WO2004035614A1 - Synthetic or partially purified peptides which can bind to specific subunits of g proteins and uses thereof - Google Patents

Abstract

Various Gα conformation specific peptides are disclosed. These may be used in assays to detect activation or deactivation of a G protein-coupled receptor.

Description

SYNTHETIC OR PARTIALLY PURIFIED PEPTIDES WHICH CAN BIND TO SPECIFIC SUBUNITS OF G PROTEINS AND USES

THEREOF

This application is a nonprovisional of U.S. Prov. Appl. 60/419,143, filed Oct. 18, 2002, and hereby incorporated by reference in its entirety. It is also a continuation in part of PCT/US01/21867, filed July 11, 2001, which is a continuation in part of USSN 09/860,688 filed May 21, 2001, which is a continuation in part of USSN 09/614,865 filed July 12, 2000, all hereby incorporated by reference.

Cross-Reference to Rela ted Applica tions

In Paige, et al., Serial No. 60/082,756 filed April 23, 1998; Serial No. 60/099,656, filed September 9, 1998; PCT/US99/06664, filed March 23, 1999, and Serial No. 09/429,431, filed October 28, 1999, a method of predicting the ability of compounds to modulate the biological activity of receptors is described. The ability of a query compound to modulate the biological activity of a receptor in a multicellular organism is predicted on the basis of its interaction with that receptor in the presence of various members of a panel of BioKeys. The BioKeys are ligands, especially peptides or nucleic acids, known to bind to a particular conformation of the receptor. This interaction data, known as a "fingerprint", is compared to the fingerprints for reference compounds with known biological activities mediated by that receptor. In the "molecular braille" (MB) embodiment of that present invention, the reference and test fingerprints are based on in vitro (cell-free) assays.

In the "cellular-braille" (CB) embodiment of that present invention, the reference and test fingerprints are based on cellular assays (but not on assays of whole multicellular organisms, or their organs or tissues) .

Example 201 of PCT/US99/06664 discussed, in a general way, peptides which bind G protein-coupled receptors (GPCRs) or their associated G-proteins, and noted that a peptide might be specific for the alpha subunit of a protein in its activated (GTP bound) or inactivated (GDP bound) state. No seguences of anti-G protein peptides were disclosed. In Paige, et al., Serial No. 09/429,431, various fluorescence assays for GPCR activity were described in which peptides specific for Gα.GDP (D-peptides) , G :GTP (T- peptides) , or specific for Gα, but binding independently of active (GTP) or inactive (GDP) conformation) (I-peptides) , were used as reagents. The sequences of many T-, D- and I- peptides which bound G_±d were disclosed.

The aforementioned Serial No. 09/429,331 was filed as a continuation-in-part of PCT/US99/06664 however, here we are not claiming §120 benefit of the filings prior to 09/429,331, merely incorporating them by reference.

In Paige, et al., Serial No. 09/614,865 filed July 12, 2000; Serial No. 09/860,688 filed May 21, 2001; and PCT/US01/21867, filed July 11, 2001, an improved method of "fingerprinting" compounds was described. Peptides which bind a cellular (surface or intracellular) receptor, such as a nuclear receptor, were identified by screening a combinatorial peptide library presented in the form of cells each of which coexpress one member peptide and the receptor, together with a signal producing system for reporting binding. A "two-hybrid" assay was of particular interest.

The screen could be carried out in the presence of a ligand, in particular, an exogenous ligand. If this screening was carried out for a plurality of different receptor conformations, then this library screening could serve to identify conformation-specific peptides for the receptor, which could then be used in a panel for "fingerprinting" query compounds as to their ability to interact with the receptor in the presence of each of the panel peptides.

These fingerprints could be compared to those of reference compounds with known biological activities mediated by that receptor.

The above applications, which all claim benefit of 09/429,331, are likewise incorporated by reference.

Thorp, Serial No. 08/904,842, METHOD OF IDENTIFYING AND DEVELOPING DRUG LEADS WHICH MODULATE THE ACTIVITY OF A TARGET PROTEIN, discloses several methods of identifying drug leads. In essence a protein of interest, in one or more states, is characterized by (a) its chemical reactivity with one or more characterizing reagents, and/or (b) its binding to one or more aptamers (especially nucleic acids) , generating an array of descriptors by which it may be characterized as more or less similar for reference proteins for which an equivalent array of descriptors have been generated, and for which one or more activity-mediating reference drugs are known. Suitable drug leads for the protein of interest are those analogous to the reference drugs for the more similar reference proteins. Fowlkes, et al. PCT/US97/19638, 08/740,671, 09/050,359 and 09/069,827, IDENTIFICATION OF DRUGS USING COMPLEMENTARY COMBINATORIAL LIBRARIES, disclose the use of a first combinatorial library, e.g., of peptides, to obtain a set of binding peptides that can serve as a surrogate for the natural ligand of a target protein. A small organic compound library (preferably combinatorial in nature) is then screened for compounds which inhibit the binding of the surrogates to the target protein. All of the above applications are hereby incorporate_d ference.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to peptides which bind the alpha subunit of G proteins, and their use in, e.g., monitoring activation or deactivation of G protein-coupled receptors. It also relates to certain chimeric G alpha subunits.

Description of the Background Art Receptor-Mediated Pharmacological Activity Many pharmacologically active substances elicit a specific physiological response by interacting with an element, known as a receptor, of the target cell. A receptor is a component, usually macromolecular, of an organism with which a chemical agent interacts in some specific fusion to cause an action which leads to an observable biological effect. For purposes of the present invention, antibodies are not considered receptors.

Hormones, growth factors, neurotransmitters and many other biomolecules normally act through interaction with specific cellular receptors. Drugs may activate or block particular receptors to achieve a desired pharmaceutical effect. Cell surface receptors mediate the transduction of an "external" signal (the binding of a ligand to the receptor) into an "internal" signal (the modulation of a pathway in the cytoplasm or nucleus involved in the growth, metabolism or apoptosis of the cell) .

In many cases, transduction is accomplished by the following signaling cascade:

An agonist (the ligand) binds to a specific protein (the receptor) on the cell surface.

As a result of the ligand binding, the receptor undergoes an allosteric change which activates a transducing protein in the cell membrane. The transducing protein activates, within the cell, production of so-called "second messenger molecules." The second messenger molecules activate certain regulatory proteins within the cell that have the potential to "switch on" or "off" specific genes or alter some metabolic process.

This series of events is coupled in a specific fashion for each possible cellular response. The response to a specific ligand may depend upon which receptor a cell expresses. For instance, the response to adrenalin in cells expressing α-adrenergic receptors may be the opposite of the response in cells expressing β-adrenergic receptors.

The above "cascade" is idealized, and variations on this theme occur. For example, a receptor may act as its own transducing protein, or a transducing protein may act directly on an intracellular target without mediation by a "second messenger".

G Protein Coupled Receptors Hundreds, if not thousands, of receptors convey messages through heterotrimeric G proteins.

Most G protein-coupled receptors (GPCRs) are comprised of a single protein chain that is threaded through the plasma membrane seven times. Such receptors are often also referred to as seven-trans embrane or serpentine receptors. The structural conservation of this class of molecules allows to predict peptide sequences to be classified as GPCRs before they are identified in a functional assay. Computer analysis of the human genome has led to predictions of around 1,000 different GPCRs, including many distinct receptors that bind the same ligand.

Based on such "in silico" approaches or through methods relying on hybridization with GPCR-specific nucleic acid probes hundreds of these predicted GPCRs have been described for which the natural ligands are unknown; these receptors are termed "orphan" G protein-coupled receptors. Examples include receptors cloned by Neote et al. Cell 72, 415

(1993); Kouba et al. FEBS Lett. 321, 173 (1993); Birkenbach et al. J. Virol. 67, 2209 (1993); Blasius et al. J. Neurochem 70 (4) :1357 (1998).

Using the so-called reverse pharmacology approach such orphan receptors can be functionally identified

("deorphanized") by screening natural compound libraries. Since many novel drug targets are suspected within this group this aspect is of great interest to the pharmaceutical industry. Examples include an MCH receptor, Chambers et al., Nature 400:261 (1999), a Urotensin II receptor, Ames et al. Nature 401:282(1999) and the latest member of the P2Y receptor family, Communi et al. J. Biol. Chem. 276:41479 (2001) .

Certain GPCRs couple to certain G-proteins and not others, as shown in Table H.

G Proteins

One family of signal transduction cascades found in eukaryotic cells utilizes heterotrimeric "G proteins", since they bind guanine nucleotides. Many different G proteins are known to interact with receptors. G protein signaling systems include three components: the receptor itself, a GTP-binding protein (G protein) , and an intracellular target protein. The cell membrane acts as a switchboard. Messages arriving through different receptors can produce a single effect if the receptors act on the same type of G protein. On the other hand, signals activating a single receptor can produce more than one effect if the receptor acts on different kinds of G proteins, or if the G proteins can act on different effectors.

In their resting state, the G proteins, which consist of alpha {a) , beta (β) and gamma (y) subunits, are complexed with the nucleotide guanosine diphosphate (GDP) and are in contact with receptors. When a hormone or pther first messenger binds to receptor, the receptor changes conformation and this alters its interaction with the G protein. This spurs the o. subunit to release GDP, and the more abundant nucleotide guanosine tri-phosphate (GTP) , replaces it, activating the G protein. The G protein then dissociates to separate the ex subunit from the still complexed beta and gamma subunits. Both the Gα subunit and the GβN complex can interact with an effector. The effector can be an enzyme which in turn converts an inactive precursor molecule into an active "second messenger," which may diffuse through the cytoplasm, triggering a metabolic cascade. After a few seconds, the Gα converts the GTP to GDP, thereby inactivating itself. The inactivated Gα may then reassociate with the Gβγ complex.

The G protein alpha-subunit family is divided by homology and function into several subgroups: G_sα G_qα (G_q, G_n, G₁₄, G₁₆)

G₁₂α and G₁₃α G-j_.0- (G_u, G_i2, G_i3, G_O1, G_o2, G₂)

The G_sα subgroup are of subunits which regulate stimulation of adenylate cyclase, and G_±α subgroup are of subunits which regulate inhibition of adenylate cyclase. The G_qα subgroup is associated with activation of β-class phospholipase C polypeptides . The downstream effects of these actions are frequently used to monitor G protein activity in cells or membrane preparations. The effect on adenylate cyclase is measured by determining cAMP concentration, which increases in response to G _s activation and decreases in response to G_t. Activation of a Gq type protein can be monitored by measuring the increase in inositol phosphates. Alternatively, the activation of Gq type proteins in a cellular context is often observed by detecting changes in intracellular calcium concentrations, because inositol trisphosphates, one of the products of the reaction catalyzed by phospholipase C β, cause a release of calcium from intracellular stores.

Conformation-Specific Gα Binding Peptides

In Paige, Hamilton, Fowlkes, Buehrer, Barnett, McDonnell and Christensen, PCT/US99/06664, filed March 26, 1999, and published October 28, 1999 as W099/54728, Example 201. suggested that one screen peptide libraries for (1) peptides which bind a GDP-treated, but not GTP-treated Gsα subunits (there called class III Biokeys, but here called D- peptides) and for (2) peptides which bind GTP-treated but not GDP-treated Gsα subunits (there called class IV Biokeys, here called T-peptides) . It further suggests that these peptides can be used as reagents in screening for agonists or antagonists to beta-2-adrenergic receptor, as an agonist would increase the signal from GTP-Gsα specific peptides, and decrease that from GDP-Gsα specific peptides, while an antagonist would have the opposite effects. There is no suggestion that one screen for peptides which bind Gα in an active conformation-independent manner (I-peptides) .

Also, no D- or T-peptides are in fact described by sequence.

Finally, there is no specific discussion of Giα as a target .

In Poster #181 at the American Society for Biochemistry and Molecular Biology (ASBMB) , June 5-8, 2000, it was reported that T-peptides, D-peptides and I-peptides for G il had been identified. However, no sequence information was provided.

Screening for Modulators of G Protein Coupled Receptor Activity

A number of assays exist which detect the activation of Gα proteins by a GPCR. One approach is to measure the incorporation of radiolabeled (e.g., ³⁵S) GTPNS into receptor-activated G proteins (Wieland and Jakobs, Methods Enzymol. 1994; 237:3-13). This assay can be carried out with G proteins and receptors expressed endogenously or exogenously but the expression level has to exceed a certain threshold which is not met by many endogenous systems. Cells or cell preparations (e.g. membranes) are incubated with receptor specific activators in the presence of

³⁵S-GTPgS which can be detected after it is separated from unbound radiation on filters (usually glass fiber) . In recent years detection methods have been developed which don't require separation of unbound material since they rely on proximity of the radioactive label to the scintillant

("scintillation proximity assays", SPA) .

There are several disadvantages: the method relies on the use of a radioactive isotope most variants require filtration, making it impossible to use in HTS; existing homogeneous methods don't have a good signal to background ratio the range of possible GTP concentrations is limited

³⁵S-GTPγS binds to numerous other membrane components, creating a large background problem signal cannot be amplified, isotope detection will always be the limit The current state of the art for screening for modulators of receptor activity involves the expression of the GPCR to be monitored in a suitable cell line (often CHO or HEK293 cells or derivatives thereof) and detection of an increase in intracellular calcium concentration. For receptors coupling through G_αq and the Phospholipase Cβ signaling pathway this generally requires successful expression of the receptor and subsequent calcium monitoring as described below. For G_αi and especially G_αs coupled receptors, however, the physiological pathway usually does not lead to a sufficient increase in intracellular Calcium. To circumvent this, Coward et al (Anal Biochem 270(2) : 242-8) devised a method using chimeric G_α proteins (see below) and the G_αl6 subunit, which couples to many receptors from multiple subgroups and has been called the "promiscuous" G alpha. The method allows these receptors to signal through the G_αq pathway and, when used in connection with a Fluorometric Imaging Plate Reader (FLIPR) , to be monitored in highthroughput fashion.

In this assay, cells expressing the appropriate receptor/G protein combination are loaded with a Calcium sensitive fluorescent dye (e.g. Fura-2) and then challenged with the test substances. If receptor activation occurs the increase in intracellular calcium leads to an increase in fluorescence which is detected by an imaging system. There are various disadvantages to this method:

• It always requires live cells and therefore a continuous cell culture which is labor intensive. The fact that the.G protein pathway has multiple connections to other cellular pathway leads to interference from many substances which have effects on other cellular components. This can be the source of interference with the consequence of both false-positive and false-negative readouts.

• For orphan receptors it is difficult to predict which G proteins they might couple to which necessitates multiple expression systems

• Cell lines contain many different endogenous receptors and G proteins which create a background and require finetuning of each receptor/G protein/cell type combination • The automated imaging systems are highly specialized and require costly equipment

Cell lines expressing these chimeric G alpha subunits are sold under the name Liveware_ product line by Molecular Devices, as well as the FLIPR machines.

For more information on Ca²⁺ imaging, see Patel et al. (2001) : "Activity of diadenosine polyphosphates at P2Y receptors stably expressed in 1321N1 cells", Eur J Pharmacol 430 (2-3) :203-10 (2001). Alternatively, reporter gene assays can be performed (See Hill et al . (2001): "Reporter-gene systems for the study of G-protein-coupled receptors", Curr Opin Pharmacol 1(5): 526). The effect of transcriptional activity is even further downstream from the second messenger systems and has therefore, despite an increase in sensitivity, bigger problems with respect to reliability.

Both of these assay formats are functional assays, that is, they reveal whether the screened substance actually activates the GPCR.

Preliminary screening assays are known in which one simply determines whether a substance binds to a GPCR. Such assays are often constructed as ligand displacement assays, that is, one screens if the molecule displaces a known ligand of the GPCR in question. However, binding the GPCR does not guarantee that the substance will modulate GPCR activity. So ligand displacement screening assays are usually used to narrow the list of candidates to be screened in functional assays.

Of course, once a substance is known to be a modulator of a GPCR, one may choose to detect or quantify it in a sample by a ligand displacement assay.

Except for the ³⁵S-GTPgS binding assays, most functional assays are carried out with live cells, which requires a lot of maintenance. In contrast, the reagents for our assays can be produced in large, quality controlled batches which leads to better uniformity overall and is more convenient than live cells.

Chimeric Gα Subunits

Liu, et al., Proc. Nat. Acad. Sci. (USA), 92:11642-6 (1995) noted that the last 3-5 amino acids of G_qα could be replaced with the corresponding sequences of G_l2α, G₀α or G_zα to allow coupling to a G_x- or G₀-specific GPCR.

Univ. of California (Conklin et al.), WO99/05177 relates to N-terminal alterations (typically of the first six a.a.), such as truncation or replacement, of G_qα. These altered G_qα can acquire the ability to be activated by one or more non-Gq receptors, while still retaining the down stream signaling capacity that is characteristic of the Gq receptor. Alternatively, they may be further modified at the C-terminal, e.g., replacing the C-terminal (4-10 a.a.) of G_qα with that of G_i2 to obtain what Conklin calls a "double chimera". Example 8 describes a mutant Gα_q with a truncated and modified amino terminal (6 a.a. removed; residues 7-10 replaced with 1-4 of G_j_α) and a substituted carboxy terminal (last 4 a.a. replaced with those of G a) . In all of Conklin' s chimera, the Gα subunit was primarily derived from G_qα. Hence, he did not know what the effects of analogous N- and C-terminal modifications on other Gα subtypes might be. Moreover, in all of his examples, the first 6 a.a. of the G_qα were truncated rather than replaced.

Liu, J. Biol. Chem., 271:6172-8 (1996) discusses the Gα binding site of m2 Muscarinic receptor, a GPCR. The receptor coupling specificity of this receptor, which is G_i/G₀-coupled, was switched by single amino acid substitutions which mimicked the G_q-coupled ml, m3 or m5 receptors.

Fong, et al., Molec. Pharmacol., 54:249-57 (1998) describes a bipartile chimeric rat G_il/G₃ Gα subunit and its activation by the human IP prostanoid receptor. Normally, agonist activation of the IP prostanoid receptor results in stimulation of adenylyl cyclase via activation of the G protein G_sα. The chimeric Gα had the backbone of rat G_uα, except that the carboxy terminal six amino acids were replaced by those of rat G_sα. The extreme carboxyl terminus of a G protein α subunit was considered a key site for functional contacts with GPCRs . There was an effective interaction between the IP prostanoid receptor and Fong's Gα chimera in HEK293 cells even though there is no detectable interaction between that receptor and wild-type G α. See also Fong and Milligan, Biochem., J. 342:457-63 (1999).

Coward, et al., Anal. Biochem., 270:242-8 (1999) used chimeric G proteins to allow Gi~ and G₀-coupled receptors to signal through G_q. The five carboxy terminal amino acids of G_qα were replaced by those of G_±α or G₀α.

Milligan and Rees, "Chimaeric Gα proteins: their potential use in drug discovery", TIPS, 20:118-124 (March 1999) explore the use of chimeric G alpha subunits, in which only the extreme C-terminus is modified, to direct GPCRs with differing G-protein-coupling specificities toward common assay endpoints. They teach that the C-terminal of Gα subunits is a key contact site with GPCRs. They review chimeric G alpha subunits with G_q (pp. 121- 123) or G_s (p. 123) backbones. However, they had only "limited success" in engrafting G_q coupling (via C-terminal modification) onto a G_± backbone.

Hamm, Proc. Nat. Acad. Sci., 98:4819-21 (April 24, 2001) suggests that, based on the published structure of rhodopsin and G_at and findings from Gobind Khorana's group using crosslinking of specific residues in G_at the amino terminal of the G alpha subunit is also involved in GPCR binding. Klein, USP 6,255,059 contemplates that if a mammalian GPCR is expressed in a yeast cell a chimeric (yeast/mammalian) G-alpha subunit may be needed to couple it to the yeast G beta-gamma. See also USPs 6,100,042; 6,001,553; 5,876,951; and 5,789,184.

Traditional Drug Screening

In traditional drug screening, natural products (especially those used in folk remedies) were tested for biological activity. The active ingredients of these products were purified and characterized, and then synthetic analogues of these "drug leads" were designed, prepared and tested for activity. The best of these analogues became the next generation of "drug leads", and new analogs were made and evaluated.

Both natural products and synthetic compounds could be tested for just a single activity, or tested exhaustively for any biological activity of the interest to the tester. Testing was originally carried out in animals, later, less expensive and more convenient model systems, employing isolated organs, tissue, or cells, or cell cultures, membrane extracts or purified receptors, were developed for some pharmacological evaluations. Testing in whole animals and isolated organs typically requires large amounts of chemical compound to test. Since the quantity of a given compound within a collection of potential medicinal compounds is limited, this requires one to limit the number of screens executed. Also, it is inherently difficult to establish structure/activity relationships (SAR) among compounds tested using whole animals, or isolated organs or tissues or, to a lesser extent, cultured cells. This is because the actual molecular target of any given compound's action may be quite different from that of other compounds scoring positive in the assay. By testing a battery of compounds on a very specific target, one can correlate the action of various chemical residues with the quantitative activity and use that information to focus ones search for active compounds among certain classes of compounds or even direct the synthesis of novel compounds having a composite of the properties shared by the active compounds tested.

Another disadvantage to whole animal, organ, tissue and cell based screening is that certain limitations may prevent an active compound from being scored as such. For instance, an inability to pass through the cellular membrane may prevent a potent inhibitor, within a tested compound library, from acting on the activated oncogene ras and giving a spurious negative score in a cell proliferation assay. However, if it were possible to test ras in an isolated system, that potent inhibitor would be scored as a positive compound and contribute to the establishment of a relevant SAR. Subsequent, chemical modifica ions could then be carried out to optimize the compound structure for membrane permeability. (In the case of cell-based assays, this problem can be alleviated to some degree by altering membrane permeability. )

Drug Discovery

The human genomics effort could yield gene sequences that code for as many as 70,000 proteins, each a potential drug target; microbial genomics will increase this number further. Unfortunately, since genomic studies identify genes, but not the biological activity of the corresponding proteins, it is likely that many of the genes will prove to encode proteins whose activation or inactivation has no effect on disease progression. (Gold, et al., J. Nature Biotech., 15:297, 1997). There is therefore a need for a method of determining which proteins are most likely to be productive targets for pharmacological intervention.

Even if one knew in advance the perhaps 10,000 proteins which could be considered interesting targets, there remains the problem of efficiently screening hundreds of thousands of possible drugs for a useful activity against these 10,000 targets .

Historically, acquiring chemical compound libraries has been a barrier to the entry of smaller firms into the drug discovery arena. Due to the large quantity of chemical required for testing on whole .animals and even on cells in culture, it was a given that whenever a compound was synthesized it should be done in fairly large quantity. Thus, there was a synthesis and purification throughput of less than 50 compounds per chemist per year. Large companies maintained their immensely valuable collections as trade barriers.^' However, with the downsizing of targets to the molecular level and the automation of screens, the quantity of a given compound necessary for an assay has been reduced to very small amounts. These changes have opened the door for the utilization of so-called combinatorial chemistry libraries in lieu of the traditional chemical libraries. Combinatorial chemistry permits the rapid and relatively inexpensive synthesis of large numbers of compounds in the small quantities suitable for automated assays directed at molecular targets. Numerous small companies and academic laboratories have successfully engineered combinatorial chemical libraries with a significant range of diversity (reviewed in Doyle, 1995, Gordon et al , 1994a, Gordon et al, 1994b) .

Combinatorial Libraries. In a combinatorial library, chemical building blocks are randomly combined into a large number (as high as 10E15) of different compounds, which are then simultaneously screened for binding (or other) activity against one or more targets . Libraries of thousands, even millions, of random oligopeptides have been prepared by chemical synthesis (Houghten et al . , Nature, 354:84-6(1991)), or gene expression (Marks et al . , J Mol Biol, 222:581-97(1991)), displayed on chromatographic supports (Lam et al . , Nature, 354:82-4(1991)), inside bacterial cells (Colas et al . , Nature, 380:548-550(1996)), on bacterial pili (Lu, Bio/Technology, 13:366-372(1990)), or phage (Smith, Science, 228:1315-7(1985)), and screened for binding to a variety of targets including antibodies (Valadon et al . , J Mol Biol, 261:11-22(1996)), cellular proteins (Schmitz et al . , J Mol Biol, 260:664-677(1996)), viral proteins (Hong and Boulanger, Embo J, 14:4714-4727(1995)), bacterial proteins (Jacobsson and Frykberg, Biotechniques, 18:878-885(1995)), nucleic acids (Cheng et al . , Gene, 171:1-8(1996)), and plastic (Siani et al . , J Chem Inf Co put Sci, 34:588- 593(1994) ) .

