A test kit supplying the reagents required to preform the method as claimed in Claim 1.