Libraries of proteins (Ladner, USP 4,664,989), peptoids (Simon et al . , Proc Natl Acad Sci U S A, 89:9367-71(1992)), nucleic acids (Ellington and Szostak, Nature, 246:818(1990)), carbohydrates, and small organic molecules (Eichler et al . , Med Res Rev, 15:481-96(1995)) have also been prepared or suggested for drug screening purposes. The first combinatorial libraries were composed of peptides or proteins, in which all or selected amino acid positions were randomized. Peptides and proteins can exhibit high and specific binding activity, and can act as catalysts. In consequence, they are of great importance in biological systems. Unfortunately, peptides per se have limited utility for use as therapeutic entities. They are costly to synthesize, unstable in the presence of proteases and in general do not transit cellular membranes. Other classes of compounds have better properties for drug candidates.

Nucleic acids have also been used in combinatorial libraries. Their great advantage is the ease with which a nucleic acid with appropriate binding activity can be amplified. As a result, combinatorial libraries composed of nucleic acids can be of low redundancy and hence, of high diversity. However, the resulting oligonucleotides are not suitable as drugs for several reasons. First, the oligonucleotides have high molecular weights and cannot be synthesized conveniently in large quantities. Second, because oligonucleotides are polyanions, they do not cross cell membranes. Finally, deoxy- and ribo-nucleotides are hydrolytically digested by nucleases that occur in all living systems and are therefore usually decomposed before reaching the target.

There has therefore been much interest, in combinatorial libraries based on small molecules, which are more suited to pharmaceutical use, especially those which, like benzodiazepines, belong to a chemical class which has already yielded useful pharmacological agents. The techniques of combinatorial chemistry have been recognized as the most efficient means for finding small molecules that act on these targets. At present, small molecule combinatorial chemistry involves the synthesis of either pooled or discrete molecules that present varying arrays of functionality on a common scaffold. These compounds are grouped in libraries that are then screened against the target of interest either for binding or for inhibition of biological activity. Libraries containing hundreds of thousands of compounds are now being routinely synthesized; however, screening these large libraries for binding or inhibition with all 10,000 potential targets cannot be reasonably accomplished with present screening technologies, and there are numerous experimental and computational strategies under development to reduce the number of compounds that must be screened for each target.

High-Throughput Screening A high-throughput screening system usually comprises

(1) suitably arrayed compound libraries, (2) an assay method configured for automation, (3) a robotics workstation for performing the method, and (4) a computerized system for handling the data.

The array may be a standard 96-well microtitre plate, or an array of compounds on chips, beads, agar plates or other solid support. The array may be a simplex array of individual compounds or a complex array in which each element is a predetermined mixture of a small number, e.g., 10-20, different compounds. In the latter case, the mixture ultimately must be deconvolved to identify the true active component (s) . For ease of automation, the assay should require as few steps as possible. Thus, homogeneous assays, which do not require fractionations, or more than a single addition of reagent, are desirable.

See generally Broach and Thorner, Nature, 384, 14 (Nov. 7, 1996); Milligan and Rees, Trends Pharmacol. Sci., 20:118- 24 (1999) .

Preferred reporter genes for high-throughput screening include bacterial beta—galactosidase, luciferase, human placental alkaline phosphatase, bacterial beta-lactamase, and jellyfish green fluorescent protein.

Gonzalez and Negulescu, Curr. Op. Biotechnol., 9:624-31 (1998), discuss intracellular detection assays suitable for high-throughput screening. Such assays are conveniently provided as optical assays, which may rely on absorbance, fluorescence, or luminescence as readouts. While absorbance assays have been useful in melanophore and beta- galactosidase reporter assays for GPCRs, such assays have relatively low sensitivity. To achieve significant absorbance changes, very high concentrations of dyes and many cells are necessary. Hence, the absorbance assays do not lend themselves as well to miniaturized formats.

In contrast, luminescence and fluorescence are more sensitive and high S/N ratios are commonplace. With regard to chemilu inescence assays, the standard substrates are luciferin and aequorin. Since high concentrations of luciferin and ATP are desirable to drive luciferase-catalyzed reactions, the luciferase assay is usually conducted in cell lysates from thousands of cells, rather than in intact cells. Membrane-impermeable luminescent substrates have been used in connection with extracellular or lysate assays. The greatest advantage of chemiluminescence assays is their extremely low background. Fluorescence can easily be detected at the single cell level. However, the process of exciting fluorescence is not absolutely selective; there is a background of unwanted fluorescence and light scattering from endogenous cellular and equipment sources. Cell-based fluorescence assays fall into three broad categories: (1) those based on changes in fluorescence intensity, such as those based on the calcium-sensitive Fluo-3 sensor; (2) those based- on energy transfer, such as FRET (where there is an energy transfer from a donor fluorophore to an acceptor fluorophore when they are in close proximity and have a spectral overlap) ; and (3) those based on energy redistribution (where a tagged molecule moves within a cell, and the change in position of the fluorescence within the individual cell is observed) . The possible signals include Ca, cAMP, voltage, enzymatic, protein interaction, and transcription. Ca and cAMP are both mentioned in the context of GPCR targets. For Ca, the suggested readout is Ca indicator dye (fluorescence) , Ca photoprotein (luminescence) , a reporter gene (fluorescence or luminescence) , and cameleon (FRET) . For cAMP, the suggested readouts are FlchR (FRET) and a reporter gene (fluorescence or luminescence) .

The authors also comment that other detection methods, such as fluorescent polarization, fluorescence correlation spectroscopy, and time-resolved detection, which are still primarily used in biochemical or binding assays, will also undoubtedly migrate into cell based assays. All references , including any pa tents or pa tent applications, cited in this specification are hereby incorporated by reference . No admission is made that any reference consti tutes prior art . The discussion of the references states wha t their authors assert and applicants reserve the right to challenge the accuracy and pertinency of the cited document .

SUMMARY OF THE INVENTION

Gα is one subunit of a heterotrimeric GTP binding protein (G protein) . G proteins are known to bind. GTP or alternatively GDP at the same binding site. The GTP bound form constitutes the active form while the GDP bound form constitutes the inactive form of the Gα subunit. Upon activation of a GPCR by ligand, one of the ways that signals are transduced is by altering GPCR's cognate G protein from a GDP bound state to a GTP bound state. This allows separation of the alpha subunit from the other two subunits that make up the G protein, beta and gamma. Consequently the signal is transduced down various cell signaling pathways due to the subunit ' s interactions with other proteins. GPCR's are relevant to many diseases, mutations have been found that relate to an array of health problems including cancer, infertility, asthma, hypertension, and endocrine disorders. Gi is one of several G proteins, and Giα is its Gα subunit. The G proteins of greatest interest are Gs, Gi, Gq and G12/13. The present invention relates to peptides which specifically bind a G-alpha subunit in either its inactive, GDP-bound conformation (D peptides) or its active GTP-bound conformation (T-peptides) , as well as to peptides which bind G-alpha without regard to its activation state (I peptides) . It will be understood that the contemplated peptides are not antibodies, nor do they comprise the variable domain of an antibody. The peptides of Tables 202, 205, 210 and 212, and peptides of similar sequence, are of particular interest. These peptides may be used for any purpose for which an oligomer of such binding characteristics is useful.

The present invention applies, mutatis mutandis, to the identification of binding molecules, other than peptides, which specifically bind a G-alpha subunit in its inactive conformation (D-molecules) or in its active conformation (T- molecules) , or which bind G-alpha in a manner indifferent to its activation state (I-molecules) . Nucleic acid molecules (aptamers) are of particular interest. The present invention likewise applies, mutatis^' mutandis, to the identification of binding molecules, including peptides and nucleic acids, which (1) specifically bind the Gβγ complex, but not the whole G protein or (2) the whole G protein, but not the Gβy or isolated Gα. Cf. Scott, et al., EMBO J. , 20: 767-76 (2001).

Use of activa tion-sensi tive G-alpha binding peptides in detecting activation or deactiva tion of a GPCR by a known or suspected modula tor. In particular, these D- and T-peptides may be used to detect the activation or deactivation of a GPCR which interacts with the aforementioned G-alpha subunits. Such assays are -useful in the identification of modulators of GPCRs, especially agonists and antagonists of GPCRs. Preferably, these modulators are small organic molecules, preferably molecules having a molecular weight of less than 500 daltons, which are pharmaceutically acceptable and potent modulators of the biological activity of the GPCR. In the contemplated assays, the analyte is the activated Gα subunit (Gα:GTP) and/or the inactivated Gα subunit (Gα:GDP) .

The contemplated assays may be whole cell assays, isolated membrane assays, or even membrane-free assays. However, it is necessary that the assay furnish the G protein coupled receptor, the Gα protein, and GDP (or analogue) or GTP (or analogue) in such form that, if an agonist of the GPCR is provided, the Gα will bind GTP, or that if an antagonist is provided, the Gα will bind GDP. The preferred assays are isolated membrane assays as in Example 204, or "two hybrid" cell-based assays in yeast or mammalian cells.

The assays may be homogeneous or heterogeneous, with homogeneous assays being preferred.

An important class of homogeneous assays are interaction assays, where the presence of a _.modulator (e.g., an agonist or antagonist) is related to the strength of interaction between two labels brought into proximity by the binding of two differently labeled binding molecules to the same molecule of analyte.

In the interaction assay, the paired interactive labels are preferably attached, respectively to (1) a T-peptide and an I-peptide, or (2) a D-peptide and an I-peptide. However, it is possible that two T-peptides, or two D-peptides, could bind simultaneously, to the G-alpha, in which case two labeled- T-peptides or two labeled D-peptides could be employed in an -interaction assay. ^{. . .}

Alternatively, instead of labeling an I-peptide, the G- alpha subunit may be labeled directly. The interaction is then between the labeled T- or D-peptide, and the labeled G- alpha subunit.

The G-alpha subunit may be labeled directly, or indirectly. Examples of indirect labeling include

Gα-biotin: avidin-label Gα-epitope tag: (anti-Gα) Ab-label

An example of an epitope tag is a His tag. Likewise, a T-, D- or I-peptide may be labeled directly or indirectly.

In one series of embodiments, the peptides are used in a "one-hybrid" assay (Example 207) for modulators, e.g., agonists or antagonists, of GPCRs. In this assay, a fusion of a T peptide and a membrane-active signaling protein is used to directly detect the activation of the receptor. Such activation converts the associated G-alpha to the GTP bound state, allowing the T peptide to bind. This causes the signaling protein to be recruited to the membrane, where it becomes active, generating a signal. Alternatively, a D- peptide is fused to the membrane-active signaling protein. In this embodiment, the signal is inversely related to the activation of the receptor.

In another series of embodiments, the peptides are used in a "two-hybrid assay" for modulators, e.g., agonists or antagonists, of GPCRs. For this purpose, a G alpha is fused to component A of a signal generating system, and a G alpha binding peptide to component B.

The binding of an agonist to the GPCR activates the GPCR, causing the G alpha hybrid to bind GTP, whereupon the first hybrid- is -bound by a T-peptide hybrid, thereby bringing A and B into proximity, generating a signal. One could similarly use the D peptide in an assay for antagonist activity, or use both, with different readout systems.

In preferred embodiments, the G-alpha binding peptides are used in fluorescent or luminescent assays for agonists and antagonists of GPCRs, as these assays lend themselves to use in a high-throughput screening assay context.

Fluorescence assays of the scintillation proximity, fluorescence polarization, or FRET formats are of particular interest (Example 204) .

In the aforementioned assays, Gα chimeras, with an N- terminal constant region and a variable C-terminal region allowing coupling to different receptors, may be used in screens for agonists or antagonists of GPCRs normally coupled to a G-alpha other than the one which is the source of the N-terminal constant region.

The BioKeys that have been identified for Giα can be categorized into four groups. BioKeys that are specific to the GTP bound form of Gα (T peptides) , those that are specific to the GDP bound form of Gα (D peptides) , those that bind independently of GTP or GDP (I peptides) , and those that have a weak specificity to either form (BT peptides) .

G-alpha binding peptides of particular interest are those which are not subsequences, or even merely conservative substitution mutants of subsequences, which are found in naturally occurring peptide agonists or antagonists of G-alpha, such as melittin, substance P, GP Antagonist-2A, MAS 7 and Mastoparan, or in G-beta, G-gamma, or GPCR. These novel peptides may be used in a variety of high throughput screening assays that take advantage of their specificity for different forms of the Gα protein to isolate novel agonists and antagonists- to GPCRs- -for use as therapeutics. These include both in vitro assays such as SPA assays, FRET assays, FP assays, and TRF assays and in vivo assays such as cell based FRET, reconstruction of enzyme activity assays, creation of enzyme activity assays, and modified two hybrid like assays. Although we have initially screened Giα and G_as, one may similarly isolate BioKeys to all the Gα's and use the resulting BioKeys to screen known and orphan GPCRs.

Use of activation-sensitive G-alpha binding peptides in monitoring the activation state of a GPCR within a cell An alternative use for the aforementioned activation- sensitive G-alpha binding peptides is in monitoring the activation state of a GPCR within a cell. An I-peptide is fused to a first fluorophore and a peptide of a different category (D or T) is fused to a different fluorophore, the two fluorophores being matched for FRET. Both peptides will bind to the G-alpha, bringing the fluorophores into interactive proximity, only if the G-alpha is inactive (in the case of the D embodiment) or active (in the case of the T embodiment) . Activation will occur as the result of agonist action on the cognate GPCR to which the G-alpha in question is functionally coupled. Inactivation will likewise occur as the result of antagonist action. Alternatively, instead of fusing an I-peptide to the first fluorophore, one could couple the G-alpha subunit itself to it. The D or T peptide would bind to the G-alpha under the appropriate condition, leading to fluorophore energy transfer.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1: Selective binding of peptide-Alkaline Phosphatase fusion constructs to purified Gαi protein. Purified G_αi protein was immobilized in 96 well plates after loading with either GDP or GTPgS. Increasing amounts of protein were added as shown on the abscissa in pmol per well. A: peptide 771; B: peptide 806; C: peptide 801.

Fig. 2: G„_±1 Peptide Probes Bind Specifically to G_Hi Subunit Class of Protein. Various hexa-histidine-tagged G_α subunits (il, i2, i3, s) were expressed in E. coli, purified by metal affinity chromatography, and immobilized in a microtiter assay plate. The assay plate was blocked with albumin and either GDP or GTPγS (50microMolar) was added to allow nucleotide charging of the G_α' subunits in a buffered Tris solution containing lOmM MgC12. Proteins were probed with peptide fused to bacterial alkaline phosphatase (BAP; #1=752, #2=757-, #3=771, #4=796, #5=801)- -by incubating at room temperature for 1 hour in the presence of the appropriate nucleotide. The assay plate was washed with TBS plus 0.5% Tween20 and bound BAP probe was detected by development with the chemiluminescent substrate CSPD (Tropix) . Plates were read on an LJL Analyst.

Fig. 3: Multivalency of Peptide Probes Reduces Background. Sf9 cells were infected with a combination of three baculovirus encoding G_α (il or s) , G_βl and G_γ2, each at an oi of 3. Cells were harvested 2 days post-infection and immobilized in microtiter assay wells by capture with Concanavalin A. After a freeze/thaw cycle to per eabilize the cells, nucleotide (GDP or GTPγS) was added at

25 microMolar in a buffered Hepes solution containing

3 milliMolar free MgC12. Galpha protein was probed with a biotinylated synthetic peptide (806) conjugated to streptavidin-alkaline phosphatase (SAAP, Pierce) . The probe was prepared by mixing 1:1, 1:2, 1:4, or 1:8 ratios of SAAP to peptide, where the SAAP concentration was lOOnanoMolar . Empty biotin-binding sites on the SAAP were blocked afterwards by incubating with 50 microMolar free biotin

(Sigma) . Peptide probes were added to the assay wells at a concentration of 2nanoMolar. After washing with TBS/0.5% Tween detergent, the bound peptide probe was detected with CSPD chemiluminescent substrate and read on a MicroBeta counter.

Fig. 4: Optimization of Peptide Probe Concentration. Assay plates were prepared as before and nucleotide-charged G_α subunits were detected with 2, 0.4,0.08, 0.016, or 0.003 nanoMolar SAAP-peptide 806 probe. Where indicated, 10 microMolar Neutravidin (Pierce) was included during the probe incubation as a potential blocking agent. The assay was developed and read as described for the Multivalency experiment.

Fig. 5: Comparison of 1^st and 2^nd generation G.,.,- peptides : Assay plates were prepared and charged with nucleotide as described in Fig. 3. The cells contained G_αi and detection was carried out with the peptides as indicated.

Fig. 6: Comparison of different evolved G^- peptides in competition binding assays with purified peptide. Purified G_ai protein was immobilized as described for figure 2. Detection was carried out with either 1755-NAAP (6a), 796- NAAP or 1746-NAAP (6b) in the presence of increasing amounts of unlabeled T peptides (1746, 1755), pre-evolved T peptide (796, 806) or unrelated peptide (Tyr2B)

Fig. 7: Phage ELISA of G_3S specific phages from first (unevolved) and second (evolved) generation peptides. Phage ELISAs were performed as described for G_ai specific phages (see Example 202 above) except that the volume of phage added was 5. ml. Peptide 95 was used as an unspecific control peptide.

Fig. 8: Comparison between phage ELISA data (see Fig.7) and capture of 35S-GTPgS bound G₃, with the corresponding synthesized peptides listed. Membrane preparations expressing the β2-Adrenergic Receptor; G_αs, . G_β and G_γ were incubated with 1 nM ³⁵S-GTPgS with or without isoproterenol. After 15 minutes the reaction mixture was added to assay wells in which the peptides had been immobilized and incubated for 1 hour before washing. Bound G protein was detected by adding scintillation fluid to the wells and counting. Peptide 1677, a D peptide, was used as control.

Fig. 9: Schematic representation of one embodiment of the AlphaKev assay. Immobilization occurs when membranes are incubated and subsequently frozen before the assay is started.

Fig. 10: AlphaKey Assay of m2 Acetylcholine Receptor S-t-imul-a-tion- and- -I-nhibi-tion. Membranes expressing m2Acetylcholine receptor, G_αil and G_PY were stimulated with drugs as indicated on the abscissa. Atropine was added to the reaction at the same time as carbachol.

Fig. 11: The AlphaKey assay is robust and can be carried out on 384 well plates. The indicated amounts (per well) of Membranes containing the β2-Adrenergic Receptor and G_αi6s, G_p and G_y proteins were immoblized as in Fig. 14 except that 384 well plates were used. After thawing the plates were left at room temperature for the times indicated before the assay was performed as in Fig. 14

Fig. 12: Test screen using whole cells immobilized on 96 well plates and a standard set of compounds (Library of Pharmacologically active Compounds) . Instead of using membrane preparations each well contained 150,000 cells which were frozen and thawed before the assay. Fig. 13: Detection of agonist promoted activation of the M2 Acetylcholine Receptor using a T-peptide with FRET. Membrane preparations (3 mg per well) containing M2AChR, a G_ai subunit which was internally His-tagged and G_b and G_g subunits were preincubated with anti-His antibody conjugated to APC for 60 minutes. Subsequently they were incubated in GPCR buffer with or without carbachol. Detection of activated G_ai was achieved by adding the Eu-labeled peptide and reading fluorescence at 620 nm and 665 nm in an LJL Analyst reader after an additional 4 hours.

Fig. 14: AlphaKey Assay of β2-Adrenergic Receptor Stimulation and Inhibition. Membranes expressing the β2AR, ^G _cu6s' G_p and G_γ proteins were activated with agonists and antagonists as indicated. Fig. 15: AlphaKey Assay on β2-Adrenergic Receptor Exhibits Expected Activation Profile for Partial Agonists. Full and partial agonists were used at concentrations indicated.

Fig. 16: AlphaKey Assay is Modular: Agonist Activation of DI Dopamine Receptor with pH dependence. Assay was performed as for Fig. 14 except that the membranes used expressed the Dl dopamine receptor.

Fig. 17: Detection of agonist promoted activation of the Beta2 Adrenergic Receptor using a T-peptide with FRET. Membrane preparations (3 mg per well) containing b2AR, a G_ai6s subunit which was internally His-tagged and G_b and G_g subunits were preincubated with anti-His antibody conjugated to APC for 30 minutes. Subsequently they were incubated in GPCR buffer with or without isoproterenol. Detection of activated G_ai6s was achieved by adding the Eu-labeled peptide and reading fluorescence at 620 nm and 665 nm in an LJL Analyst reader after an additional 4 hours.

Fig. 18: Diagram of G_{2rj i} chimeric molecules. The amino acid residues are: 1-6 from G_aq (accession number AAB64301.1) and 1-354 from G_ai (accession number P04898)in No. 175; 1-6 from G_aq/ 1-348 from G_ai and 354-359 from G_aq in No. 176; 1-348 from G_ai and 354-359 from G_aq in No. 211; 1-341 from G_ai and 347-359 from G_aq in No. 450; 1-331 from G_ai and 337-359 from G_aq in No. 450; 1-40 from G_aq, 35- 341 from G_ai and 347-359 from G_aq in No. 452; 1-40 from G_aq, 35- 331 from G_ai and 337-359 from G_aq in No. 453.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

1. Target G Protein Coupled Receptor The target GPCR may be a naturally occurring substance, or a subunit or domain thereof, from any natural source, including a virus, a microorganism (including bacterial, fungi, algae, and protozoa) , an invertebrate (including insects and worms) , or the normal or cancerous cells of a vertebrate (especially a mammal, bird or fish and, among mammals, particularly humans, apes, monkeys, cows, pigs, goats, llamas, sheep, rats, mice, rabbits, guinea pigs, cats and dogs) .

Alternatively, the receptor protein may be a modified form of a natural receptor. Modifications may be introduced to facilitate the labeling or immobilization of the target receptor,- or to alter its biological activity (An inhibitor of a mutant receptor may be useful to selectively inhibit an undesired activity of the mutant receptor and leave other activities substantially intact) .

In the case of a protein, modifications include mutation (substitution, insertion or deletion of a genetically encoded amino acid) and derivatization (including glycosylation, phosphorylation, and lipidation) . The target GPCR may be a chimera of two receptors, e.g., a mammalian and a yeast receptor, or two receptors of different functions, so as to combine the ligand binding function of one receptor with the signal transduction function of another, or create a binding site with altered properties different from those of either receptor the chimera is derived from.

For further information of how to construct a mutant receptor, including a chimeric receptor, please see the discussion of "mutant proteins" and "chimeric proteins" in Serial No. 09/429,331.

The functional groups of the receptor which participate in the ligand-binding interactions together form the ligand binding site, or paratope, of the receptor. Similarly, the functional groups of the ligand which participate in these interactions together form the epitope of the ligand. In the case of a protein, the binding sites are typically relatively small surface patches. The binding characteristics of the protein may often be altered by local modifications at these sites, without denaturing the protein.

While it is possible for a chemical reaction to occur between a functional group on a receptor and one on a ligand, resulting in a covalent bond, receptor protein- ligand binding normally occurs as a result of the aggregate effects of several noncovalent interactions. Electrostatic interactions include salt bridges-, ^•• hydrogen -bonds, and van der Waals .forces. What is called the hydrophobic interaction is actually the absence of hydrogen bonding between nonpolar groups and water, rather than a favorable interaction between the nonpolar groups themselves. Hydrophobic interactions are important in stabilizing the conformation of a receptor protein and thus indirectly affect ligand binding, although hydrophobic residues are usually buried and thus not part of the binding site.

The receptor may have more than one paratope and they may be the same or different. Different paratopes may interact with epitopes of different binding partners. An individual paratope may be specific to a particular binding partner, or it may interact with several different binding partners. A receptor can bind a particular binding partner through several different binding sites. The binding sites may be continuous or discontinuous (e.g., vis-a-vis the primary sequence of a receptor protein) .

Some GPCRs, and their agonists and/or antagonists, are listed in Table A. Suitable receptors include, but are not limited to, dopaminergic, muscarinic, cholinergic, α-adrenergic, β- adrenergic, opioid (including delta and mu) , cannabinoid, serotoninergic, and GABAergic receptors. Other suitable receptors are listed in Table 2 of Fowlkes USP 5,789,184. The term "receptor," as used herein, encompasses both naturally occurring and mutant receptors.

The homology of GPCRs is discussed in Dohlman et al., Ann. Rev. Biochem., 60:653-88 (1991). When GPCRs are compared, a distinct spatial pattern of homology is discernable. The transmembrane domains are often the most similar, whereas the N- and C-terminal regions, and the cytoplasmic loop connecting transmembrane segments V and VI are -more divergent.

The functional significance of different GPCR regions has been studied by introducing point mutations (both substitutions and deletions) and by constructing chimeras of different but related GPCRs. Synthetic peptides corresponding to individual segments have also been tested for activity. Affinity labeling has been used to identify ligand binding sites.

2. Conformation-specific G Protein subunit Binding Molecules

Substances which modulate GPCR activity may be identified in an assay in which a change in GPCR activity is detected by a change in the binding of a G protein subunit conformation-specific binding molecule. The molecule may be one which, e.g., binds substantially more strongly to Gα alone than to the Gαβγ complex, binds substantially more strongly to Gα as part of the Gαβγ complex than to Gα alone, binds substantially more strongly to Gβ or Gγ as part of the heterodimeric Gβγ complex than as part of the heterotrimeric Gαβγ complex, or binds substantially more strongly to Gβ or Gγ as part of the heterotrimeric Gαβγ complex than as part of heterodimeric Gβγ complex. ..

The binding molecule may be a peptide, especially one of those set forth below; a peptoid, peptidomimetic or other analogue of a binding peptide; or any other kind of binding molecule, such as a nucleic acid. Insofar as the present invention relates to the binding molecules per se, it will be appreciated that these molecules should not be mere products of nature, i.e., they should either be "non-natural-ly occurring" a-s- de-fined in section 9.2 below, or they should be purified sufficiently to differentiate them from the molecules in the natural state. Insofar as the present invention relates to the use of the binding molecules for screening or other purposes, while it is preferred that they be non-naturally occurring and/or purified, it is not required. The molecules may be at least partially purified by any art-recognized means. If their target is available in substantial purity, then they may be purified by affinity chromatography. The molecules may be synthesized initially with a view to minimizing impurities from which it would be relatively difficult to separate them, e.g., peptides of similar size or affinity.

2.1. Preferred Gαi :GTP Binding Peptides Based on the data in table 202B, we have identified several distinct motifs for .preferred Gαi: GTP specific (T- peptide) binding.

In the T-l motif, the peptide comprises the sequence Cys-Xaa-Gly-Trp-Xaa-Cys-Tyr (residues 3-9 of extended T-l, SEQ ID NO: 125) where Xaa is any amino acid. In the four peptides from which this motif was deduced, the first Xaa was Phe, Leu or

Gin, and the second was Ala, Gin or His. Several peptides were sequenced from.the second generation T-l (792) library (#3) . In the first Xaa position, they featured Arg, lie, Gin, Lys, Val, Leu or Phe

(Lys favored) , and in the second, they showed predominantly

Gly, but also Gin, Glu, and Ser. More preferably, the peptide comprises the extended T-l sequence

Xaaj-Xaaa-Cys-Xaa-Gly-Trp-Xaa-Cys-Tyr (SEQ ID

NO:125) where Xaa_x is a charged residue and Xaa₂ is any amino acid. All of the second generation peptides had acidic residues at Xaa_x of the extended T-l motif, and lie, Val,

His, Phe, Tyr, Val and Ser at the Xaa₂.

In the T-2 motif, the peptide comprises the sequence Gly-Val-Trp-Xaa-Gly (residues 2-6 of extended T-2,

SEQ ID NO: 126) where Xaa is any amino acid.

More preferably, it comprises the extended T-2 sequence Gly-Gly-Val-Trp-Xaa^Gly (SEQ ID NO: 126), where Xaa_x is any amino acid, but is preferably hydrophobic. Preferably, a Pro immediately follows the final Gly in the sequence above.

Out of the second generation library (#4) evolved from T-2 peptide 796, only two peptides were sequenced. These revealed that GVW could be replaced by GIW. They confirmed the preference that Xaa_x be hydrophobic. While they both featured an acidic residue after the final Gly of T-2, the first generation peptides favored Pro at that position.

They did not feature Gly-Gly, but 1753 had a Tyr just before that position, similar to 408(796), and G22/G25.

In the T-3 motif, the peptide comprises the sequence Pro-Trp-Xaa (residues 6-8 of SEQ ID NO: 128) where Xaa is a charged acidic residue, e.g., Asp or Glu.

More preferably, the peptide comprises the extended T-3 sequence

Xaa^Xaas-Pro-Trp-Xaa where Xaa_x and Xaa₂ are hydrophobic residues; Xaa_x is most preferably lie.

From the second generation library #3, based on T-3 peptide 806, the following consensus sequence (T-3') may be deduced:

Cys-Pro-Xaa.-Xaa₂-Cys-Pro-Trp-Xaa₃-Xaa. ₍SE_Q ID NO:128)

where Xaa_x is any amino acid (Glu, Arg, Gin and Pro were observed) , Xaa₂ is any amino acid (preferably hydrophobic like the observed Trp and Phe, but Thr also seen) , Xaa₃ is acidic (Asp and Glu observed) , and Xaa₄ is amino acid (preferably hydrophobic, especially aromatic, like the observed Tyr and Trp, but Ser also seen) .

2.2. Preferred Gαi : GDP Binding Peptides One motif common to many of the preferred peptides which bound Gαi: GDP was the DI-1 sequence Xaa₄-Xaa₅-Xaa₆ (Neg-Hyd-AliphHyd) where Xaa₄ is a negatively charged amino acid, such as Asp or Glu, or is Val or Gin,

Xaa₅ is a hydrophobic amino acid, such as Phe, Tyr, Trp, Met,

Leu, lie, Val (presumably can also be Ala) Xaa₆ is a aliphatic hydrophobic amino acid, such as Met, Leu, lie, Val or Ala.

Note that this consensus sequence covers both D- peptides (which bind Gα:GDP in preference to Gα:GTP) and I- peptides (which bind Gα.-GDP and Gα:GTP about equally well). In most cases (peptides 387 and 386 excepted) , the D-l and 1-1 peptides were further characterized as comprising the extended DI-1 sequence

Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆ (Any-Hyd-Any-Neg-Hyd-

Alphi Hyd) where

Xaa_x is any amino acid

Xaa₂ is usually a hydrophobic amino acid; preferably it is

Leu, lie, T-rp, - Phe or Pro

Xaa₃ is any amino acid, and Xaa₄, Xaa₅ and Xaa₆ are as previously stated.

Scrutiny of the D-l peptides of Table 202C and the 1-1 peptides of Table 202A reveals the following preferences

Xaa_:: Ser, Thr, Tyr, Phe, His or Pro, with Thr and Ser more preferred Xaa₂: Trp or Leu, with Trp more preferred

Xaa₃: Glu, Gin, Tyr, Trp, Val, Ser, Ala, His, Arg, Lys

Xaa₄: Glu

Xaa₅: Phe, Tyr or Trp', especially Phe

Xaa₆: Leu Among the D-l peptides, the following combinations were seen for Xaa_x-Xaa₂ (peptides showing this combination are identified by clone ID) :

TW: G4, 314 (757), 73, 343, 217, 93 (740), 62, 193, 289, 265, 273, 272/282/6R2

SW: 324, 400, 281

SL: 359/161, 380/381/140

TL: 176 HW: 230, 213, 266

SW: 237

RW: 126/128/133/242/248

PW: 379

YW: 196 TF: 117, 192

TP: 179

Among the 1-1 peptides, Xaa₁-Xaa₂ were

TW: 99, 103 (743), 107, 361, 388/391, 45, 397/401/402, 15R2/B01/394

SL: 380/381/140

TL: 16 -YW-: 360

FW: 101 (779)

Among the D-peptides, the following combinations were seen for Xaa₄-Xaa₅-Xaa₆:

EFL: G4, 314 (357), 73, 343, 93, 400, 281, 359/161, 176,

380/381/140, 409/24r2, 126/128/133/242/248, 289, 273, 272/282/6R2

EFI: 217

EYL: 62, 265

DFL: 193

EFM: 324 EVL: 320, 213

EMM: 230

EYI: 266

QYL: 237 VWL : 379 DWL : 196, 117 ELM : 92 EWL : 179 Among the I-peptides , Xaa₄-Xaa₅-Xaa₆ were

EFL: 99, 103 (743), 107, 361, 45, 15r2/301/394,^' 380/381/140, 16, 360 DFL: 388/391 EYL: 397/401/412 EYA: 101 (779)

DWF: 387 (801), 386 (816)

Among the D-peptides, Xaa₃ was most often Trp, followed by Tyr, Lys and His. Among the I-peptides, Xaa₃ was most often Tyr, followed by Trp. Another common motif for Gαi : GDP binding was the sequence

Trp-Xaa-Trp This -de-fines the 1-2 group of I-peptides and the D-2 group of D-peptides. More preferably, we have the sequences

Pro-Trp-Xaa-Trp, Pro-Pro-Trp-Xaa-Trp (SEQ ID NO: 130),

Pro-Trp-Xaa-Trp-Trp/Tyr and Pro-Pro-Trp-Xaa-Trp-Trp/Tyr (SEQ ID NO: 131).

In all of the above D-2 and 1-2 sequences, Xaa may be any amino acid. In the three 1-2 peptides, it was Ala, Val or Lys, and the overall consensus sequence was Pro-Trp-Xaa- Trp. In the more numerous D-2 peptides, Xaa was Met, Leu, Ala, Tyr or Gin.

It should also be noted that there is potential overlap between the D-l and D-2, and 1-1 and 1-2, motifs. We can define a "supermotif" for Gαi: GDP as the DI supermotif sequence

Trp-Asp/Glu-Trp-Hyd where Hyd is any hydrophobic amino acid.

This supermotif does not include all D-l and I-l peptides, only those in which Xaa₃ is Trp. It also includes certain D-2 and 1-2 peptides which are not in D-l or I-l if insofar as Hyd may be an aromatic AA.

To better understand the criteria for D- and I-peptides against Gαi, one might screen a heptalibrary of the form Hyd-Ser/Thr-His-Trp-Any-Acidic-Hyd-Hyd which has a sequence space of 2xl0⁵.

There are three D-peptides in table 202C (115, 197, 347) which do not correspond to the D-l or D-2 motifs, and hence may individually define motifs which are Gα:GDP- specific; for example, by allowing only highly conservative substitutions (D/E, R/K/H, N/Q, V/I/L/M, S/T) within those sequences .- . . .. . . ^.

2.3. Preferred Gas: GTP Binding Peptides

These preferred peptides comprise the consensus sequence

Xaa^Trp-Gly-Leu-Ala (residues 1-5 of SEQ ID NO:129) where Xaa_x is a hydrophobic amino acid (preferably Leu) . There is an extended consensus sequence of the form

Xaai-Trp-Gly-Leu-Ala-Xaag-Pro-Xaae (SEQ ID NO: 129) where Xaa₈ is any amino acid (but preferably hydrophilic) and Xaa₈ is a hydrophobic amino acid (preferably Trp or Val) .

2.4. Screening of Peptide Libraries

As previously noted, when GPCR is activated, the G protein dissociates into Gα and Gβγ, and the GDP bound to the Gα is replaced by GTP. By screening a peptide library, one may identify additional .peptides which bind Gα in a conformation-specific manner, e.g., in the presence or absence of GDP or GTPγS, essentially as set forth in Example 202.

Likewise, one could screen specifically for the release of Gβγ. Peptides which bind Gβγ may be useful in certain assays of the present invention. Methods for the purification of βγ subunits are known to those skilled in the art, for references see Kozasa and Gilman, J. Biol. Chem. 270(4): 1734-41 (1995). A method of purifying biotinylated Gβγ protein, immobilizing it and using it to select peptides displayed on phages is described in Scott et al., Embo Journal 20(4): 767-76 (2001). In short, a construct with an avidity tag, e.g., one identical to the one described for G_αi, could be designed for Go and coexpressed with G_v in a baculovirus expression system. Also present would be a G_αi which could be epitope tagged with a His-tag. In the presence of excess GDP these proteins can be bound to a Ni- column according to the method by Kozasa and Gilman. The Gβγ subunit can then be eluted specifically in the presence of A1F₄ ^" and biotinylated with the procedure described by Scott et al. Subsequently the protein can be immobilized and used for phage display as described here for G . The present invention is not limited to any particular method of screening for peptides which bind Gβγ.

2.5. Screening of Other Libraries Other classes of binding molecules may be identified, as in §2.4, but by screening other libraries, such as peptoid, nucleic acid, or small organic compound libraries. 2.6 Identi ication of Analogues

The binding molecule may be an analogue of one of the aforementioned peptides, in which case it preferably is a peptoid or other peptidomimetic, or it may. be an analogue of a binding molecule identified per 2.5.

3. Receptor Agonists , Antagonists , and Ligands

Ligands are substances which bind to a receptor, and which are not part of its signal transduction apparatus (thus, the G protein of a GPCR is not a, ligand as here defined) .

If such binding can affect the activity of the receptor (alone or in conjunction with the binding of other ligands), the ligand is said to be a modula tor. The most important classes of modulators are "agonists" and "antagonists". (Some modulators change roles, acting as agonists or antagonists, depending on circumstances.)

-The substances which- are able -to- elieit -t-he r-e-sponse are called "agonists", and if the mechanism is by specific interaction with a receptor site, are known as pharmacological agonists. (In general, when we refer to an "agonist" without qualification, we mean a pharmacological agonist.) Typically, increasing the concentration of the agonist at the receptor site leads to an increasingly larger response, until a maximum response is achieved. A substance able to elicit the maximum response is known as a full agonist, and one which elicits only, at most, a lesser (but discernible) response is a partial agonist. In general, the term "agonist" applies to ligands which are capable of eliciting some response even when no other ligand is bound to the receptor.

Substances which have an effect opposite that of agonists are called antagonists. A pharmacological antagonist is a compound which interacts with the receptor, at a specific site, without . eliciting a response, and by doing so inhibits the receptor from responding to an agonist A competitive antagonist is one whose effect can be overcome by increasing the agonist concentration; a noncompetitive antagonist is one whose action is unaffected by agonist concentration. A sequestering antagonist is one which inhibits a ligand: receptor interaction by binding to the ligand in such a way that it can no longer bind the receptor. A competitive sequestering antagonist competes with the receptor for the ligand, whereas a competitive pharmacological antagonist competes with the ligand for the receptor. Generally speaking, a competitive pharmacological antagonist and a pharmacological agonist compete for the same binding site on the receptor.

An "inverse agonist" is a substance which influences the equilibrium between active receptor and inactive receptor in the direct-ion of inactive receptor. If a receptor has no basal activity in the absence of agonist, an "inverse agonist" is difficult to distinguish from a conventional antagonist. However, if a receptor has a basal activity in the absence of agonist, a substance which can partially or totally inhibit that basal activity is called an "inverse agonist". It should be noted that for the purpose of definition, "pharmacological antagonists" include "inverse agonists". An antagonist which is not an inverse agonist may be termed a "strict antagonist".

Natural modulators are those which, in nature, without human intervention, are responsible for agonizing or antagonizing a natural receptor. A natural modulator may be produced by the organism to which the receptor is native. One native to a pathogen or parasite may bind to a receptor native to a host. Or one native to a host may bind to a receptor native to a pathogen or parasite. All of these are natural modulators.

The clinical concept of drug antagonism is broader than the pharmacological concept, including phenomena that do not involve direct inhibition of agonist : receptor binding. A "physiological" antagonist could be a substance which directly or indirectly inhibits the production, release or transport to the receptor site of the natural agonist, or directly or indirectly facilitates its elimination .(whether physical, or by modification to an inactive form) from the receptor site, or inhibits the production or increases the rate of turnover of the receptor, or interferes with signal transduction from the activated receptor. A physiological antagonist (functional antagonist) of one receptor (e.g., an estrogen receptor) may be a pharmacological antagonist of another, e.g., a transcription factor. Or it- maybe a pharmacological -ag-onist- of -another receptor, such as one which activates an enzyme which degrades the natural ligand of the first receptor.

Similarly, one may speak of a physiological agonist, which is a substance which directly or indirectly enhances the production, release or transport to the receptor site of the natural agonist, or directly or indirectly inhibits its elimination from the receptor site, or enhances the production or reduces the rate of turnover of the receptor, or in some way facilitates signal transduction from the activated receptor.

It follows that there are both "pharmacological" and "physiological" modulators. Outside this section, all references to modulators (including specific references to agonists, antagonists, inverse agonists, coactivators and coinhibitors) , if unqualified, should be considered references to pharmacological, rather than physiological, modulators .

If a disease state is the result of inappropriate activation of a receptor, the disease may be prevented or treated by means of a physiological or pharmacological antagonist. Other disease states may arise through inadequate activation of a receptor, in which case the disease may be prevented by means of a suitable physiological or pharmacological agonist. An important class of receptors are proteins embedded in the phospholipid bilayer of cell membranes. The binding of an agonist to the receptor (typically at an extracellular binding site) can cause an allosteric change at an intracellular site, altering the receptor's interaction with other biomolecules . The physiological response is initiated by the interaction with this "second messenger" (the agonist is the "first messenger") or "effector" molecule.

Enzymes are special -types of receptors. Receptors interact with agonists to form complexes which elicit a biological response. Ordinary receptors then release the agonist intact. With enzymes, the agonists are enzyme substrates, and the enzymes catalyze a chemical modification of the substrate. Thus, enzyme substrates are "ligands". Enzymes are not necessarily integral membrane proteins; they may be secreted, or intracellular, proteins. Often, enzymes are activated by the action of a receptor's second messenger, or, more indirectly, by the product of an "upstream" enzymatic reaction.

Thus, drugs may also be useful because of their interaction with enzymes. The drug may serve as a substrate for the enzyme, as a coenzyme, or as an enzyme inhibitor. (An irreversible inhibitor is an "inactivator" . ) Drugs may also cause, directly or indirectly, the conversion of a proenzyme or apoenzyme into an enzyme. Many disease states are associated with inappropriately low or high activity of particular enzymes.

The concept of a pharmacological "modulator" includes allosteric modulators. An allosteric modulator is one that binds to a receptor at a site which is not the binding site of an agonist, and which causes a conformational change in the receptor. Allosteric modulators include coactivators (positive allosteric modulators) and coinhibitors (negative allosteric modulators) .

Examples of allosteric modulators (coactivators, coinhibitors) include:

Both agonists and co-activators bind to a receptor, and increase its level of activation (signal transduction; enzymatic activity; etc.). However, an agonist binds to a ligand binding site which is exposed even in the absence of a co-activator. A co-activator binds a receptor only after an agonist binds the receptor, causing a change in conformation which opens up the co-activator's binding site.

A co-inhibitor competitively inhibits the binding of a co-activator to the co-activator binding site. Unlike an antagonist, it has no effect in the absence of a co- activator.

One may also recognize the possibility of modulators which are co-agonists. These are molecules which, in combination, activate the receptor, but which, acting alone, cannot do so .

-The present invention- may be used to identify modulators (including agonists, antagonists, and coactivators and coinhibitors) of receptors. To identify a coactivator, one must first know or identify an agonist. To identify a co-inhibitor, one must first know or identify a co-activator.

The present invention can be used to identify allosteric modulators of GPCRs by providing a receptor, a G protein interacting with said receptor, and a known agonist at a concentration which activates the receptor at a submaximal level. If test substances are added to the described system, a positive allosteric modulator can be identified as a substance which increases the signal generated by the known agonist, but elicits no signal in the absence of this agonist. A negative allsoteric modulator may be identified by an assay employing the agonist as well as a positive allosteric modulator.

It is not unusual for a relatively small structural change to convert an agonist into a pharmacological antagonist, or vice versa. Therefore, even if the drugs known to interact with a reference protein are all agonists, the drugs in question may serve as leads to the identification of both agonists- and antagonists of the reference protein and of related proteins. Similarly, known antagonists may serve as drug leads, not only to additional antagonists, but to agonists as well.

4. Use of G protein Subunit Conformation-Specific Binding Molecules in Assays for the Detection of the Activation or Inactivation of a GPCR

- The G protein subunit conformation—specific- binding molecules of the present invention may be used in assays for the detection of the activation or inactivation of a

"target" GPCR. The assays may be preliminary screening assays, used to determine whether a compound is an agonist, antagonist, coactivator or coinhibitor of a GPCR, or diagnostic assays, used to evaluate the presence or amount of a known GPCR modulator, or of a known GPCR. The assays may be in vitro or in vivo. If in vivo, they may be cell- based or organismic.

4.1. Preliminary Screening Assays Preliminary screening assays are used to determine the binding, pharmacological or biological activity of a substance of uncertain activity. They will typically be either in vitro (cell-free) assays (for binding to an immobilized receptor) or cell-based assays (for alterations in the phenotype of the cell) . They will not involve screening of whole multicellular organisms, or isolated organs. The comments on biological assays apply mutatis mutandis to preliminary screening cell-based assays.

4.2. Diagnostic Assays

While preliminary screening assays are used to determined the activity of a compound of uncertain activity, diagnostic assays employ a binding molecule of known binding activity, or a conjugate or derivative thereof, as a diagnostic reagent.

4.3. In Vitro vs . In Vivo Assays The term "in vivo" is descriptive of an event, such as binding or enzymatic action, which occurs within a living organism. The organism in question may, however, be genetically modified. The -term -"-in -vitro" refers to an event which occurs outside a living organism. Parts of an organism (e.g., a membrane, or an isolated biochemical) are used, together with artificial substrates and/or conditions. For the purpose of the present invention, the term in vitro excludes events occurring inside or on an intact cell, whether of a unicellular or multicellular organism. In vivo assays include both cell-based assays, and organismic assays. The term cell-based assays includes both assays on unicellular organisms, and assays on isolated cells or cell cultures derived from multicellular organisms. The cell cultures may be mixed, provided that they are not organized into tissues or organs. The term organismic assay refers to assays on whole multicellular organisms, and assays on isolated organs or tissues of such organisms.

"Biological assays" include both in vivo assays, and in vitro assays on subcellular multimolecular components of cells such as membranes.

4.4. Preferred In Vitro Screening Assays 4. 4. 1 . Scintilla tion Proximity Assay (SPA) :

An SPA is a homogeneous assay which relies on the short penetration range in solution of beta particles from certain isotopes, such as ³H, ¹²⁵I, ³P and ³⁵S .

In a competitive SPA, the scintillant (which emits light when a beta particle passes .close by) is conjugated to an analyte binding molecule. The analyte is allowed to compete with a short range beta particle-emitting radiolabeled analyte analogue for binding to the ABM. If the analyte analogue binds, the beta particles emitted by its label come close enough to stimulate the scintillant. Usually, the scintillant is embedded in beads, or in the -walls of the wells of a microtiter plate.

^■ -I-n~ a s-andwieh---SPA, the -scintili-ant-ABM- conj-ug-ate -binds the analyte, and a second radiolabeled ABM also binds the analyte, thereby forming a ternary complex.

There are practical reasons for using, instead of a scintillant-ABM conjugate, a primary simple ABM reagent, and a scintillant- (anti-ABM) conjugate acting as a secondary reagent which binds the primary reagent. The ABM of the primary reagent could then be a mouse monoclonal antibody, and the anti-ABM of the secondary reagent a cheaper polyclonal anti-mouse antibody, usable in assays for different analytes.

4. 4. 2. Fluorescence Polariza tion (FP) :

A method for detection of ligand binding that results in a change of the rotational relaxation time of the fluorescent label reflecting in a change in the total molecular mass of the complex containing the fluorescent ligand. A measurement is taken by excitation of the fluorescent moiety on the ligand by light of the proper wavelength that has passed through a polarizing filter and performing two measurements on the emitted light. The first measurement is performed by passing the light through a polarizing filter that is parallel to the polarization of the excitation polarizer. The second measurement is performed by passing the light through a polarizing filter that is perpendicular to the polarization of the excitation polarizer. The intensities of the emitted light from the parallel and perpendicular measurements are used to determine the polarization of the fluorescent ligand by the following equation mP = [ ( I_parallel - Iperpendicu_lar) / ( Iparallel + Iperpe_ndic_ular) xlOOO] . An increase in mP indicates that more polarized light is being emitted and corresponds to the formation of a complex.

4. 4. 3. Fluorescence Resonance Energy Transfer (FRET) : A method for detection of complex formation, such as ligand-receptor binding, that relies upon the through-space interactions between two fluorescent groups. A fluorescent molecule has a specific wavelength for excitation and another wavelength for emission. Pairs of fluorophores are selected that have an overlapping emission and excitation wavelength. Paired fluorophores are detected by a through- space interaction referred to as resonance energy transfer. When a donor fluorophore is excited by light, it would normally emit light at a higher wavelength; however, during FRET energy is transferred from the donor to the acceptor fluorophore allowing the excited donor to relax to the ground-state without emission of a photon. The acceptor fluorophore becomes excited and release energy by emitting light at its emission wavelength. This means that when a donor and an acceptor fluorophore are held in close proximity (<100 Angstroms) , such as when one fluorophore is attached to a ligand and one is attached to a receptor and the ligand binds to the receptor, excitation of the donor is coupled with emission from the acceptor. Conversely, if no complex is formed the excitation of the donor results in no emission from the acceptor. A common modification of this technique, sometimes referred to as fluorescence quenching, is accomplished using an acceptor group that is not fluorescent but efficiently accepts the energy from the donor fluorophore. In this case, when a complex is formed the excitation of the donor fluorophore is not accompanied by light emission at any wavelength. When this complex is dissociated the excitation of the donor results in emission of light at the wavelength of the donor.

4 -. 4. 4-. Time-Resolved Fluorescence-

The basic fluorescence assays can be modified to increase the signal to noise ratio. If there is a difference in the temporal behavior of signal fluorescence and background fluorescence, then "time-resolved fluorescence" may be used to better distinguish the two. One may measure the decay of the total fluorescence intensity, or the decay of the polarization anisotropy.

In a time-resolved form of a FRET assay, Europium cryptate (EuK) serves as the donor fluorophore. The cryptate protects the europium ion from fluorescence quenching. The acceptor fluorophore is XL665, a modified allophycocyanine. The efficiency of FRET is 50% at a distance of 9 nm in serum, and the emission is at 665 nm. The XL665 emission is measured after a 50 microsec time delay (hence the name) which eliminates background (e.g., from free XL665 not stimulated by EuK) . This is possible because the XL665 emission is relatively long-lived.

Fluorescence assays may be used in both cell-free and cell-based formats. Of course, for cell-based assays, the fluorophore labeled probes must be introduced into the cells in question.

For more information on fluorescence assays, see Szollosi, et al., Comm. Clin. Cytometry, 34:159-179 (1998); Millar, Curr. Op. Struct. Biol., 6:637-42 (1996); Mitra, et al., Gene, 173:13-17 (1996), Alfano, et al., Ann. N.Y. Acad. Sci., 838:14-28 (1998); Lundblad, et al . , Mol. Endocrinol . , 10:607-12 (1996); Gonzalez and Negulescu, Curr. Op. Biotechnology, 9:624-31 (1998). For bioluminescence assays, see Stables, et al., Anal. Biochem., 252:115-126 (1997).

4.5. Preferred In Vitro Assay Protocols

While there are many possible assay formats, certain -preferred- protocols are -described below.

Definitions :

D-peptide: A peptide that binds preferentially to the GDP/Gα protein complex compared to the GTP/Gα or the GTPγS/Gα complex.

T-peptide: A peptide that binds preferentially to the GTP/Gα or the GTPγS/Gα protein complex compared to the GDP/Gα complex.

I-peptide: A peptide that binds to Gα independent of the activation state. Summary of Protocols

Abbrevia tions : SPA, scintillation proximity assay; FP, fluorescence polarization assay; Het, heterogeneous assay (usually with radioactive or fluorescent label) ; Ag, agonist; Antag, antagonist; D, D-peptide; T, T-peptide; (r) , radioactive label; (f) , fluorophore label; (fl) and (f2), fluorophores 1 and 2, respectively, matched for FP or for FRET; T' , labeled T-peptide, label not specified; D' , labeled D-peptide, label not specified; +, signal increases if analyte present; -, signal decreases if analyte present; c, signal is complex. Note that here we are referring to the ultimate analyte (the agonist or antagonist) and not the immediate target of the T-, D- or I-peptide, the G alpha subunit.

Detailed Protocols

Type 1: Scintillation Proximity Assay (SPA)

Protocol 1.1. Detection of agonist binding using a T-peptide

1. Membranes containing the GPCR and G protein to be assayed are attached to the surface of a scintillant plate, such as a FlashPlate (NEN) , or to scintillant beads, such as SPA-beads (Amersham) .

2. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the

Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A radiolabeled T-peptide can then bind to the activated Gα/GTP or Gα/GTPγS complex. 4. The binding of the T-peptide results in production of light by the scintillant that is in close proximity to the membrane/Gα/T-peptide complex. This light is detected by a scintillation counter.

Protocol 1.2. Detection of an antagonist in the presence of an agonist using a T-peptide

1. Membranes containing the GPCR and G protein to be assayed are at-t-ached--to the surface of a- sci-ntillan-t ρla-t-e>- sueh--as a FlashPlate (NEN) , or to scintillant beads, such as SPA-beads (Amersham) .

2. An agonist is added at subsaturating levels to the assay to activate the GPCR, when no antagonist is present, resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A radiolabeled T-peptide can then bind to the activated Gα/GTP or Gα/GTPγS complex.

4. Compounds to be assayed are incubated to allow an antagonist to bind to the GPCR. 5. The binding of the T-peptide results in production of light by the scintillant that is in close proximity to the membrane/Gα/T-peptide complex. This light is detected by a scintillation counter. An antagonist is detected by a reduction in the scintillation signal.

Protocol 1.3. Detection of agonist binding using a D-peptide

1. Membranes containing the GPCR to be assayed are attached to the surface of a scintillant plate, such as a

FlashPlate (NEN) , or to scintillant beads, such as SPA-beads (Amersham) .

2. A radiolabeled D-peptide is bound to the inactive heterotrimeric G protein/GDP complex. 3. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation and the D-peptide is released from the activated Gα/GTP or Gα/GTPγS complex. 4. The presence of an agonist is detected by a decrease in the production of light by the scintillant that is in close proximity to the membrane/heterotrimeric G protein/GDP/D- peptide comple -by a -scintillation - counting.

Protocol 1.4. Detection of an antagonist in the presence of an agonist using a D-peptideProtocol 1.4. Detection of an antagonist in the presence of an agonist using a D-peptide

1. Membranes containing the GPCR to be assayed are attached to the surface of a scintillant plate, such as a FlashPlate (NEN) , or to scintillant beads, such as SPA-beads (Amersham) .

2. A radiolabeled D-peptide is bound to the inactive heterotrimeric G protein/GDP complex.

3. An agonist is added to the assay to activate the GPCR, when no antagonist is present, resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation 4. When no antagonist is present, the D-peptide is released from the activated Gα/GTP or Gα/GTPγΞ complex resulting in a decrease in the production of light by the scintillant that is in close proximity to the membrane/Gα/D- peptide complex as detected by a scintillation counting.

5. Compounds to be assayed are incubated in the well to allow an antagonist to bind to the GPCR.

6. The presence of an antagonist is detected by the increased production of light by the membrane/heterotrimeric G protein/GDP/D-peptide complex.

Type 2: FP Assays

Protocol 2.1. Detection of agonist binding using a T- peptideProtocol 2.1. Detection of agonist binding using a T- peptide

1. Membranes containing the GPCR to be assayed are incubated with possible agonist compounds.

2-. -Activation of the- GPCR y -an- a-g-on-i-st- ^■-re-s lts in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A fluorescently labeled T-peptide can then bind to the activated Gα/GTP or Gα/GTPγS complex. Many fluorescent labels can be used such as, but not limited to, fluorescein, rhodamine, texas red, Cy-3, and oregon green.

4. The binding of the peptide results in polarization of the fluorescent light emitted by the fluorescent peptide in the membrane/Gα/T-peptide complex.

5. An agonist is detected by an increase in the polarization of emitted light.

Protocol 2.2. Detection of antagonist binding using a T- peptide 1. Membranes containing the GPCR to be assayed are incubated with possible antagonist compounds.

2. An agonist is added to produce and activation of the GPCR resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A fluorescently labeled T-peptide can then bind to the activated Gα/GTP or Gα/GTPγS complex.

4. The binding of the peptide results in polarization of the fluorescent light emitted by the fluorescent peptide in the membrane/Gα/T-peptide complex.

5. An antagonist is detected by the lack of polarization of the T-peptide indicating that the Gα was not activated.

Protocol 2.3. Detection of agonist binding using a D-peptide 1. A membrane/heterotrimeric G protein/GDP complex containing the GPCR to be assayed is incubated with possible agonist co p-ounds- and a fluorescently labeled D-peptide -that will bind to to the Gα/GDP complex. 2. The bound peptide will produce emitted light that is polarized.

3. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

4. The fluorescently labeled D-peptide will not bind to the activated Gα/GTP or Gα/GTPγS complex and no longer emits polarized light.

5. An agonist is detected by the loss of polarized light.

Protocol 2.4. Detection of antagonist binding using a D- peptide

1. A membrane/heterotrimeric G protein/GDP complex containing the GPCR to be assayed is incubated with possible antagonist compounds .

2. An agonist is added to activate the GPCR resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. The fluorescently labeled D-peptide that will bind to the Gα/GDP complex is then added to the activated receptor/G protein in membranes. When an antagonist is present, the fluorescently labeled D-peptide is not released from the membrane/heterotrimeric G protein/GDP complex.

4. An antagonist is detected by the production of polarized light indicating that the Gα was not activated.

Protocol 2.5. Detection of agonists using both a D-peptide and T-peptide simultaneouslyProtocol 2.5. Detection of agonists using both a D-peptide and T-peptide simultaneously

1. A membrane/heterotrimeric- - -G proteirr-GDF -complex containing the GPCR to be assayed is incubated with possible agonist compounds, a fluorescently labeled D-peptide that will bind to the Gα/GDP complex and a T-peptide that is labeled with a fluorescent group with excitation and emission wavelengths that are different from the D-peptide label.

2. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the

Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. When an agonist is present, the fluorescently labeled D-peptide is released from the membrane/Gα/GTP complex with the concurrent binding of the T-peptide to the membrane/Gα/GTP complex.

4. An agonist is detected by the loss of polarized light at the emission wavelength of the D-peptide and the gain in polarization at the emission wavelength of the T-peptide.

Protocol 2.6. Simultaneous detection of antagonists using both a D-peptide and T-peptide 1. A membrane/heterotrimeric G protein/GDP complex containing the GPCR to be assayed is incubated with possible antagonist compounds, a fluorescently labeled D-peptide that will bind to the Gα/GDP complex and a T-peptide that is labeled with a fluorescent group with excitation and emission wavelengths that are different from the D-peptide label.

2. An agonist is added to activate the GPCR resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation. 3. When an antagonist is present, the fluorescently labeled D-peptide binds to the membrane/Gα/GDP complex and the T-peptide does not bind to that complex.

4. Antagonists are detected by retention of polarized light at the emission wavelength of the D-peptide and no gain in polarization at the emission wavelength of the T-peptide.

Type 3: Ligand/Surrogate Ligand Binding Assays

This type differs from Type 2 only in the detection method.

Protocol 3.1. Detection of agonist binding using a T-peptide (Figs. 9- 12 and 14-16)

1. Membranes containing GPCR to be assayed are immobilized on the surface of a microtiter plate or immobilized by another method (ie beads) and incubated with possible agonist compounds .

2. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A labeled T-peptide is added and incubated so that it can bind to the activated Gα/GTP or Gα/GTPγS complex, many labels can be used such as, but not limited to, fluorescent moieties, radioactivity, or enzymes such as alkaline phosphatase or β-galactosidase.

4. After incubation of the immobilized membrane/GPCR/Gα complex is washed and the amount of labeled peptide that binds to the plate is quantitated by fluorescence, scintillation counting, luminescence, or spectrophotometry.

5. An agonist is detected by an increase in the quantified signal indicating that the GPCR was activated. (Fig. 9)

Membranes of Sf9 cells infected with the M2 Muscarinic

Acetylcholine Receptor (M2AChR) , G_ai, G_b and G_g were used to

^{• ■}establish detection of agonist -promoted- -n-ucl-eot-ide ^■ e-xehange.

Binding of a G_ai specific peptide in the presence of the agonist carbachol was significantly higher than in its absence and could be inhibited by the receptor specific antagonist atropine, a clear indication that the effect was receptor promoted (Fig. 10) .

In order to investigate the properties of the assay shown in figure 10 under conditions more closely resembling a high throughput situation the assay was adapted to 384 well plates. The time during which the plates with membranes were exposed to room temperature was varied as well as the amount of protein per well. As seen in figure 11 there was only a slight variation in the signal to background ratio when 1 , 3 or 9 mg of membrane were immobilized per well and the results were also quite stable when plates were exposed to room temperature up to 3 hours. This shows that detection of GPCR activation by binding of peptides is a very robust method.

An actual small scale screen was designed in a slightly varied assay format. Instead of preparing membranes whole cells were immobilized in wells and the assay was carried out with the same parameters as described before. The test compounds were taken from a commercially available chemical library which contains many well-known pharmacologically active substances. Members of this collection activate numerous different cellular pathways and there use can therefore be very informative with respect to susceptibility of an assay system to interference. It also contains 12 known M2 Acetylcholine Receptor agonists. All 12 of these substances triggered signals above the cutoff. An additional group of 4 compounds also scored positive (Fig. 12) . These results proved that the assay can truly be used as a highthroughput GPCR assay.

Protocol 3. 2. Detection of antagonist binding using a T—eptide (Figs. 10 and -14)-

1. Membranes containing GPCR to be assayed are immobilized on the surface of a microtiter plate or immobilized by another method (ie beads) and incubated with possible antagonist compounds .

2. After incubation with possible antagonists, an agonist and a labeled T-peptide are added to allow for activation of the GPCR and activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. The labeled T-peptide can then bind to the activated Gα/GTP or Gα/GTPγS complex, many labels can be used such as, but not limited to, fluorescent moieties, radioactivity, or enzymes such as alkaline phosphatase or β-galactosidase.

4. After incubation of the immobilized membrane/GPCR/Gα complex is washed and the amount of labeled peptide that binds to the plate is quantitated by fluorescence, scintillation counting, luminescence, or spectrophotometry.

5. An antagonist is detected by a decrease in the quantified signal indicating that the GPCR was not activated. As described above, the experiments carried out with membranes containing the M2AChR, G_ai and G_b and G_g (Fig. 10) prove that antagonists specifically inhibit_. the signal generated by a specific agonist. This is clear indication that the assay can be used to screen GPCRs for potential antagonists.

Protocol 3.3. Detection of agonist binding using a D-peptide

1. Membranes containing the GPCR to be assayed are immobilized on the surface of a microtiter plate or immobilized by another method (ie. beads) and incubated with possible agonist compounds .

2. Activation of the GPCR by an agonist results in activation o-f- the heterotrimeric -G -protei -to d-i-s-s-oe-i-a-te^■ the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A labeled D-peptide is added and incubated so that it can bind to the inactive heterotrimeric G protein complex, many labels can be used such as, but not limited to, fluorescent moieties, radioactivity, or enzymes such as alkaline phosphatase or β-galactosidase.

4. After incubation of the immobilized membrane/GPCR/Gα complex is washed and the amount of labeled peptide that binds to the plate is quantitated by fluorescence, scintillation counting, luminescence, or spectrophotometry. 5. An agonist is detected by a decrease in the quantified signal indicating that the GPCR was activated.

Protocol 3.4. Detection of antagonist binding using a D- peptide

1. Membranes containing GPCR to be assayed are immobilized on the surface of a microtiter plate or immobilized by another method (ie beads) and incubated with possible antagonist compounds .

2. After incubation with possible antagonists, an agonist and a labeled D-peptide are added to allow for activation of the GPCR and activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A labeled D-peptide is added and incubated so that it can bind to the inactive heterotrimeric G protein complex, many labels can be used such as, but not limited to, fluorescent moieties, radioactivity, or enzymes such as alkaline phosphatase or β-galactosidase.

4. After incubation of the immobilized membrane/GPCR/Gα complex is washed and the amount of labeled peptide that binds te- the -plate is quantitated- by -fluorescence, -scintillation counting, luminescence, or spectrophotometry. 5. An antagonist is detected by an increase in the quantified signal indicating that the GPCR was not activated.

Type 4 : FRET Assays

Protocol 4.1. Detection of agonist binding using a T-peptide and an I-peptide

1. Membranes containing the GPCR to be assayed are incubated with possible agonist compounds.

2. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the G subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A fluorescently labeled T-peptide and a fluorescently labeled I-peptide with fluorophores matched for FRET are added. 4. The T-peptide will bind to the activated Gα/GTP or Gα/GTPγS complex and the I-peptide will bind to the either the Gα/GTP, Gα/GTPγS or Gα/GDP complex.

5. When both peptides are bound to the Gα/GTP or Gα/GTPγS complex excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore.

6. An agonist is detected by an increase in the emission of light from the acceptor fluorophore.

Protocol 4.2. Detection of antagonist binding using a T- peptide and an I-peptide

1. Membranes containing the GPCR to be assayed are incubated with possible antagonist compounds.

2. An agonist is added to produce and activation of the GPCR resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A fluorescently labeled T-peptide and a fluorescently labeled I-peptide with fluorophores matched for FRET are added.

4. The T-peptide will bind to the activated Gα/GTP or Gα/GTPγS complex and the I-peptide will bind to the either the

Gα/GTP, Gα/GTPγS or Gα/GDP complex.

5. When both peptides are bound to the Gα/GTP or Gα/GTPγS complex excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore. 6. An antagonist is detected when no emission of light from the acceptor fluorophore occurs.

Protocol 4.3. Detection of agonist binding using a T-peptide and a Gα/fluorescent protein fusion 1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein containing a Gα/fluorescent protein fusion are incubated with possible agonist compounds.

2. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A T-peptide that is fluorescently labeled with a fluorophore whose excitation or emission wavelength matches the fluorescent protein fused to the Gα for FRET is added.

4. The T-peptide will bind to the activated Gα/GTP or Gα/GTPγS complex.

5. When the T-peptide is bound to the Gα/GTP or Gα/GTPγS complex, excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore.

6. An agonist is detected by an increase in the emission of light from the acceptor fluorophore.

Protocol 4.4. Detection of antagonist binding using a T- peptide and a Gα/fluorescent protein fusion

1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein containing a Gα/fluorescent protein fusion are incubated with possible antagonist compounds. 2. An agonist is added to activate the GPCR resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A fluorescently labeled T-peptide and a fluorescently labeled I-peptide with fluorophores matched for FRET are added.

4. A T-peptide that is fluorescently labeled with a fluorophore whose excitation or emission wavelength matches the fluorescent protein fused to the Gα for FRET is added.

5. The T-peptide will bind to the activated Gα/GTP or Gα/GTPγS complex.

6. When the T-peptide is bound to the Gα/GTP or Gα/GTPγS complex, excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore. 7. An antagonist is detected when no emission of light from the acceptor fluorophore occurs.

Protocol 4.5. Detection of agonist binding using a T-peptide and a biotinylated Gα protein

1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein containing a biotinylated Gα protein

(produced by either in vi tro or in vivo biotinylation methods) are incubated with possible agonist compounds. 2. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the

Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A fluorescently labeled T-peptide and fluorescently labeled streptavidin (can also include neutravidin or avidin) with fluorophores matched for FRET are added.

-4. The T-peptide will bind to the activated Gα/GTP or

Gα/GT-PγS complex and the steptavidin will bind to the biotinylated Gα. 5. When the T-peptide is bound to the Gα/GTP or Gα/GTPγS complex, excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore.

6. An agonist is detected by an increase in the emission of light from the acceptor fluorophore. A variation of this principle is the use of a G_a protein fused to an epitope-tag and an antibody directed at this tag.

The fluorescent moiety exchanging energy with the fluorophore- peptide construct is conjugated to the antibody in this case.

Protocol 4.6. Detection of an antagonist binding using a T- peptide and a biotinylated Gα protein

1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein containing a biotinylated Gα protein (produced by either in vi tro or in vivo biotinylation methods) are incubated with possible antagonist compounds.

2. An agonist is added to activate the GPCR resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A fluorescently labeled T-peptide and fluorescently labeled streptavidin (can also include neutravidin or avidin) with fluorophores matched for FRET are added. 4. The T-peptide will bind to the activated Gα/GTP or

Gα/GTPγS complex.

5. When the T-peptide is bound to the Gα/GTP or Gα/GTPγS complex, excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore. 6. An antagonist is detected when no emission of light from the acceptor fluorophore occurs.

A variation of this principle is the use of a G_a protein fused to an epitope-tag and an antibody directed at this tag.

The fluorescent moiety exchanging energy with the fluorophore- peptide construct is conjugated to the antibody in this case.

Protocol 4.7. Detection of agonist binding using a T-peptide and fluorescent membranes

1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein are labeled with a fluorescent dye and then incubated with possible agonist compounds.

2. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A T-peptide labeled with a fluorophore matched for FRET with the membrane dye is added.

4. The T-peptide will bind to the activated Gα/GTP or Gα/GTPγS complex.

5. When the T-peptide is bound to the Gα/GTP or Gα/GTPγS complex, excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore. 6. An agonist is detected by an increase in the emission of light from the acceptor fluorophore.

Protocol 4.8. Detection of an antagonist binding using a T- peptide and fluorescent membranes 1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein are labeled with a fluorescent dye and then incubated with possible antagonist compounds.

2. An agonist is added to activate the GPCR resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A T-peptide labeled with a fluorophore matched for FRET with the membrane dye is added.

4. The T-peptide will bind to the activated Gα/GTP or Gα/GTPγS complex.

5. When the T-peptide is bound to the Gα/GTP or Gα/GTPγS complex, excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore.

6. An antagonist is detected when no emission of light from the acceptor fluorophore occurs.

Protocol 4.9. Detection of agonist binding using a D-peptide and an I-peptide.

1. Membranes containing the GPCR to be assayed are incubated with possible agonist compounds.

2. A fluorescently labeled D-peptide and a fluorescently labeled I-peptide with fluorophores matched for FRET are added.

3. The D-peptide will bind to the inactive Gα/GDP complex and the I-peptide will bind to the either the Gα/GTP, Gα/GTPγS or Gα/GDP complex.

4. When both peptides are bound to the inactive Gα/GDP complex excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore.

5. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits, conversion to the activated Gα-GTP conformation, displacement of the D-peptide and loss of FRET.

6. An agonist is detected by a decrease in the emission of light from the acceptor fluorophore.

Protocol 4.10. Detection of antagonist binding using a D- peptide and an I-peptide

1. Membranes containing the GPCR to be assayed are incubated with possible antagonist compounds.

-2-. An agonist is added- to produce and activation of the GPCR resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A fluorescently labeled D-peptide and a fluorescently labeled I-peptide with fluorophores matched for FRET are added.

4. The D-peptide will bind to the inactive Gα/GDP complex and the I-peptide will bind to the either the Gα/GTP, Gα/GTPγS or Gα/GDP complex.

5. When both peptides are bound to the inactive Gα/GDP complex excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore. 6. Activation of the GPCR by the agonist results in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits, conversion to the activated Gα-GTP conformation, displacement of the D-peptide and loss of FRET.

7. An antagonist is detected when light is emission from the acceptor fluorophore occurs.

Protocol 4.11. Detection of agonist binding using a D-peptide and a Gα/fluorescent protein fusion

1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein containing a Gα/fluorescent protein fusion are incubated with possible agonist compounds. 2. A D-peptide that is fluorescently labeled with a fluorophore whose excitation or emission wavelength matches the fluorescent protein fused to the Gα for FRET is added.

3. The D-peptide will bind to the inactive Gα/GDP complex. 4. When the D-peptide is bound to the inactive fluorescent-Gα/GDP complex excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore.

5. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits, conversion to the activated Gα-GTP conformation, displacement of the D-peptide and loss of FRET.

6. An agonist is detected by a decrease in the emission of light from the acceptor fluorophore.

Protocol 4.12. Detection of antagonist binding using a D- peptide and a Gα/fluorescent protein fusion

1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein containing a Gα/fluorescent protein fusion are incubated with possible antagonist compounds.

2. An agonist is added to activate the GPCR resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A D-peptide that is fluorescently labeled with a fluorophore whose excitation or emission wavelength matches the fluorescent protein fused to the Gα for FRET is added. 4. The D-peptide will bind to the inactive Gα/GDP complex.

5. When D-peptide is bound to the inactive fluorescent- Gα/GDP complex excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore. 6. Activation of the GPCR by the agonist results in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits, conversion to the activated Gα-GTP conformation, displacement of the D-peptide and loss of FRET. 7. An antagonist is detected when light is emission from the acceptor fluorophore occurs.

Protocol 4.13. Detection of agonist binding using a D-peptide and a biotinylated Gα protein 1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein containing a biotinylated Gα protein (produced by either in vi tro or in vivo biotinylation methods) are incubated with possible agonist compounds.

2. A fluorescently labeled D-peptide and fluorescently labeled streptavidin (can also include neutravidin or avidin) with fluorophores matched for FRET are added.

3. The D-peptide will bind to the inactive Gα/GDP complex.

4. When both peptides are bound to the inactive Gα/GDP complex excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore.

5. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits, conversion to the activated Gα-GTP conformatipn, displacement of the D-peptide and loss of FRET.

6. An agonist is detected by a decrease in the emission of light from the acceptor fluorophore.

A variation of this principle is the use of a G_a protein fused to an epitope-tag and an antibody directed at this tag. The fluorescent moiety exchanging energy with the fluorophore- peptide construct is conjugated to the antibody in this case.

Protocol 4.14. Detection of an antagonist binding using a D- peptide and a biotinylated Gα protein

1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein containing a biotinylated Gα protein (produced by either in vitro or in vivo biotinylation methods) are incubated with possible antagonist compounds.

2. An agonist is added to activate the GPCR resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A fluorescently labeled D-peptide and fluorescently labeled streptavidin (can also include neutravidin or avidin) with fluorophores matched for FRET are added.

4. The D-peptide will bind to the inactive Gα/GDP complex.

5. When both peptides are bound to the inactive Gα/GDP complex excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore.

6. Activation of the GPCR by the agonist results in activation of the heterotrimeric G protein to dissociate the

Gα subunit from the Gβ and Gγ subunits, conversion to the activated Gα-GTP conformation, displacement of the D-peptide and loss of FRET. 7. An antagonist is detected when light is emission from the acceptor fluorophore occurs.

A variation of this principle is the use of a G_a protein fused to an epitope-tag and an antibody directed at this tag. The fluorescent moiety exchanging energy with the fluorophore- peptide construct is conjugated to the antibody in this case.

Protocol 4.15. Detection of agonist binding using a D-peptide and fluorescent membranes 1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein are labeled with a fluorescent dye and then incubated with possible agonist compounds.

2. A D-peptide labeled with a fluorophore matched for FRET with the membrane dye is added. 3. The D-peptide will bind to the inactive Gα/GDP complex.

4. When both peptides are bound to the inactive Gα/GDP complex excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore. 5. Activation of the GPCR by an agonist results in activation of the heterotrimeric G protein to dissociate the

Gα subunit from the Gβ and Gγ subunits, conversion to the activated Gα-GTP conformation, displacement of the D-peptide and loss of FRET. 6. An agonist is detected by a decrease in the emission of light from the acceptor fluorophore.

Protocol 4.16. Detection of an antagonist binding using a D- peptide and fluorescent membranes 1. Membranes containing the GPCR to be assayed and a heterotrimeric G protein are labeled with a fluorescent dye and then incubated with possible antagonist compounds.

2. An agonist is added to activate the GPCR resulting in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits and conversion to the activated Gα-GTP conformation.

3. A D-peptide labeled with a fluorophore matched for FRET with the membrane dye is added.

4. The D-peptide will bind to the inactive Gα/GDP complex.

5. When both peptides are bound to the inactive Gα/GDP complex excitation of the donor fluorophore results in the emission of light from the acceptor fluorophore.

6. Activation of the GPCR by the agonist results in activation of the heterotrimeric G protein to dissociate the Gα subunit from the Gβ and Gγ subunits, conversion to the activated Gα-GTP conformation, displacement of the D-peptide and loss of FRET.

7. An antagonist is detected when light is emission from the acceptor fluorophore occurs.

4.6. Cell-Based Assays In a preferred cell-based assay, the receptor is functionally connected to a signal (biological marker) producing system, which may be endogenous or exogenous to the cell.

4. 6. 1 . "Zero-Hybrid" Systems

In these systems, the binding of a peptide to the target protein results in a screenable or selectable phenotypic change, without resort to fusing the target protein (or a ligand binding moiety thereof) to an endogenous protein. It may be that the target protein is endogenous to the host cell, or is substantially identical to an endogenous receptor so that it can take advantage of the latter' s native signal transduction pathway. Or sufficient elements of the signal transduction pathway normally associated with the target protein may be engineered into the cell so that the cell signals binding to the target protein.

4. 6.2. "One-Hybri d " Systems

In some of these systems, a chimeric receptor, a hybrid of the target protein and an endogenous receptor, is used. The chimeric receptor has the ligand binding characteristics of the target protein and the signal transduction characteristics of the endogenous receptor. Thus, the normal signal transduction pathway of the endogenous receptor is subverted.

Preferably, the endogenous receptor is inactivated, or the conditions of the assay avoid activation of the endogenous receptor, to improve the signal-to-noise ratio. See Fowlkes USP 5,789,184 for a yeast system.

Another type of "one-hybrid" system combines a peptide: DNA-binding domain fusion with an unfused target receptor that possesses an activation domain.

4. 6. 3. "Two-Hybrid" System

In a preferred embodiment, the cell-based assay is a two hybrid system. This term implies that the ligand is incorporated into a first hybrid protein, and the receptor into a second hybrid protein. The first hybrid also comprises component A of a signal generating system, and the second hybrid comprises component B of that system. Components A and B, by themselves, are insufficient to generate a signal. However, if the ligand binds the receptor, components A and B are brought into sufficiently close proximity so that they can cooperate to generate a signal.

Components A and B may naturally occur, or be substantially identical to moieties which naturally occur, as components of a single naturally occurring biomolecule, or they may naturally occur, or be substantially identical to moieties which naturally occur, as separate naturally occurring biomolecules which interact in nature.

4. 6. 3. 1 . Two-Hybrid System : Transcription Factor Type

In a preferred "two-hybrid" embodiment, one member of a peptide ligand: receptor binding pair is expressed as a fusion to a DNA-binding domain (DBD) from a transcription factor (this fusion protein is called the "bait") , and the other is expressed as a fusion to a transactivation domain (TAD) (this fusion protein is called the "fish", the "prey", or the "catch") . The transactivation domain should be complementary to the DNA-binding domain, i.e., it should interact with the latter so as to activate transcription of a specially designed reporter gene that carries a binding site for the DNA-binding domain. Naturally, the two fusion proteins must likewise be complementary.

This complementarity may be achieved by use of the complementary and separable DNA-binding and transcriptional activator domains of a single transcriptional activator protein, or one may use complementary domains derived from different proteins. The domains may be identical to the native domains, or mutants thereof. The assay members may be fused directly to the DBD or TAD, or fused through an intermediated linker.

The target DNA operator may be the native operator sequence, or a mutant operator. Mutations in the operator may be coordinated with mutations in the DBD and the TAD. An example of a suitable transcription activation system is one comprising the DNA-binding domain from the bacterial repressor LexA and the activation domain from the yeast transcription factor Gal4, with the reporter gene operably linked to the LexA operator. It is not necessary to employ the intact target receptor; just the ligand-binding moiety is sufficient.

The two fusion proteins may be expressed from the same or different vectors. Likewise, the activatable reporter gene may be expressed from the same vector as either fusion protein (or both proteins), or from a third vector.

Potential DNA-binding domains include Gal4, LexA, and mutant domains substantially identical to the above.

Potential activation domains include E. coli B42, Gal4 activation domain II, and HSV VP16, and mutant domains substantially identical to the above.

Potential operators include the native operators for the desired activation domain, and mutant domains substantially identical to the native operator. The fusion proteins may comprise nuclear localization signals .

The assay system will include a signal producing system, too. The first element of this system is a reporter gene operably linked to an operator responsive to the DBD and TAD of choice. The expression of this reporter gene will result, directly or indirectly, in a selectable or screenable phenotype

1

(the signal) . The signal producing system may include, besides the reporter gene, additional genetic or biochemical elements which cooperate in the production of the signal. Such an element could be, for example, a selective agent in the cell growth medium. There may be more than one signal producing system, and the system may include more than one reporter gene.

The sensitivity of the system may be adjusted by, e.g., use of competitive inhibitors of any step in the activation or signal production process, increasing or decreasing the number of operators, using a stronger or weaker DBD or TAD, etc.

When the signal is the death or survival of the cell in question, or proliferation or nonproliferation of the cell in question, the assay is said to be a selection. When the signal merely results in a detectable phenotype by which the signaling cell may be differentiated from the same cell in a nonsignaling state (either way being a living cell), the assay is a screen. However, the term "screening assay" may be used in a broader sense to include a selection. When the narrower sense is intended, we will use the term "nonselective screen".

Various screening and selection systems are discussed in Ladner, USP 5,198,346. Screening and selection may be for or against the peptide: target protein or compound: target protein interaction.

Preferred assay cells are microbial (bacterial, yeast, algal, protozooal), invertebrate (esp. mammalian, particularly human) . The best developed two-hybrid assays are yeast and mammalian systems.

Normally, two hybrid assays are used to determined whether a protein X and a protein Y interact, by virtue of their ability to reconstitute the interaction of the DBD and the TAD.

However, augmented two-hybrid assays have been used to detect interactions that depend on a third, non-protein ligand.

For more guidance on two-hybrid assays, see Brent and Finley, Jr., Ann. Rev. Genet., 31:663-704 (1997); Fremont- Racine, et al., Nature Genetics, 277-281 (16 July 1997); Allen, et al., TIBS, 511-16 (Dec. 1995); LeCrenier, et al., BioEssays, 20:1-6 (1998); Xu, et al., Proc. Nat. Acad. sci. (USA), 94:12473-8 (Nov. 1992); Esotak, et al., Mol. Cell. Biol., 15:5820-9 (1995); Yang, et al . , Nucleic Acids Res., 23:1152-6 (1995); Bendixen, et al., Nucleic Acids Res., 22:1778-9 (1994); Fuller, et al., BioTechniques, 25:85-92 (July 1998); Cohen, et al., PNAS (USA) 95:14272-7 (1998); Kolonin and Finley, Jr., PNAS (USA) 95:14266-71 (1998). See also Vasavada, et al., PNAS (USA), 88:10686-90 (1991) (contingent replication assay), and Rehrauer, et al . , J. Biol. Chem., 271:23865-73 91996) (LexA repressor cleavage assay) .

4. 6. 3.2. Two-Hybrid Systems : Reporter Enzyme type

In another embodiment, the components A and B reconstitute an enzyme which is not a transcription factor. It may, for example, be DHFR, or one of the other enzymes identified in WO98/34120.

As in the last example, the effect of the reconstitution of the enzyme is a phenotypic change which may be a screenable change, a selectable change, or both.

Universite de Montreal, WO98/34120 describes the use of protein-fragment complementation assays to detect biomolecular interactions in vivo and in vitro. Fusion peptides respectively comprising N and C terminal fragments of murine DHFR were fused to GCN4 leucine zipper sequences and co- expressed in bacterial cells whose endogenous DHFR activity was inhibited. DHFR is composed of three structural- fragments forming two domains; the discontinuous 1-46 and 106-186 fragments form one domain and the 47-105 fragment forms the other. WO98/34120 cleaved DHFR at residue 107. GCN4 is a homodimerizing protein. The homodi erization of GCN4 causes reassociation of the two DHFR domains and hence reconstitution of DHFR activity.

WO98/34120 suggest that fragments of other enzyme reporter molecules could be used in place of DHFR.

See also, Pelletier, et al . , Proc. Nat. Acad. Sci. USA, 95: 12141-6 (1998) (same system);

Karimova et al., Proc. Nat. Acad. Sci. USA 95:5752-6

(1998) discloses a bacterial two-hybrid system, in which the catalytic domain of Bordetella pertussis adenylate cyclase reconstituted as a result of interaction of two proteins, leading to cAMP synthesis) . 4. 6. 3. 3. Two Hybrid Systems : Miscellaneous

In a similar system, designed to distinguish heterodimerization as distinct from homodimerization, one test protein was fused to native LexA and the other to a mutant of LexA with altered DNA specificity. Normally, LexA dimerizes to bind its target operator. Because of the mutation, and the use of a hybrid operator, only a heterodimer could achieve DNA binding. See Dmitrova, et al., Mol. Gen. Genet., 57: 205-212 (1998) . Stanford U., WO98/44350 describes a reporter subunit complementation assay which employs fusion proteins each compromising one of a pair of weakly complementing, singly inactive, beta galactosidase mutants, which complement each other to produce an active beta galactosidase. See also Rossi, et al., Proc. Nat. Acad. Sci. USA, 94:8405-10 (1997); Mohler and Blau, Proc. Nat. Acad. Sci. USA, 93: 12423-7 (1996).

Cornell U., W098/34948 describes a strategy for the identification of small peptides that activate or inactivate a G protein coupled receptor. The peptides of a combinatorial peptide library are tethered to a GPCR of interest in a cell, and the cell is monitored to determine whether the peptide is an agonist or an antagonist. The peptide is tethered to the GPCR by replacing the N-terminal of the GPCR with the N- terminus of a self-activating receptor, and replacing the natural peptide ligand present therein with the library peptide. An example of a self-activating receptor would be the thrombin receptor.

Sadee, USP 5,882,944 discloses a cell-based assay for the effect of test compounds on ml receptors in which the cells are incubated with an ml agonist to constitutively activate them, the agonist is removed, the baseline activity of the receptor is determined, the cells are exposed to the test compound, and the receptor activity is compared to the baseline level. The activity measured may be directed to cAMP, GTPase, or GTP exchange.

Martin, et al., J. Biol. Chem., 271: 361-6 (1996) describes the screening of a combinatorial peptide-on-plasmid library based on the C terminus of the alpha subunit of Gsubt (340-350) for peptides which bind rhodopsin. In the library, the library peptides are fused to the C terminus of the DNA binding protein lad, which binds to lacO DNA sequences on the vector expressing the peptide. In the random DNA, the base mix was chosen so as to yield roughly a 50% chance that a given codon would be mutated to yield a different amino acid.

Stables, et al., Anal. Biochem., 252: 115-126 (1997) describes a cell-based bioluminescent assay for GPCR agonist activity. The GPCR is co-expressed with apoaequorin, a calcium-sensitive photoprotein. Agonist binding to a receptor which activates certain G-alpha subunits, such as G-alphal6, results in an increase in intracellular calcium concentration and subsequent bioluminescence.

4.7. Use of GDP and GTP Analogues

If desired, the assay may employ a GTP or GDP analogue in place of GTP or GDP. The analogue must be able to fulfill the functional role of GTP or GDP, respectively, in the GPCR/G protein signal transduction system. Either the analogue must be recognized by the BioKey peptide specific for the wild-type GTP or GDP, as appropriate, or a complementary BioKey specific for the analogue must be developed and used. A list of GTP analogues follows: Guanosine 5 ' -0- (3-thiotriphosphate) /GTP gamma S

Guanyl-5 ' -ylimidodiphosphate (GMPPNP) Guanyl-5 ' -yl ethylenediphosphate Guano s ine 5 ' - [ bet a , gamma - imido] triphosphate

Guanosine 5 ' - [gamma-thio] triphosphate

2 ' , 3 ' -dideoxyguanosine 5' triphosphate (ddGTP)

2' ,3'-dialdehyde GTP (oGTP) gaunyl-5-yl imidodiphosphate (GppNHp) cytidylyl (5 ' -3 ' ) guanosine 5 ' -triphosphate (pppGpc) There are GDP analogues, too:

* GDPbS (Guanosine 5 ' - [b-thio] diphosphate)

* Periodate oxidized GDP (Guanosine 5*- diphosphate-2*, 3*-dialdehyde)

* GDP-Mannose * GDP-Fucose

* GDP-Glucose

The following 5 compounds were synthesized in an attempt to obtain nonhydrolyzable GDP analogues for the analysis of transducin function (J. Org. Chem., 1998, 63, 7244-7257) * 9-[5> >-0- (Malonyl) -b-D-ribofuranosyl] guanine

* 9- [5> ^λ >-0- ( Phosphonoacetyl ) -b-D- ribofuranosyl] guanine

* 9-{5> ^λ>-0- [ (Carboxymethyl) dhdroxyphoshinyl] -b- D-ribof uranosyl } guanine * 9-[5> ^λ>-0- (Methylenebisphoshonate) -b-D- ribofuranosyl] -guanine 9- [5> ^Λ>-0- (Imidodiphosphate) -b-D-ribofuranosyl] guanine 4.8. Use of Peptides in Conjunction with Chimeric Gα Subunits

4. 8. 1 . Chimeric Subtypes

The G.α-specific peptide probes, besides being used to detect activation or inactivation of a GPCR which couples in nature to G_{ι r} may also be used, in conjunction with a chimeric

Gα subunit, to detect activation or inactivation of a GPCR which couples in nature to a different G protein, such as G_q.

In essence, the chimeric subunit must be structured so it is recognized by the G_Lα-specific peptide but couples to the GPCR specific for a Gα other than G₁α. In general, this will necessitate modification (replacement or substitution) of one or more amino acids of the carboxy terminal region of the G_xα subunit. In a preferred embodiment, that region is replaced by the corresponding region of the alternative Gα subunit that has the desired coupling characteristic.

According to Sprang, Ann. Rev. Biochem. 66:639 (1997), Table 1, there are five conserved nucleotide binding motifs (G- 1 to G-5) recognizable by comparison of human Ras, Ra-IA, Ran, and ARF-1A, E. coli EF-Tu, T. thermophilus EF-G, bovine Gsα, Giα, Gtα, and Goα, human Gzα, and mouse Gqα. In general, it is desirable that the modified carboxy terminal region lie downstream of the G-5 motif identified by Sprang.

In the case of Giα, this motif is the THFTCAT at residues 321-7. The corresponding Gsα and Gqα are PHFTCAV (361-7) and SHFTCAT (320-6), respectively.

The area of high homology actually extends downstream of the Sprang G-5 motif, as shown in Table NC. Using the numbering of Giα, it extends to the residue 24^th from the C- terminal of Giα. Hence, it is especially preferred that the modified region be not more than the last 23 amino acids of the Giα.

The C-terminal modification may be combined with an N- terminal modification. Sprang' s G-l motif is the GAGESGKS (40- 47) for bovine Giα. Preferably, any N-terminal modification does not extend as far as the G-l motif. More preferably, it also avoids the highly conserved region of five amino acids (usually KLLLL or KILLL) upstream of the G-l motif. As shown in Table NC, this means that it is desirable to limit the modification to the first 34 amino acids of Gilα.

4. 8. 1 . 1 . Gq-Coupled Chimeric Ga Subuni ts

In these embodiments, the chimeric subunit is structured so it is recognized by the Giα-specific peptide but couples to the G_q-specific GPCR.

In certain of the preferred chimera of the present invention, the carboxy terminus of the chimeric Gα subunit is derived from a G_qα, whereas the backbone is derived from a G_±α subunit. We will call these "i/i/q chimera", the first "i" denoting a Gjα-derived amino terminal.

In other preferred chimera of the present invention, both the amino- and carboxy- termini of the chimeric Gα subunit are derived from a G_qα, while the backbone is derived from a Gi , subunit. We will call these "q/i/q chimera".

If a foreign amino terminal is provided, it preferably corresponds to residues 1 to i of a Gqα subunit, where i is in the range of 6 to 40.

If the amino terminal of the G_t L subunit is truncated, the truncation is preferably of residues 1 to j_, where j_ is in the range of 6 to 34.

There may be both a foreign amino terminal and a truncation of the old one, and, if so, i may, but need not, equal ±. In the contemplated chimeric subunits, the C-terminal is modified. The truncation of the old Gi subunit is of the last m residues, where m is in the range of 6 to 23. Also, a new, Gα-derived terminal is provided, corresponding to the last n residues of a G_qα subunit, where n is in the range of 6 to 23. Values of about 6, 13 and 23, for each of m and n, are especially preferred, Normally, n will equal m.

The "corresponding" sequence may be absolutely identical to the sequence from which it is derived, or substantially but less than perfectly identical. In the latter case, it is more similar to G_q sequence than to the equivalent G_A sequence.

Human G_uα has the amino acid sequence set forth in SWISS- PROT entry P04898.

Human G_qα has the amino acid sequence set forth in SWISS- PROT entry P50148.

The following i/q chimeric Gα subunits are of particular interest:

#211 (i/i/q) a.a. 1-348 from human G_nα (348 a.a.) a.a. 354-359 from human G_qα (6 a.a.) total length 354 a.a.

#175 (q/i/i) a.a. 1-6 from human G_qα (6 a.a.) a.a. 1-354 from human G_uα (354 a.a.) total length 360 a.a.

(#175 was constructed as a control; it has only an N-terminal modification)

#176 (q/i/q) a.a. 1-6 from human G_qα (6 a.a.) a.a. 1-348 from human G_±1α (348 a.a.) a.a. 354-359 from human G_qα (6 a.a.) total length: 360 a.a. il-13q (#450) a.a. 1-341 from human G α (341 a.a.) a.a. 347-359 from human G_qα (13 a.a.) total length 354 a.a.

il-23q (#451) a.a. 1-331 from human G_ixα (331 a.a.) a.a. 337-359 from human G_qα (23 a.a.) total length 354 a.a.

iN40C13q (#452) a.a. 1-40 from human G_qα (40 a.a.) a.a. 35-341 from human G_uα (307 a.a.) a.a. 347-359 from human G_qα (13 a.a.) total length 360 a.a.

iN40c23q (#453) a.a. 1-40 from human G_qα (40 a.a.) a.a. 35-331 from human G_uα (297 a.a.) a.a. 337-359 from human G_qα (23 a.a.) total length: 360 a.a.

The preferred probe is peptide 1755,

4.8.1.2. Gs-Coupled Chimeric G Subunits

In a like manner, one may construct chimeras of the Gia? and Gscr subunits. i6s chimera: The last 6 residues in G_αil (KDCGLF, SEQ ID NO: 134)) were replaced with the last 6 residues in G_αs (RQYELL

SEQ ID NO:135)): aa 1-348 from G_αil and aa 349-354 are 389-394 in G_αsL. This i/s chimera was superior to any of the i/q chimeras . 4. 8.2. Cross-Species Chimeric Subunits

Because mammalian GPCRs are of particular interest, but mammalian cells are relatively difficult to maintain in culture, there has been some interest in developing yeast cell- based assays for modulators of mammalian GPCRs, or indeed of any GPCR which is exogenous to the wild-type yeast cell. This receptor may be a plant or animal cell receptor. Screening for binding to plant cell receptors may be useful in the development of, e.g., herbicides. In the case of an animal receptor, it may be of invertebrate or vertebrate origin. If an invertebrate receptor, an insect receptor is preferred, and would facilitate development of insecticides. The receptor may also be a vertebrate, more preferably a mammalian, still more preferably a human, receptor. The exogenous receptor is also preferably a seven transmembrane segment receptor.

It is possible that the endogenous yeast Gα subunit (e.g., GPA) will be sufficiently homologous to the "cognate" Gα subunit which is natively associated with the exogenous receptor for coupling to occur. More likely, it will be desirable to genetically engineer the yeast cell to produce a foreign Gα subunit which can properly interact with the exogenous receptor. For example, the Gα subunit of a yeast G protein may be replaced by a Gα subunit natively associated with the exogenous receptor (or with a mutant of said subunit) . Dietzel and Kurjan, Cell, 50:1001 (1987) demonstrated that rat Gas functionally coupled to the yeast Gβγ complex. However, rat Gαi2 complemented only when substantially overexpressed, while GαO did not complement at all. Kang, et al., Mol. Cell. Biol., 10:2582 (1990). Consequently, with some foreign Gα subunits, it is not feasible to simply replace the yeast Gα. If the exogenous G protein coupled receptor is not adequately coupled to yeast Gβγ by the Gα subunit natively associated with the receptor, the Gα subunit may be modified to improve coupling. These modifications often will take the form of mutations which increase the resemblance of the Gα subunit to the yeast Gα while decreasing its resemblance to the receptor-associated Gα. For example, a residue may be changed so as to become identical to the corresponding yeast Gα residue, or to at least belong to the same exchange group of that residue. After modification, the modified Gα subunit might or might not be "substantially homologous" to the foreign and/or the yeast Gα subunit. The modifications are preferably concentrated in regions of the Gα which are likely to be involved in Gβγ binding.

In some embodiments, the modifications will take the form of replacing one or more segments of the receptor-associated Gα with the corresponding yeast Gα segment (s), thereby forming a chimeric Gα subunit. (For the purpose of the appended claims, the term "segment" refers to three or more consecutive amino acids.) In other embodiments, point mutations may be sufficient. This chimeric Gα subunit will interact with the exogenous receptor and the yeast Gβγ complex, thereby permitting signal transduction. While use of the endogenous yeast Gβγ is preferred, if a foreign or chimeric Gβγ is capable of transducing the signal to the yeast effector, it may be used instead. See more generally Fowlkes, USP 5,789,184.

It is conceivable that a foreign receptor which is expressed in yeast will functionally integrate into the yeast membrane, and there interact with the endogenous yeast G protein. More likely, either the receptor will need to be modified (e.g., by replacing its V-VI loop with that of the yeast STE2 or STE3 receptor) , or a compatible G protein should be provided. If the wild-type exogenous G protein-coupled receptor cannot be made functional in yeast, it may be mutated for this purpose. A comparison would be made of the amino acid sequences of the exogenous receptor and of the yeast receptors, and regions of high and low homology identified. Trial mutations would then be made to distinguish regions involved in ligand or G protein binding, from those necessary for functional integration in the membrane. The exogenous receptor would then be mutated in the latter region to more closely resemble the yeast receptor, until functional integration was achieved. If this were insufficient to achieve functionality, mutations would next be made in the regions involved in G protein binding. Mutations would be made in regions involved in ligand binding only as a last resort, and then an effort would be made to preserve ligand binding by making conservative substitutions whenever possible.

Preferably, the yeast genome is modified so -that it is unable to produce the endogenous a- and α-factor receptors in functional form. Otherwise, a positive assay score might reflect the ability of a peptide to activate the endogenous G protein-coupled receptor, and not the receptor of interest.

For further information of how to design a chimeric subunit, please see the discussion of "chimeric proteins" in Serial. No. 09/429,331.

5. Substances Testable as Modulators of GPCR Activity 5.1. Substances

A "substance" may be either a pure compound, or a mixture of compounds. Preferably it is at least substantially pure, that is, sufficiently pure enough to be acceptable for clinical use. If it is a mixture, then it comprises at least an effective amount (i.e., able to give rise to a detectable biological response in a biological assay) of a biologically active compound, or it comprises a substantial amount of a compound which is suspected, of being biologically active and is suitable as a drug lead if so active.

The preferred sources of the substances to be screened for activity in the assay may be divided into three main groups :

( l) Noncombinatorial synthetic libraries, consisting of compounds that were synthesised using conventional chemical synthesis approaches in small numbers (less than 50 at a time, and most often individually) . The compounds are typically at least 10% pure .

( 2 ) Combina torial libraries, collections of compounds synthesized using techniques of combinatorial synthesis where large number of related compounds are synthesized in parallel.

( 3 ) Na tural product libraries, consisting of extracts from natural sources such as plants, fermentation broths, fungi for example. In some cases these are crude extracts containing thousands of components, and in others the crude extract is fractionated into partially or fully purified components. In each case the chemical entities being searched for are small molecules .

The compounds that can be screened include peptides or molecules related to peptides. Many receptors have peptides as their natural ligands and thus synthetic and naturally occurring collections of peptides or peptide-like molecules are good choices to be screened for molecules which modulate the activity of receptors. In addition, other naturally occurring cellualar components (lipids, sugars, polysaccharides, amino acids, proteins, nucleotides and nucleic acids) have been shown to be ligands for receptors and any collection of samples which contain these components may be used to screen for receptor modulators.

5.2. Test substances and Drug Leads

A test substance comprises an effective amount of a compound which is not already known to have the pharmacological activity of interest. Preferably, it is a member of a structural class which is generally suitable, in terms of physical characteristics (e.g., solubility), as a source of drugs. A test substance may be a member of a combinatorial library, especially one of small organic compounds.

A drug lead is a former test substance which has either been predicted to have desirable pharmacological activity, or in fact has been shown to have such activity, and which therefore could serve effectively as a starting point for the design of analogues and derivatives which are useful as drugs. The "drug lead" may be a useful drug in its own right, or it may be a substance which is deficient as a drug because of inadequate potency or undesirable side effects. In the latter case, analogues and derivatives are sought which overcome these deficiencies. In the former case, one seeks to improve the already useful drug.

Preferably, a drug lead is a compound with a molecular weight of less than 1,000, more preferably, less than 750, still more preferably, less than 600, most preferably, less than 500. Preferably, it has a computed log octanol-water partition coefficient in the range of -4 to +14, more preferably, -2 to +7.5. Analogues and derivatives of a drug bead may be identified by rational drug design, or by screening of combinatorial or noncombinatorial libraries of analogues and derivatives.

A small organic compound library is a library of compounds each of which has a molecular weight of less than 1000, and which are not peptides or nucleic acids.

The compounds screened may be (1) ones which were publicly known at the time of filing, (2) compounds not publicly known at the time of filing, but analogues of compounds so known, or (3) compounds which satisfy neither (1) nor (2) above. For the definition of "analogues", see §8.3 below. Publicly known compounds may be identified by review of suitable databases, such as chemical Abstracts database, and patent databases.

5.3. Potency

The potency of an antagonist of a receptor may be expressed as an IC50, the concentration of the antagonist which causes a 50% inhibition of a receptor's binding or biological activity in an in vitro or in vivo assay system. A pharmaceutically effective dosage of an antagonist depends on both the IC50- of the antagonist, and the effective concentrations of the receptor and its clinically significant binding partner (s) . Potencies may be categorized as follows: Category IC50

Very Weak >1 i moles

Weak 100 n moles to 1 i mole

Moderate 10 n moles to 100 n moles Strong 1 p mole to 10 n moles

Very Strong <1 p mole

Preferably, the antagonists identified by the present invention are in one of the four higher categories identified above.

Preferably, the antagonists are more potent than any antagonist known for the protein in question at the time of filing of this application. In a similar manner, the potency of an agonist may be quantified as the dosage resulting in 50% of its maximal effect on a receptor, and categorized as shown above for antagonists. The preferred agonists are analogous.

5.4. Other Pharmacological Properties

Besides potency, other relevant pharmacological properties of an agonist or antagonist of a receptor include toxicity, solubility, and stability. A drug may be preferred to one of higher potency because it is less toxic, has a longer half- life, etc.

6. Uses of Modulators

Once a GPCR modulator is identified by the assays disclosed herein, it may be put to use, i.e., as a diagnostic or therapeutic agent, or in purification.

The G protein subunit conformation-specific binding molecules may also be used as diagnostic or therapeutic agents, or in purification, and this section applies to them, too, mutatis mutandis.

6.1. In Vitro Diagnostic Use

A labeled or insolubilized GPCR modulator may be used in an assay to detect or quantify (1) the corresponding GPCR, (2) the corresponding modulator, or (3) another modulator, in a sample.

6. 1. 1 . Samples

The sample may be any material suspected of containing the analyte, or known to contain it but in an amount which is unknown and therefore to be quantified by the assay. It will normally be a biological fluid, such as blood, urine, lymph, semen, milk, or cerebrospinal fluid, or a fraction or derivative thereof, or a biological tissue, in the form of,^' e.g., a tissue section or homogenate. However, the sample conceivably could be (or derived from) a food or beverage, a pharmaceutical or diagnostic composition, soil, or surface or ground water. If a biological fluid or tissue, it may be taken from a human or other mammal, vertebrate or animal, or from a plant. The preferred sample is blood, or a fraction or derivative thereof.

6. 1 .2. In Vi tro Assay Methods and Reagents

In vitro assays may be diagnostic assays (using a known binding molecule to detect or measure an analyte) or screening assays (determining whether a potential binding molecule in fact binds a target) . The format of these two types of assays is very similar and, while the description below refers to diagnostic assays for analytes, it applies, mutatis mutandis, to the screening of molecules for binding to targets. The in vitro assays of the present invention may be applied to any suitable analyte-containing sample, and may be qualitative or quantitative in nature. In order to detect the presence, or measure the amount, of an analyte, the assay must provide for a signal producing system (SPS) in which there is a detectable difference in the signal produced, depending on whether the analyte is present or absent (or, in a quantitative assay, on the amount of the analyte) . The detectable signal may be one which is visually detectable, or one detectable only with instruments. Possible signals include production of colored or luminescent products, alteration of the characteristics

(including amplitude or polarization) of absorption or emission of radiation by an assay component or product, and precipitation or agglutination of a component or product. The term "signal" is intended to include the discontinuance of an existing signal, or a change in the rate of change of an observable parameter, rather than a change in its absolute value. The signal may be monitored manually or automatically. A combination of two or more signals may itself be a signal. The combination may be boolean, arithmetic (e.g., sum, difference, multiplicand, ratio) , or vector (a point in an n- dimensional space, where n is the number of raw signals) . An arithmetic combination may be linear or nonlinear.

The component of the signal producing system which is most intimately associated with the diagnostic reagent is called the "label". A label may be, e.g., a radioisotope, a fluorophore, an enzyme, a co-enzyme, an enzyme substrate, an electron-dense compound, or an agglutinable particle. One form of diagnostic reagent is a conjugate, direct or indirect, covalent or noncovalent, of a label with a binding molecule of the invention.

The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. Isotopes which are particularly useful for the purpose of the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S, ¹C, and, preferably, ¹²⁵I.

It is also possible to label a compound with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine.

Alternatively, fluorescence-emitting metals such as ¹²⁵Eu, or others of the lanthanide series, may be attached to the binding protein using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) of ethylenediamine- tetraacetic acid (EDTA) .

The binding molecules also can be detectably labeled by coupling to a chemiluminescent compound. The presence of the chemiluminescent compound is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction after a suitable reactant is provided. Examples of particularly useful chemiluminescent labeling compounds are luminol, isolumino, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the binding molecule. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

Enzyme labels, such as horseradish peroxidase and alkaline phosphatase, are preferred. When an enzyme label is used, the signal producing system must also include a substrate for the enzyme. If the enzymatic reaction product is not itself detectable, the SPS will include one or more additional reactants so that a detectable product appears.

Assays may be divided into two basic types, heterogeneous and homogeneous. In heterogeneous assays, the interaction between the affinity molecule and the analyte does not affect the signal provided by the label, hence, to determine the amount or presence of analyte, bound label must be separated from free label. In homogeneous assays, analyte levels can be deduced without the need for a separation step.

In some homogeneous assays, the interaction of the affinity molecule and the analyte affects the signal provided by the label. For example, in a fluorescent polarization assay, the signal is the amount of polarized light emitted by the fluorophore, which changes when a complex is formed between the affinity molecule and the analyte. (Or, the signal is simply the change in the amount of polarized light emitted.)

In others, two labels are used which, when brought into proximity, interact, resulting in a new or altered signal. In the simplest form of this kind of "interaction" assay, one label is attached to the affinity molecule and the other to the target. More often, two affinity molecules, capable of simultaneous binding to the same target, are used. Each binding molecule is labeled, and the labels are chosen so that they interact when brought into proximity through binding to the target. The fluorescence resonance energy transfer (FRET) assay is an interaction assay. Naturally, it is possible for binding to the target to be indirect, that is, a binding molecule may bind an intermediate reagent which in turn itself binds the target.

In general, a target-binding molecule of the present invention may be used diagnostically in the same way that a target-binding antibody is used. Thus, depending on the assay format, it may be used to assay the target, or by competitive inhibition, other substances which bind the target.

In one embodiment, the binding molecule is insolubilized by coupling it to a macromolecular support, and target in the sample is allowed to compete with a known quantity of a labeled or specifically labelable target analogue. (The conjugate of the binding molecule to a macromolecular support is another diagnostic agent within the present invention.) The "target analogue" is a molecule capable of competing with target for binding to the binding molecule, and the term is intended to include target itself. It may be labeled already, or it may be labeled subsequently by specifically binding the label to a moiety differentiating the target analogue from authentic target. The solid and liquid phases are separated, and the labeled target analogue in one phase is quantified. The higher the level of target analogue in the solid phase, i.e., sticking to the binding molecule, the, lower the level of target analyte in the sample.

In a "sandwich assay", both an insolubilized target- binding molecule, and a labeled target-binding molecule are employed. The target analyte is captured by the insolubilized target-binding molecule and is tagged by the labeled target- binding molecule, forming a tertiary complex. The reagents may be added to the sample in either order, or simultaneously. The target-binding molecules may be the same or different, and only one need be a target-binding molecule according to the present invention (the other may be, e.g., an antibody or a specific binding fragment thereof) . The amount of labeled target- binding molecule in the tertiary complex is directly proportional to the amount of target analyte in the sample.

The two embodiments described above are both heterogeneous assays. However, homogeneous assays are conceivable. The key is that the signal be affected by whether or not the complex is formed. A label may be conjugated, directly or indirectly (e.g., through a labeled anti-target-binding molecule antibody) , covalently (e.g., with SPDP) or noncovalently, to the target- binding molecule, to produce a diagnostic reagent. Similarly, the target binding molecule may be conjugated to a solid-phase support to form a solid phase ("capture") diagnostic reagent. Suitable supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to its target. Thus the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or. the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc.

6.2. Pharmaceutical Methods and Preparations

The preferred animal subject of the present invention is a mammal. By the term "mammal" is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects, although it is intended for veterinary uses as well. Preferred nonhuman subjects are of the orders Primata (e.g., apes and monkeys), Artiodactyla or Perissodactyla (e.g., cows, pigs, sheep, horses, goats), Carnivora (e.g., cats, dogs), Rodenta (e.g., rats, mice, guinea pigs, hamsters), Lagomorpha (e.g., rabbits) or other pet, farm or laboratory mammals.

The term "protection", as used herein, is intended to include "prevention," "suppression" and "treatment." "Prevention" involves administration of the protein prior to the induction of the disease (or other adverse clinical condition) . "Suppression" involves administration of the composition prior to the clinical appearance of the disease. "Treatment" involves administration of the protective composition after the appearance of the disease. Protection, including prevention, need not be absolute.

It will be understood that in human and veterinary medicine, it is not always possible to distinguish between "preventing" and "suppressing" since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, it is common to use the term "prophylaxis" as distinct from "treatment" to encompass both "preventing" and "suppressing" as defined herein. The term "protection," as used herein, is meant to include "prophylaxis." It should also be understood that to be useful, the protection provided need not be absolute, provided that it ^■ is sufficient to carry clinical value. An agent which provides protection to a lesser degree than do competitive agents may still be of value if the other agents. are ineffective for a particular individual, if it can be used in combination with other agents to enhance the level of protection, or if it is safer than competitive agents. The drug may provide a curative effect, an ameliorative effect, or both.

At least one of the drugs of the present invention may be administered, by any means that achieve their intended purpose, to protect a subject against a disease or other adverse condition. The form of administration may be systemic or topical. For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. Parenteral administration can be by bolus injection or by gradual perfusion over time.

A typical regimen comprises administration of an effective amount of the drug, administered over a period ranging from a single dose, to dosing over a period of hours, days, weeks, months, or years.

In general, for a human adult, the dose of a drug will usually be in the range of 10 micrograms to 10 grams per day. It is understood that the suitable dosage of a drug of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. However, the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This will typically involve adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight. Prior to use in humans, a drug will first be evaluated for safety and efficacy in laboratory animals. In human clinical studies, one would begin with a dose expected to be safe in humans, based on the preclinical data for the drug in question, and on customary doses for analogous drugs (if any) . If this dose is effective, the dosage may be decreased, to determine the minimum effective dose, if desired. If this dose is ineffective, it will be cautiously increased, with the patients monitored for signs of side effects. See, e.g., Berkow et al, eds., The Merck Manual, 15th edition, Merck and Co., Rahway, N.J., 1987; Goodman et al., eds., Goodman and Gilman ' s The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery 's Drug Treatment : Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, MD. (1987), Ebadi, Pharmacology, Little,

Brown and Co., Boston, (1985), which references and references cited therein, are entirely incorporated herein by reference.

The total dose required for each treatment may be administered by multiple doses or in a single dose. The protein may be administered alone or in conjunction with other therapeutics directed to the disease or directed to other symptoms thereof.

The appropriate dosage form will depend on the disease, the protein, and the mode of administration; possibilities include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments and parenteral depots. See, e.g., Berker, supra, Goodman, supra , Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, including all references cited therein.

In the case of peptide drugs, the drug may be administered in the form of an expression vector comprising a nucleic acid encoding the peptide, such a vector, after in corporation into the genetic complement of a cell of the patient, directs synthesis of the peptide. Suitable vectors include genetically engineered poxviruses (vaccinia) , aden.oviruses, adeno- associated viruses, herpesviruses and lentiviruses which are or have been rendered nonpathogenic. In addition to at least one drug as described herein, a pharmaceutical composition may contain suitable pharmaceutically acceptable carriers, such as excipients, carriers and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. See, e.g., Berker, supra, Goodman, supra , Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, included ail references cited therein.

6.3. In Vivo Diagnostic Uses Analyte-binding molecules can be used for in vivo imaging.

Radio-labelled binding molecule may be administered to the human or animal subject. Administration is typically by injection, e.g., intravenous or arterial or other means of administration in a quantity sufficient to permit subsequent dynamic and/or static imaging using suitable radio-detecting devices. The preferred dosage is the smallest amount capable of providing a diagnostically effective image, and may be determined by means conventional in the art, using known radio- imaging agents as a guide. Typically, the imaging is carried out on the whole body of the subject, or on that portion of the body or organ relevant to the condition or disease under study. The radio- labelled binding molecule has accumulated. The amount of radio-labelled binding molecule accumulated at a given point in time in relevant target organs can then be quantified.

A particularly suitable radio-detecting device is a scintillation camera, such as a gamma camera. A scintillation camera is a stationary device that can be used to image distribution of radio-labelled binding molecule. The detection device in the camera senses the radioactive decay, the distribution of which can be recorded. Data produced by the imaging system can be digitized. The digitized information can be analyzed over time discontinuously or continuously. The digitized data can be processed to produce images, called frames, of the pattern of uptake of the radio-labelled binding protein in the target organ at a discrete point in time. In most continuous (dynamic) studies, quantitative data is obtained by observing changes in distributions of radioactive decay in target organs over time. In other words, a time- activity analysis of the data will illustrate uptake through clearance of the radio-labelled binding molecule by the target organs with time. Various factors should be taken into consideration in selecting an appropriate radioisotope. The radioisotope must be selected with a view to obtaining good quality resolution upon imaging, should be safe for diagnostic use in humans and animals, and should preferably have a short physical half-life so as to decrease the amount of radiation received by the body. The radioisotope used should preferably be pharmacologically inert, and, in the quantities administered, should not have any substantial physiological effect.

The binding molecule may be radio-labelled with different isotopes of iodine, for example ¹²³I, ¹²⁵I, or ¹³¹I (see for example, U.S. Patent 4,609,725). The extent of radio-labeling must, however be monitored, since it will affect the calculations made based on the imaging results (i.e. a diiodinated binding molecule will result in twice the radiation count of a similar monoiodinated binding molecule over the same time frame) .

In applications to human subjects, it may be desirable to use radioisotopes other than ¹²⁵I for labelling in order to decrease the total dosimetry exposure of the human body and to optimize the detectability of the labelled molecule (though this radioisotope can be used if circumstances require) . Ready availability for clinical use is also a factor. Accordingly, for human applications, preferred radio-labels are for example, ^99mTc, ⁶⁷Ga, ⁶⁸Ga, ⁹⁰Y, ^luIn, ^113raIn, ¹²³I, ¹⁸⁶Re, ¹⁸⁸Re or ²¹¹At .

The radio-labelled binding molecule may be prepared by various methods. These include radio-halogenation by the chloramine - T method or the lactoperoxidase method and subsequent purification by HPLC (high pressure liquid chromatography) , for example as described by J. Gut owska et al in "Endocrinology and Metabolism Clinics of America: (1987) 16 (1) :183. Other known method of radio-labelling can be used, such as IODOBEADSO. There are a number of different methods of delivering the radio-labelled binding molecule to the end-user. It may be administered by any means that enables the active agent to reach the agent's site of action in the body of a mammal. If the molecule is digestible when administered orally, parenteral administration, e.g., intravenous, subcutaneous, or intramuscular, would ordinarily be used to optimize absorption. 6.4. Other Uses

The binding molecules of the present invention may also be used to purify target from a fluid, e.g., blood. For this purpose, the target-binding molecule is preferably immobilized on a solid-phase support. Such supports include those already mentioned as useful in preparing solid phase diagnostic reagents .

Peptides, in general, can be used as molecular weight markers for reference in the separation or purification of peptides by electrophoresis or chromatography. In many instances, peptides may need to be denatured to serve as molecular weight markers. A second general utility for peptides is the use of hydrolyzed peptides as a nutrient source. Hydrolyzed peptide are commonly used as a growth media component for culturing microorganisms, as well as a food ingredient for human consumption. Enzymatic or acid hydrolysis is normally carried out either to completion, resulting in free amino acids, or partially, to generate both peptides and amino acids. However, unlike acid hydrolysis, enzymatic hydrolysis (proteolysis) does not remove non-amino acid functional groups that may be present. Peptides may also be used to increase the viscosity of a solution.

The peptides of the present invention may be used for any of the foregoing purposes, as well as for therapeutic and diagnostic purposes as discussed further earlier in this specification.

7. Combinatorial Libraries

The term "library" generally refers to a collection of chemical or biological entities which are related in origin, structure, and/or function, and which can be screened simultaneously for a property of interest.

The term "combinatorial library" refers to a library in which the individual members are either systematic or random combinations of a limited set, of basic elements, the properties of each member being dependent on the choice and location of the elements incorporated into it. Typically, the members of the library are at least capable of being screened simultaneously. Randomization may be complete or partial; some positions may be randomized and others predetermined, and at random positions, the choices may be limited in a predetermined manner. The members of a combinatorial library may be oligomers or polymers of some kind, in which the variation occurs through the choice of monomeric building block at one or more positions of the oligomer or polymer, and possibly in terms of the connecting linkage, or the length of the oligomer or polymer, too. Or the members may be nonoligomeric molecules with a standard core structure, like the 1, 4-benzodiazepine structure, with the variation being introduced by the choice

- of substituents at particular variable sites on the core structure. Or the members may be nonoligomeric molecules assembled like a jigsaw puzzle, but wherein each piece has both one or more variable moieties (contributing to library diversity) and one or more constant moieties (providing the functionalities for coupling the piece in question to other pieces) .

The ability of one or more members of such a library to recognize a target molecule is termed "Combinatorial Recognition". In a "simple combinatorial library", all of the members belong to the same class of compounds (e.g., peptides) and can be synthesized simultaneously. A "composite combinatorial library" is a mixture of two or more simple libraries, e.g., DNAs and peptides, or benzodiazepine and carbamates. The number of component simple libraries in a composite library will, of course, normally be smaller than the average number of members in each simple library, as otherwise the advantage of a library over individual synthesis is small.

A biased combinatorial library is one in which, at one or more positions in the library member, only one of the possible basic elements is allowed for all members of the library, i.e., the biased positions are invariant.

The term "amplifiable combinatorial library" refers to a library in which the individual members, after found to bind to a target, may be amplified in vivo or in vitro, using elements already present in the library as starting materials. There are two classes of amplifiable members. First, nucleic acids may be amplified in vivo through natural replicative processes, or in vitro through techniques such as polymerase chain reaction (PCR) . Second, peptides, when presented on phage, or otherwise associated with an encoding nucleic acid, may be amplified indirectly by in vivo or in vitro amplification of the associated nucleic acid encoding the peptide, the amplified nucleic acid being expressed to produce the peptide.

The term "biopolymeric library" refers to a library composed of peptides (together with peptoids), nucleic acids, and/or oligosaccharides . (It is not necessary that they be composed of naturally occurring amino acids, bases, or sugars, respectively.) However, because of the greater complexity of carbohydrate synthesis, peptides and nucleic acids are of greater interest.

A "panel of combinatorial libraries" is a collection of different (although possibly overlapping) and separately screenable simple or composite combinatorial libraries. A "panel" differs from a composite library in that the component simple libraries have not been mixed together, that is, they may still be screened separately.

A "structured panel" is a panel as defined above where there is some structural relationship between the member libraries. For example, one could have a panel of 20 different biased peptide libraries where, in each library, the middle residue is held constant as a given amino acid, but, in each library the constant residue is different, so, collectively, all 20 possible genetically encoded amino acids are explored by the panel.

A "scanning residue library" refers to the preparation of panel of biased combinatorial peptide libraries such that the position of the constant residue shifts from one library to the next. For example, in library 1, residue 1 is held constant as a particular residue AA, in library, residue 2 is, and so forth through two or more (usually all) positions of the peptide.

One may have structured panels of libraries in which one may define subpanels, too. For example, in one subpanel, the middle residue AA_X may be the same for all libraries, but the libraries also have a constant residue AA₂ which is scanned through all other residue positions.

A library screening program is a program in which one or more libraries (e.g., a structured panel of biased peptide libraries) are screened for activity. The libraries may be screened in parallel, in series, or both. In serial screening, the results of one screening may be used to guide the design of a subsequent library in the series. The size of a library is the total number of molecules in it, whether they be the same or different. The diversity of a library as the number of different molecules in it. "Diversity" does not measure how different the structures of the library; the degree of difference between two structures is referred to here as "disparity" or "dispersion". The "disparity" is quantifiable in some respects, e.g., size, hydrophilicity, polarity, thermostability, etc. The average sampling frequency of a library is the ratio of size to diversity. The sampling frequency should be over the detection limit of the assay in order to assure that all members are screened.

The combinatorial libraries usually will have a diversity of at- least 10³ different structures. Preferably, the initial, surrogate-generating library is of high diversity, e.g., preferably at least about 10⁶, more preferably at least about 10^s different members. While a peptide library is preferred, a library composed of a different class of compounds (e.g., peptoids or nucleic acids) is acceptable if there would be a detectable preference for binding the activity-mediating binding sites of the target protein.

7.1. Oligonucleotide (Nucleic acid) Libraries An oligonucleotide library is a combinatorial library, at least some of whose members are single-stranded oligonucleotides having three or more nucleotides connected by phosphodiester or analogous bonds. The oligonucleotides may be linear, cyclic or branched, and may include non-nucleic acid moieties. The nucleotides are not limited to the nucleotides normally found in DNA or RNA. For examples of nucleotides modified to increase nuclease resistance and chemical stability of aptamers, see Chart 1 in Osborne and Ellington, Chem. Rev., 97: 349-70 (1997). For screening of RNA, see Ellington and Szostak, Nature, 346: 818-22 (1990).

There is no formal minimum or maximum size for these oligonucleotides. However, the number of conformations which an oligonucleotide can assume increases exponentially with its length in bases. Hence, a longer oligonucleotide is more likely to be able to fold to adapt itself to a protein surface. On the other hand, while very long molecules can be synthesized and screened, unless they provide a much superior affinity to that of shorter molecules, they are not likely to be found in the selected population, for the reasons explained by Osborne and Ellington (1997) . Hence, the libraries of the present invention are preferably composed of oligonucleotides having a length of 3 to 100 bases, more preferably 15 to 35 bases. The oligonucleotides in a given library may be of the same or of different lengths.

Oligonucleotide libraries have the advantage that libraries of very high diversity (e.g., 10¹⁵) are feasible, and binding molecules are readily amplified in vitro by polymerase chain reaction (PCR) . Moreover, nucleic acid molecules can have very high specificity and affinity to targets.

In a preferred embodiment, this invention prepares and screens oligonucleotide libraries by the SELEX method, as described in King and Famulok, Molec. Biol. Repts., 20: 97-107 (1994) ; L. Gold, C. Tuerk. Methods of producing nucleic acid ligands, US#5595877; Oliphant et al. Gene 44:177 (1986).

The term "aptamer" is conferred on those oligonucleotides which bind the target protein. Such aptamers may be used to characterize the target protein, both directly (through identification of the aptamer and the points of contact between the aptamer and the protein) and indirectly (by use of the aptamer as a ligand to modify the chemical reactivity of the protein) .

7.2. Amino Acids and Peptides

Amino acids are the basic building blocks with which peptides and proteins are constructed. Amino acids possess both an amino group (-NH₂) and a carboxylic acid group (-COOH) . Many amino acids, but not all, have the structure NH₂-CHR-COOH, where R is hydrogen, or any of a variety of functional groups. Twenty amino acids are genetically encoded: Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamic Acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine. Of these, all save Glycine are optically isomeric, however, only the L-form is found in humans. Nevertheless, the D-forms of these amino acids do have biological significance; D-Phe, for example, is a known analgesic.

Many other amino acids are also known, including: 2- Aminoadipic acid; 3-Aminoadipic acid; beta-Aminopropionic acid; 2-Aminobutyric acid; 4-Aminobutyric acid (Piperidinic acid) ; 6-Aminocaρroic acid; 2-Aminoheptanoic acid; 2-Aminoisobutyric acid, 3-Aminoisobutyric acid; 2-Aminopimelic acid; 2,4- Diaminobutyric acid; Desmosine; 2, 2 ' -Diaminopimelic acid; 2,3- Diaminopropionic acid; N-Ethylglycine; N-Ethylasparagine; Hydroxylysine; allo-Hydroxylysine; 3-Hydroxyproline; 4- Hydroxyproline; Isodesmosine; allo-Isoleucine; N-Methylglycine (Sarcosine) ; N-Methylisoleucine; N-Methylvaline; Norvaline; Norleucine; and Ornithine.

Technically speaking, proline is not an amino acid at all, but rather, a cyclic i ino acid, wherein the side chain - (CH₂)₃- is linked, not only to the alpha carbon, but also to the peptide bond' s amide nitrogen, forming a five-membered pyrrolidine ring. There is no amide hydrogen for proline residues. Following convention, if we refer to an "amino acid", it should be assumed to include proline (and substituted forms such as 3-hydroxyproline) unless these are expressly excluded.

A charged amino acid is one whose side chain is at least 5% ionized at neutral pH (7) . Among the genetically encoded amino acids, Asp, Glu, Arg, His and Lys are considered charged amino acids. Asp and Glu are considered negatively charged ("acidic") amino acids; they are deprotonated at pH7. Arg, Lys and His are considered positively charged (basic) amino acids; they are protonated at pH7. In the case of His (pKa 6.5), only about 10% is protonated at pH7. While Tyr is also capable of ionization, it traditionally is not classified as a charged amino acid.

Among non-genetically encoded amino acids, an amino acid will be considered charged if it is at least 5% ionized at physiological pH.

An aliphatic amino acid is one whose side chain does not include any aromatic moieties. An aromatic amino acid is one whose side chain is wholly or partially aromatic in character. Peptides are constructed by condensation of amino acids and/or smaller peptides. The amino group of one amino acid (or peptide) reacts with the carboxylic acid group of a second amino acid (or peptide) to form a peptide (-NHCO-) bond, releasing one molecule of water. Therefore, when an amino acid is incorporated into a peptide, it should, technically speaking, be referred to as an amino acid residue.

The core of that residue is the moiety which excludes the -NH and -CO linking functionalities which connect it to other residues. This moiety consists of one or more main chain atoms (see below) and the attached side chains.

The main chain moiety of each AA consists of the -NH and -CO linking functionalities and a core main chain moiety. Usually the latter is a single carbon atom. However, the core main chain moiety may include additional carbon atoms, and may also include nitrogen, oxygen or sulfur atoms, which together form a single chain. In a preferred embodiment, the core main chain atoms consist solely of carbon atoms.

The side chains are attached to the core main chain atoms. For alpha amino acids, in which the side chain is attached to the alpha carbon, the C-l, C-2 and N-2 of each residue form the repeating unit of the main chain, the word "side chain" refers to the C-3 and higher numbered carbon atoms and their substituents . It also includes H atoms attached to the main chain atoms.

Amino acids may be classified according to the number of carbon atoms which appear in the main chain inbetween the carbonyl carbon and amino nitrogen atoms which participate in the peptide bonds. Among the 150 or so amino acids which occur in nature, alpha, beta, gamma and delta amino acids are known. These have 1-4 intermediary carbons. Only alpha amino acids occur in proteins.

For beta and higher order amino acids, there is a choice as to which main chain core carbon a side chain other than H is attached to. The preferred attachment site is the C-2 (alpha) carbon, i.e., the one adjacent to the carboxyl carbon of the -CO linking functionality. It is also possible for more than one main chain atom to carry a side chain other than H. However, in a preferred embodiment, only one main chain core atom carries a side chain other than H.

A main chain carbon atom may carry either one or two side chains; one is more common. A side chain may be attached to a main chain carbon atom by a single or a double bond; the former is more common.

A peptide is composed of a plurality of amino acid residues joined together by peptidyl (-NHCO-) bonds. A biogenic peptide is a peptide in which the residues are all genetically encoded amino acid residues; it is not necessary that the biogenic peptide actually be produced by gene expression.

The peptides of the present invention include peptides whose sequences are disclosed in this specification, or sequences differing from the above solely by no more than one nonconservative substitution and/or one or more conservative substitutions, preferably no more than a single conservative substitution. The substitutions may be of non-genetically encoded (exotic) amino acids, in which case the resulting peptide is nonbiogenic. Preferably, the peptides are biogenic. If the peptide is being expressed in a cell, all of its amino acids must be biogenic (unless the cell is engineered to alter certain amino acids post-expression, or the peptide is recovered and modified in vitro) . If it is produced nonbiologically (e.g., Merrifield-type synthesis) or by semisynthesis, it may include nonbiogenic amino acids.

Additional peptides within the present invention may be identified by systematic mutagenesis of the lead peptides, e.g. (a) separate synthesis of all possible single substitution (especially of genetically encoded AAs) mutants of each lead peptide, and/or

(b) simultaneous binomial random alanine-scanning mutagenesis of each lead peptide, so each amino acids position may be either the original amino acid or alanine (alanine being a semi-conservative - substitution for all other amino acids) , and/or

(c) simultaneous random mutagenesis sampling conservative substitutions of some or all positions of each lead peptide, the number of sequences in total sequences space for a given experiment being such that any sequence, if active, is within detection limits (typically, this means not more than about 10¹⁰ different sequences) . Substitutions are preferably at sites shown to tolerate mutation by the mutagenic strategies set forth above.

The mutants are tested for activity, and, if active, are considered to be within "peptides of the present invention".

Even inactive mutants contribute to our knowledge of structure- activity relationships and thus assist in the design of peptides, peptoids, and peptidomimetics .

The core sequences of the peptides may be identified by systematic truncation, starting at the N-terminal, the C- terminal, or both simultaneously or sequentially. The truncation may be one amino acid at a time, but preferably, to speed up the process, is of 10-50% of the molecule at one time. If a given truncation is unsuccessful, one retreats to a less dramatic truncation intermediate between the last successful truncation and the last unsuccessful truncation.

Most extensions should be tolerated. However, if one is not, it may be helpful to introduce a linker, such as one made primarily of amino acids such as Glycine (introduces flexibility), and Proline (introduce a rigid extension), or other amino acids favored in protein turns, loops and interdomain boundaries. Indeed, the sequences of such segments may be used directly as linkers.

Preferably, substitutions of exotic amino acids for the original amino acids take the form of

(I) replacement of one or more hydrophilic amino acid side chains with another hydrophilic organic radical, not more than twice the volume of the original side chain, or (II) replacement of one or more hydrophobic amino acid side chains with another hydrophobic organic radical, not more than twice the volume of the original side chain.

The exotic amino acids may be alpha or non-alpha amino acids (e.g., beta alanine) . They may be alpha amino acids with

2 R groups on the Cα, which groups may be the same or different. They may be dehydro amino acids (HOOC-C (NH₂) =CHR) . 7.3. Cyclic Peptides

Many naturally occurring peptide are cyclic. Cyclization is a common mechanism for stabilization of peptide conformation thereby achieving improved association of the peptide with its ligand and hence improved biological activity. Cyclization is usually achieved by intra-chain cystine formation, by formation of peptide bond between side chains or between - and C- terminals. Cyclization was usually achieved by peptides in solution, but several publications have appeared recently that describe cyclization of peptides on beads.

7.4. Peptide Library

A peptide library is a combinatorial library, at least some of whose members are peptides having three or more amino acids connected via peptide bonds. Preferably, they are at least five, six, seven or eight amino acids in length. Preferably, they are composed of less than 50, more preferably less than 20 amino acids.

The peptides may be linear, branched, or cyclic, and may include nonpeptidyl moieties. The amino acids are not limited to the naturally occurring amino acids.

A biased peptide library is one in which one or more (but not all) residues of the peptides are constant residues. The individual members are referred to as peptide ligands (PL) . In one embodiment, a single internal residue is constant.

The biased residue may be at a terminal, or in an interior residue. When the biased residue is at a terminal the libraries of the form (Xaa)m-Cys, where m is 4-40 and Cys-

(Xaa)n, where n is 4-40, are of particular interest. Alternatively, the biased residue may be an interior residue, so that the peptide sequence may be written as

(Xaa)_m-AA₁-(Xaa)_n where Xaa is either any naturally occurring amino acid, or any amino acid except cysteine, m and n are chosen independently from the range of 1 to 20, m + n >= 5, the Xaa may be the same or different, and AA_: is the same naturally occurring amino acid for all peptides in the library but may be any amino acid. Preferably, m and n are chosen independently from the range of 4 to 9.

Preferably, AA_X is located further away from the termini, e.g., m and n both >2. More preferably, it is at or near the center of the peptide. More specifically, it is desirable that m and n are not different by more than 2; more preferably not more than 1, still more preferably m and n are equal. Even if the chosen A₂ is required (or at least permissive) of the target protein (TP) binding activity, one may need particular flanking residues to assure that it is properly positioned. If AA_X is more or less centrally located, the library presents numerous alternative choices for the flanking residues. If AA_X is at an end, this flexibility is diminished.

The most preferred libraries are those in which AA_X is tryptophan, proline or tyrosine. Second most preferred are those in which AAj_. is phenylalanine, histidine, arginine, aspartate, leucine or isoleucine. Third most preferred are those in which AA_X is asparagine, serine, alanine or methionine. The least preferred choices are cysteine and glycine. These preferences are based on evaluation of the results of screening random peptide libraries for binding to many different TPs.

Ligands that bind to functional domains tend to have both constant as well as unique features. Therefore, by using "biased" peptide libraries, one can ease the burden of finding ligands. Either "biased" or "unbiased" libraries may be screened to identify "BioKey" peptides for use in developing reactivity descriptors, and, optionally, peptide aptamer descriptors and additional drug leads. In other embodiments, the number of biased residue is greater than one. If so, then preferably at least one is positioned within the middle 50% (round downward to nearest integer number of amino acids) , more preferably the middle 25%, still more preferably the middle residue (or any of the two middle residues, if the peptide is of even length) .

7.5. Small Organic Compound Library

The small organic compound library ("compound library", for short) is a combinatorial library whose members are suitable for use as drugs if, indeed, they have the ability to mediate a biological activity of the target protein.

Peptides have certain disadvantages as drugs. These include susceptibility to degradation by serum proteases, and difficulty in penetrating cell membranes. Preferably, all or most of the compounds of the compound library avoid, or at least do not suffer to the same degree, one or more of the pharmaceutical disadvantages of peptides.

The design of a library may be illustrated by the example of the benzodiazepines . Several benzodiazepine drugs, including chlordiazepoxide, diazepam and oxazepam, have been used on anti-anxiety drugs. Derivatives of benzodiazepines have widespread biological activities; derivatives have been reported to act not only as anxiolytics, but also as anticonvulsants, cholecystokinin (CCK) receptor subtype A or B, kappa opioid receptor, platelet activating factor, and HIV transactivator Tat antagonists, and GPIIblla, reverse transcriptase and ras farnesyltransferase inhibitors.

The benzodiazepine structure has been disjoined into a 2- aminobenzophenone, an amino acid, and an alkylating agent. See Bunin, et al., Proc. Nat. Acad. Sci. USA, 91:4708 (1994). Since only a few 2-aminobenzophenone derivatives are commercially available, it was later disjoined into 2- aminoarylstannane, an acid chloride, an amino acid, and an alkylating agent. Bunin, et al., Meth. Enzymol., 267:448 (1996) . The arylstannane may be considered the core structure upon which the other moieties are substituted, or all four may be considered equals which are conjoined to make each library member.

A basic library synthesis plan and member structure is shown in Figure 1 of Fowlkes, et al . , U.S. Serial No. 08/740,671, incorporated by reference in its entirety. The acid chloride building block introduces variability at the R¹ site. The R² site is introduced by the amino acid, and the R³ site by the alkylating agent. The R⁴ site is inherent in the arylstannane. Bunin, ^' et al. generated a 1, 4-benzodiazepine library of 11,200 different derivatives prepared from 20 acid chlorides, 35 amino acids, and 16 alkylating agents. (No diversity was introduced at R⁴; this group was used to couple the molecule to a solid phase.) According to the Available Chemicals Directory (HDL Information Systems, San Leandro CA) , over 300 acid chlorides, 80 Fmoc-protected amino acids and 800 alkylating agents were available for purchase (and more, of course, could be synthesized) . The particular moieties used were chosen to maximize structural dispersion, while limiting the numbers to those conveniently synthesized in the wells of a microtiter plate. In choosing between structurally similar compounds, preference was given to the least substituted compound.

The variable elements included both aliphatic and aromatic groups. Among the aliphatic groups, both acyclic and cyclic

(mono- or poly-) structures, substituted or not, were tested. (While all of the acyclic groups were linear, it would have been feasible to introduce a branched aliphatic) . The aromatic groups featured either single and multiple rings, fused or not, substituted or not, and with heteroatoms or not. The secondary substitutents included -NH₂, -OH, -OMe, -CN, -Cl, -F, and - COOH. While not used, spacer, moieties, such as -0-, -S-, -OO-, -CS-, -NH-, and -NR-, could have been incorporated.

Bunin et al. suggest that instead of using a 1, 4- benzodiazepine as a core structure, one may instead use a 1, 4-benzodiazepine-2, 5-dione structure.

As noted by Bunin et al., it is advantageous, although not necessary, to use a linkage strategy which leaves no trace of the linking functionality, as this permits construction of a more diverse library.

Other combinatorial nonoligomeric compound libraries known or suggested in the art have been based on carbamates, mercaptoacylated pyrrolidines, phenolic agents, aminimides, N- acylamino ethers (made from amino alcohols, aromatic hydroxy acids, and carboxylic acids) , N-alkylamino ethers (made from aromatic hydroxy acids, amino alcohols and aldehydes) 1, 4- piperazines, and 1, 4-piperazine-6-ones .

DeWitt, et al., Proc. Nat. Acad. Sci. (USA), 90:6909-13 (1993) describes the simultaneous but separate, synthesis of 40 discrete hydantoins and 40 discrete benzodiazepines. They carry out their synthesis on a solid support (inside a gas dispersion tube) , in an array format, as opposed to other conventional simultaneous synthesis techniques (e.g., in a well, or on a pin) . The hydantoins were synthesized by first simultaneously deprotecting and then treating each of five amino acid resins with each of eight isocyanates. The benzodiazepines were synthesized by treating each of five deprotected amino acid resins with each of eight 2-amino benzophenone imines. Chen, et al . , J. Am. Chem. Soc, 116:2661-62 (1994) described the preparation of a pilot (9 member) combinatorial library of formate esters. A polymer bead-bound aldehyde preparation was "split" into three aliquots, each reacted with one of three different ylide reagents. The reaction products were combined, and then divided into three new aliquots, each of which was reacted with a different Michael donor. Compound identity was found to be determinable on a single bead basis by gas chromatography/mass spectroscopy analysis.

Holmes, USP 5,549,974 (1996) sets forth methodologies for the combinatorial synthesis of libraries of thiazolidinones and metathiazanones . These libraries are made by combination of amines, carbonyl compounds, and thiols under cyclization conditions.

Ellman, USP 5,545,568 (1996) describes combinatorial synthesis of benzodiazepines, prostaglandins, beta-turn mimetics, and glycerol-based compounds. See also Ellman, USP 5,288,514. Sum erton, USP 5,506,337 (1996) discloses methods of preparing a combinatorial library formed predominantly of morpholino subunit structures.

Heterocylic combinatorial libraries are reviewed generally in Nefzi, et al., Chem. Rev., 97:449-472 (1997). For pharmacological classes, see, e.g., Goth, Medical Pharmacology: Principles and Concepts (C.V. Mosby Co.: 8th ed. 1976) ; Korolkovas and Burckhalter, Essentials of Medicinal Chemistry (John Wiley & Sons, Inc. : 1976) . For synthetic methods, see, e.g., Warren, Organic Synthesis: The Disconnection Approach (John Wiley & Sons, Ltd.: 1982); Fuson,

Reactions of Organic Compounds (John Wiley & Sons: 1966); Payne and Payne, How to do an Organic Synthesis (Allyn and Bacon,

Inc. : 1969) ; Greene, Protective Groups in Organic Synthesis

(Wiley-Interscience) . For selection of substituents, see e.g., Hansch and Leo, Substituent Constants for Correlation Analysis, in Chemistry and Biology (John Wiley & Sons: 1979).

The library is preferably synthesized so that the individual members remain identifiable so that, if a member is shown to be active, it is not necessary to analyze it. Several methods of identification have been proposed, including:

(1) encoding, i.e., the attachment to each member of an identifier moiety which is more readily identified than the member proper. This has the disadvantage that the tag may itself influence the activity of the conjugate.

(2) spatial addressing, e.g., each member is synthesized only at a particular coordinate on or in a matrix, or in a particular chamber. This might be, for example, the location of a particular pin, or a particular well on a microtiter plate, or inside a "tea bag". The present invention is not limited to any particular form of identification.

However, it is possible to simply characterize those members of the library which are found to be active, based on the characteristic spectroscopic indicia of the various building blocks. Solid phase synthesis permits greater control over which derivatives are formed. However, the solid phase could interfere with activity. To overcome this problem, some or all of the molecules of each member could be liberated, after synthesis but before screening. Examples of candidate simple libraries which might be evaluated include derivatives of the following: Cyclic Compounds Containing One Hetero Atom Heteronitrogen pyrroles pentasubstituted pyrroles pyrrolidines pyrrolines prolines indoles beta-carbolines pyridines dihydropyridines 1, 4-dihydropyridines pyrido [2, 3-d] pyrimidines tetrabydro-3H-imidazo [4 , 5-c] pyridines Isoquinolines tetrahydroisoquinolines quinolones beta-lactams azabicyclo [4.3.0] nonen-8-one amino acid Heterooxygen furans tetrahydrofurans

2, 5-disubstituted tetrahydrofurans pyrans hydroxypyranones tetrahydroxypyranones gamma-butyrolactones

Heterosulfur sulfolenes Cyclic Compounds with Two or More Hetero atoms Multiple heterσnitrogens imidazoles pyrazoles piperazines diketopiperazines arylpiperazines benzylpiperazines benzodiazepines 1, -benzodiazepine-2, 5-diones hydantoins 5-alkoxyhydantoins dihydropyrimidines

1, 3-disubstituted-5, 6-dihydopyrimidine-2, 4- diones cyclic ureas cyclic thioureas quinazolines chiral 3-substituted-quinazoline-2, 4-diones triazoles

1, 2, 3-triazoles purines Heteronitrogen and Heterooxygen dikelomorpholines isoxazoles isoxazolines Heteronitrogen and Heterosulfur thiazolidines

N-axylthiazolidines dihydrothiazoles

2-methylene-2, 3-dihydrothiazates 2-aminothiazoles thiophenes

3-amino thiophenes 4-thiazolidinones

4-melathiazanones benzisothiazolones

For details on synthesis of libraries, see Nefzi, et al., Chem. Rev., 97:449-72 (1997), and references cited therein.

8. Analogues 8.1. Peptoid A peptoid is an analogue of a peptide in which one or more of the peptide bonds are replaced by pseudopeptide bonds, which may be the same or different. A pseudopeptide bond connects two adjacent residues by a structure of the form -A-B-, where A and B are atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur. The carbon or nitrogen may be unsubstituted (i.e., bonded to H) or substituted with an aliphatic group. The total molecular weight of the pseudopeptide bond usually will not exceed 200 daltons, including any substitutions.

Such pseudopeptide bonds may be: Carba 0(CH₂-CH₂) Depsi 0(CO-O)

Hydroxyethylene 0(CHOH-CH₂) Ketomethylene 0(CO-CH₂)

Methylene-ocy CH₂-0- Reduced CH₂-NH Thiomethylene CH₂-S- Thiopeptide CS-NH N-modified -NRCO-

8.2. Peptidomimetic

A peptidomimetic is a molecule which mimics the biological activity of a peptide, by substantially duplicating the pharmacologically relevant portion of the conformation of the peptide, but is not a peptide or peptoid as defined above. Preferably the peptidomimetic has a molecular weight of less than 700 daltons.

Designing a peptidomimetic usually proceeds by: (a) identifying the pharmacophoric groups responsible for the activity; (b) determining the spatial arrangements of the pharmacophoric groups in the active conformation of the peptide; and

(c) selecting a pharmaceutically acceptable template upon which to mount the pharmacophoric groups in a manner which allows them to retain their spatial arrangement in the active conformation of the peptide. Step (a) may be carried out by preparing mutants of the active peptide and determining the effect of the mutation on activity. One may also examine the 3D structure of a complex of the peptide and the receptor for evidence of interactions, e.g., the fit of a side chain of the peptide into a cleft of the receptor; potential sites for hydrogen bonding, etc.).

Step (b) generally involves determining the 3D structure of the active peptide, in the complex, by NMR spectroscopy or X-ray diffraction studies. The initial 3D model may be refined by an energy minimization and molecular dynamics simulation.

Step (c) may be carried out by reference to a template database, see Wilson, et al. Tetrahedron, 49:3655-63 (1993). The templates will typically allow the mounting of 2-8 pharmacophores, and have a relatively rigid structure. For the latter reason, aromatic structures, such as benzene, biphenyl, phenanthrene and benzodiazepine, are preferred. For orthogonal protection techniques, see Tuchscherer, et al., Tetrahedron, 17:3559-75 (1993) . For more information on peptoids and peptidomimetics, see USP 5,811,392, USP 5,811,512, USP 5,578,629, USP 5,817,879, USP 5,817,757, USP 5,811,515.

8.3. Analogues Also of interest are analogues of the disclosed peptides, and other compounds with activity of interest.

Analogues may be identified by assigning a hashed bitmap structural fingerprint to the compound, based on its chemical structure, and determining the similarity of that fingerprint to that of each compound in a broad chemical database. The fingerprints are determined by the fingerprinting software commercially distributed for that purpose by Daylight Chemical Information Systems, Inc., according to the software release current as of January 8, 1999. In essence, this algorithm generates a bit pattern for each atom, and for its nearest neighbors, with paths up to 7 bonds long. Each pattern serves as a seed to a pseudorandom number generator, the output of which is a set of bits which is logically ored to the developing fingerprint. The fingerprint may be fixed or variable size.

The database may be SPRESI'95 (InfoChe GmbH), Index Chemicus (ISI) , MedChem (Pomona/Biobyte) , World Drug Index (Derwent) , TSCA93 (EPA) Maybridge organic chemical catalog (Maybridge) , Available Chemicals Directory (MDLIS Inc.), NCI96 (NCI), Asinex catalog of organic compounds (Asinex Ltd.), or IBIOScreen SC and NP (Inter BioScreen Ltd.), or an inhouse database. A compound is an analogue of a reference compound if it has a daylight fingerprint with a similarity (Tanamoto coefficient) of at least 0.85 to the Daylight fingerprint of the reference compound.

A compound is also an analogue of. a reference compound id it may be conceptually derived from the reference compound by isosteric replacements.

Homologues are compounds which differ by an increase or decrease in the number of methylene groups in an alkyl moiety.

Classical isosteres are those which meet Erlenmeyer's definition: "atoms, ions or molecules in which the peripheral layers of electrons can be considered to be identical". Classical isosteres include Monovalents Bivalents Trivalents Tetra Annular F, OH , NH₂ , CH₃ -0- -N= =C= -CH=CH- =Si=

Cl , SH , PH₂ -S- -P= -N+= -S-

Br -Se- -As- =P+= -0- i -Te- -Sb- =As+= -NH- -CH= =Sb+=

Nonclassical isosteric pairs include -CO- and -S0₂-, -COOH and -S0₃H, -S0₂NH₂ and -PO(OH)NH₂, and -H and -F, -OC(=0)- and C(=0)0-, -OH and -NH₂.

9. Miscellaneous Disclosure

9.1. Relative Affinity

Where this specification indicates that a molecule B binds a target TI substantially more strongly than a target T2, or that a molecule Bl binds a target T substantially more strongly than an alternative molecule B2 binds the same target T, it means that the difference in binding is detectable and is manifest to a useful degree in the relevant context, e.g., screening, diagnosis, purification, or therapy. Generally speaking, a tenfold difference in binding will be considered substantial, however this is not necessarily required.

9.2. "No -Naturally Occurring" Reference to a peptide or protein as "non-naturally occurring" means that it does not occur, as a unitary molecule, in non-genetically engineered cells or viruses. It may be biologically produced in genetically engineered cells, or genetically engineered virus-transfected cells, and it may be a segment of a larger, naturally occurring protein.

If it is disclosed that a peptide preferably is not naturally occurring, it more preferably is not conservatively identical to any naturally occurring peptide. Non-naturally occurring binding molecules, including peptides, peptoids, and nucleic acids, and small organic compounds, are of particular interest.

9.3. Relationship of Peptides to Antibody Variable Domain

Immunoglobulin molecules of the most common class, IgG, are composed of two identical "heavy chain" polypeptides (~ 53,000 D each) and two identical "light chain" polypeptides (~ 23,000 D each) . Each light chain is linked to one heavy chain by a covalent disulfide bridge, as well as by noncovalent associations. The heavy chain can be subdivided into variable

(V-H) and constant (C-H) regions. The latter may further be subdivided into the C-Hl, C-H2 and C-H3 homology units. The light chain can be subdivided into variable (V-L) and constant (C-L) regions.

There are six homology units (V-H, C-Hl, C-H2, C-H3, V-L and C-L) , each about 110 amino acids in length. There are sequence homologies between V-H and V-L, and among the four C- region homology units. While there is no substantial sequence homology between the V-region and the C-region homology units, they do show similarities in their three-dimensional conformation, notably the so-called immunoglobulin fold. Immunoglobulins of other classes also possess V-region and C- region homology units.

The V-H and V-L are characterized by hypervariable regions, in which most of antibody diversity is focused, and framework regions of relatively low diversity which join them together and hold them in a particular relationship to each other.

There are two antigen-binding sites in IgG, each formed by a paired V-H and V-L. (The V-H and V-L regions may have some antigen-binding activity even by themselves.) Thus, each of the two variable domains is composed of one V-H and one V-L. There are also four constant domains in IgG (C-H3 & C-H3, C-H2 & C-H2, and two domains composed of C-Hl & C-L) .

The peptides of the present invention do not comprise a V-H or V-L homology unit of an antibody. Preferably, they have less than 50% identity, more preferably less than 25% identity, with the V-H or V-L homology unit of any naturally occurring antibody. (If the peptide is shorter than the homology unit, then each unmatched amino acid of the homology unit should count as a mismatch for the purpose of calculating the % identity) . Preferably, they have less than 50% identity with the framework residues of the V-H or V-L homology unit of any naturally occurring antibody. Preferably, they do not comprise an immunoglobulin fold.

The peptides of the present invention are preferably less than 100 amino acids, more preferably less than 50 amino acids, still more preferably less than 30 amino acids in length.

JEXflMPIES

Example 10 IX

For the application of molecular and cellular braille to estrogen and other nuclear receptors, see PCT/US99/06664, filed March 26, 1999, published as W099/54728 on October 28, 1999, hereby incorporated by reference.

Example 201

The contents of Example 201 of the prior applications, to the extent applicable to this case, has been relocated outside of the examples.

Example 202: Discovery of GDP-dependent, GTP-dependent and GDP/GTP-independent Peptides Specific for Giαl using Phage Affinity Selection and Phage Display Peptide Libraries

Phage display was performed on biotinylated Giαl in buffer A in the presence or absence of GDP or GTPγS. The deduced amino acid sequences of Giαl-binding phage peptides formed several different sequence clusters (a group of peptides with amino acid sequence similarity) .

All phage which were isolated by affinity selection on biotinylated Giαl and subjected to DNA sequence analysis were examined for Giαl specificity against a panel of other biotinylated proteins (alkaline phosphatase, glucose-6- phosphate, β-galactosidase, hexokinase) . The phage were found to be specific for Giαl protein.

Giαl protein amounts and phage volumes were titrated to determine where the optimal GDP/GTP or GTP/GDP differential binding occurs for the phage. It was determined that one p ole of Giαl and 5 il of phage in an ELISA yielded the optimal differential binding of phage to Giαl. All phage containing peptides of known sequence were categorized into GDP-dependent, GTP-dependent or GDP/GTP-independent Giαl binders.

GDP/GTP-dependent phage differentiation assays were also performed with Giαl in the presence of GDP and A1F₄ ^", a complex that mimics the transition state for GTP hydrolysis of the Gα subunit. A1F₄ ^" is an activator of GDP-bound Gα subunits that is believed to substitute for and mimic the γ- phosphate of GTP. Only GTP-specific phage were found to bind in the presence of A1F4- and GDP. GTP-dependent peptides (T-peptides), GDP-dependent peptides (D-peptides) and GDP/GTP-independent peptides (I- peptides) were synthesized, and biotinylated or fluorescently labeled.

Materials and Methods

Materials . GOP and GTPγS were purchased from Sigma. Immulon 4 plates were purchased from Dynatech. Purified Giαl was kindly provided by Merck. Sequencing of single-strand M13 DNA was conducted by Sequetech Corporation (Mountain View, CA)

Phage Affini ty Selection . Giαl specific peptides were identified using the following procedures. Phage affinity selection of biotinylated Giαl was performed in the- presence of GDP, GTPγS or buffer A alone, essentially as previously described (Sparks, A.B., Adey, N.B., Cwirla, S. & Kay, B.K. (1996) in Phage Display of Peptides and Proteins, A Laboratory Manual, eds. Kay, B.K., Winter, J. & McCafferty, J. (Academic, San Diego) , pp. 227-253) . The nonhydrolyzable form of GTP, GTPγs, was used in all experiments to prevent hydrolysis of GTP to GDP. Briefly, Immulon 4 plates were coated with streptavidin in 0.1 M sodium bicarbonate followed by blocking with 1.0% BSA in 0.1M sodium bicarbonate. The plates were then incubated for lh at room temperature (RT) with 10 pmoles of purified biotinylated Giαl protein in Buffer A (20 mM Hepes, pH 7.5 buffer containing 1 mM EDTA, 16 mM MgCl_2/ 1 mM DTT and 0.05% Tween 20) alone or Buffer containing either 5 iM GDP or 5 iM GTPγS. Streptavidin binding sites not containing a bound Giαl protein were blocked with a Buffer A solution containing 0.5 mM biotin. Twenty different phage libraries were tested, one library per well. After incubation of the phage libraries with the immobilized protein for 3h at RT, nonspecifically bound phage were removed by washing with TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 0.5 mM biotin. Phage that bound specifically to the Giαl protein were then eluted sequentially with a low pH glycine buffer and a high pH ethanolamine buffer. The pH of the eluted phage was then neutralized and the phage were amplified and repanned using iterative rounds of the affinity selection procedure. Each phage library was panned until the amplified pool of phage bound to immobilized Giαl significantly above no-protein controls as judged by phage ELISA. At this point, phage were plated out and individual phage were picked and examined by ELISA for specific binding to Giαl and a panel of other unrelated biotinylated proteins. Individual phage displaying peptides specifically binding to Giαl were then plaque purified and DNA was isolated from these phage. The DNA sequence of the segment encoding the peptide of interest was then determined along with the deduced amino acid sequence of the peptide.

Phage ELISA. Giαl was immobilized in the presence of either GDP or GTPγS as described for phage affinity selection. Phage (50 il) from either a 5h or overnight culture grown in DH5αF' cells was added directly to wells containing immobilized Gial and incubated for 45 min at RT. Unbound phage were removed by TBST washes and bound phage were detected using an anti-M13 antibody conjugated to horse radish peroxidase. Assays were developed for 10 min at. RT by adding 2, 2-azinobis (3- ethylbenzothiazoline) -6 sulfonic acid and hydrogen peroxide. The development was stopped by adding SDS to a final concentration of 1%. Absorbance was measured at 405 nm in a Molecular Devices microplate reader.

GD /GTP -Dependent Phage Differentiation . Phage were categorized into GDP-dependent, GTP-dependent or GDP/GTP- independent Giαl binders as described for Phage ELISAs with the following modifications. Giαl was incubated overnight in buffer A containing either 100 iM GDP or GTPγS, and then one pmol of this protein was immobilized on plates as previously described. Phage (5 il) was added to the wells in the presence of buffer A containing either 100 iM GDP or GTPγS and incubated for 30 min at RT. Phage was also added to no protein control wells in the presence of Buffer A.

Expression and test of peptide-Alkaline Phospha tase fusion constructs : Peptide encoding sequences were PCR amplified and subcloned into pNPC-AP vector (derivative of pFLAG-BAP from Sigma-Aldrich, without tags) . The AP-fusion proteins were isolated by (NH₄)₂S0₄ precipitation and ion-exchange chromatography. The purity was confirmed by SDS-PAGE and the activity established by enzyme assays using the AP-substrate p-nitrophenyl phosphate (pNPP) from Sigma BioSciences. Serial dilutions of biotinylated G_ail were prepared in a buffer (20 mM Hepes pH7.5; 1 mM EDTA; 16 mM MgCl₂; 1 mM DTT; 0.05 % Tween 20) with 100 mM GTPgS or GDP. Biotinylated G_all was immobilized on BSA blocked, streptavidin-coated, 96- or 384-well plates. Bound fusion proteins were detected by adding CSPD substrate and Sapphire II enhancement solution (Tropix) and reading on an LJL Analyst Multimode Detector. Results

The phage sequences obtained are shown in Tables 202A-

202C, below. The properties of these "first generation" peptides were also analyzed with Alkaline Phosphatase (AP-) fusion constructs (Fig. 1) and with conjugates of synthesized biotinylated peptides and streptavidin-AP (Figs. 2-5) to confirm and extend the information obtained from the phage

ELISA experiments. Based on these results, "second generation" peptide phage libraries were planned and produced. The second generation libraries were inspired by various first generation peptides, see Table 203. The designs of the second generation libraries appears in Table 204; bracketed amino acids are alternatives for a single position.

The results of the screening of the second generation libraries is shown in Table 205. While the gα oligo 1 and 2 libraries were screened, and phage were selected, no sequencing was done and hence no gα oligo 1 or 2 sequences appear in that table.

Binding affinities, to the extent known, are set forth in Table 206. The fluorescence polarization was determined for peptides 388, 213, and 801 in triplicate in a series of experiments where the peptide concentration was kept constant at 200 pM and the protein concentration was generally varied 10 μM to 57 pM. K_d values were determined by non-linear regression analysis fitting the data to the simple, single site ligand binding equation. (Fig. 1)

The peptides of the various libraries are screened as fusions to a phage coat protein. However, we have synthesized several of these peptides as independent entities. These include Giα binding T peptides 792, 796, 866, 1746, 1753 and 1755; D-peptides 752 and 771, and I-peptide 801.

A group of phage sequences were selected for expression as Alkaline Phosphatase fusion constructs which were used to test nucleotide dependent binding (Fig. 1) . It was demonstrated that fusion constructs of peptides of the D (IA), T (IB), or I (1C) class all bound with high specificity and binding was dependent on the protein amount immobilized per well.

Several of the peptides expressed as fusion proteins were also tested for their G_a subclass specificity (Fig. 2) . It was established that peptides 752, 757, 771, 796 and 801 all recognize G_ail, G_ai2 and G_ai3 but not G_as.

In an effort to establish conditions to detect G_a protein within membranes, Sf9 cells were coinfected with either G_ai or G_as, resp. and G_b and G_g (i.e., triple infections) . After loading with either GTPgS or GDP the membranes were probed with the synthesized, biotinylated form of peptide 806 conjugated at various ratios to streptavidin-alkaline phosphatase (Fig. 3) . Detection was best at a 1:8 ratio of enzyme to peptide and 806 was, like 796, specific for G_ai over G_as. The concentration of probe that is necessary to get the optimal signal to background ratio was determined in an experiment using similar conditions as for the valency experiment above. In the same experiment it was tested whether there is a significant contribution to background by nonspecific streptavidin binding. This was assessed by adding excess Neutravidin during binding of the probe (Fig. 4). The best results were achieved using 0.4 nM peptide-SA-AP and blocking with Neutravidin only slightly improved the background. 'Using the optimized assay conditions a comparison between G_al specific peptides from the first and second generation was performed. The results confirmed that signal to background ratios are much improved with second generation peptides (Fig. 5) .

Example 202b: Development of 2^nd generation G_ai specific peptides with higher affinity Competition binding assays were performed with G_ai specific T peptides of the first and second generation to establish specificity of binding (Fig. 6). .Even though there was a clear difference between the inhibition of binding caused by specific versus unspecific peptides, this method gave little insight into relative binding affinities. All T peptides seemed to compete with 1755 roughly at an equal level. However, as figures 6a and 6b show the signal obtained with 1755 was much stronger than the one obtained with 1746 or 796. This was confirmed in parallel assays with these probes on Sf9 membranes (data not shown) . While all 2^nd generation peptides seemed to be superior over the first generation peptides (see Fig. 5) , 1753 and 1755 turned out to be the preferred probes for most subsequent experiments.

Example 203: Discovery of GDP-dependent, GTP-dependent and GDP/GTP-independent Peptides Specific for G_as using Phage Affinity Selection and Phage Display Peptide Libraries

Phage affinity selection and analysis of G_as specific peptides was carried out essentially as described for G_ai. _specifics of the purification and deviations from the G_ai protocol are described below.

G_as cloning, purification, and phage affinity selection Cloning: G_as was PCR amplified from the Invitrogen GeneStorm Clone which corresponds to the clone called alpha-S2, (genbank accession # BC002722) and subsequently cloned into a pAN-5 vector (Avidity) . For ease of purification, a His6 tag was later inserted between the avitag (already present in pAN-5) and the initiator ethionine of Gas. The sequence of the construct was confirmed prior to purification and subsequent phage affinity selections.

Expression and Purification:

For expression purposes G_as-His6/pAN-5 was transformed into BL21 (DE3) strain carrying the pBirA plasmid (encoding Biotin Ligase) . An overnight inoculum was diluted 1:10 into 2xYT with 50 mg/ml Amp and 25 mg/ml Chloramphenicol, and allowed to grow at 37 °C and reach an OD (A600) of around 1.2, at which point the temperature was decreased to 25 °C and the culture induced with 0.3 mM IPTG and supplemented with 50 mM Biotin. After an induction of 2.5 hours, the cells were pelleted by centrifugation and stored at -80 °C. For purification, the frozen pellet was lysed in 50 mM Tris, pH 8.0, 50 mM GDP, 10 mM b-ME, 0.1 mg/ml lysozyme, and protease inhibitor cocktail (without EDTA) , with 4-10 second bursts of sonication, all at 4 °C. The lysate was then clarified by a high-speed spin (ultra-centrifugation) , filtered through a 0.45 mm filter, and loaded onto a Ni-NTA column. The column was washed extensively with 50 mM Tris, 0.5 M NaCl, 10 M b-ME, 50 mM GDP, pH 8.0 + 10 mM Imidazole, and the bound protein was eluted with 50 M Tris, pH 8.0, 50 mM NaCl, 10 mM b-ME, 50 mM GDP, 0.2 M Imidazole, and 0.1 mM PMSF. The protein was kept on ice.

Phage affinity selections: The phage affinity selections were carried out as per the standard BioKey protocol, with the following 20 phage libraries :

D, F, H, K, L, M, N, P, R, W, X10C, CWL, PHD7, PHD12, X14C<W>, X7WX7<P>, E, Y, AroP, and L-Box.

The phage selection buffers were as follows:

"GDP-selection": 10 mM Tris, 100 mM NaCl, 0.1 % Tween- 20, 10 mM MgC12, 1 mM EDTA, 2 mM DTT, 50 mM' GDP

"GTP-selection": 10 mM Tris, 100 mM NaCl, 0.1 % Tween- 20, 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 50 mM GTPgS .

Approximately 60 pmol of G_as-GDP or G_as-GTPgS was used for each round of selection.

First generation Gαs-binding T-peptides were identified in the W, K and E libraries. The sequences of these peptides are set forth in Table 210. Using phage ELISA data comparable to what's been described for G_ai peptides above, G_as specific peptides 1686 and 1687 were chosen as the best binders.

Example 203b: Development of 2^nd generation G_aS specific peptides with higher affinity

Based on the sequences described above libraries were designed with a more restricted repertoire which were used to screen for 2^nd generation peptides. These screens were also carried out under more stringent conditions than for the first generation. In two sets of conditions the following amounts of G_as protein per well were used:

All other conditions were as described before. The second generation libraries are shown in Table 211, and the binding peptides therefrom in Table 212.

When G_as specific peptides were tested in assays using membranes in the way described for G_ai peptides ^"above, no signal over background was detected (data not shown) . Since specific binding to purified G_as protein had been established, the configuration of the assay had to be responsible for this observation. Thus, a different type of assay was developed. As described below, peptides were immobilized and used to capture ³⁵S-GTPgS labeled G_as activated in Sf9 membrane suspensions.

When this "peptide on plate" (POP) assay was used the results clearly showed that G_as specific peptides could detect receptor activated G_as in membrane preparations. Furthermore, this assay as well as the phage ELISAs demonstrated convincingly that the use of second generation peptides led to a higher signal to background ratio when compared to their first generation parent peptides (Figs. 7 and 8) .

Example 204: In Vitro Assays Using GDP-dependent, GTP-dependent and/or GDP/GTP-independent Peptides for the Detection of Agonists and Antagonists of G-Protein Coupled Receptors The contents of Example 204 of the prior applications has been relocated out of the Examples.

Example 205 :Cell-Based Domain Complementation Assays Using GDP- dependent, GTP-dependent and/or GDP/GTP-independent Peptides for the Detection of Agonists and Antagonists of G-Protein Coupled Receptors

Here we describe a domain complementation assay. The basic premise for the domain complementation approach is to take the gene that codes for a reporter molecule (in most cases an enzyme), split it into two fragments (A' and B' ) and encode them in separate cistrons. This is done in such a way that the encoded fragments A and B do not by themselves interact and reconstitute a functional protein. To assemble an active protein, a gene encoding a fusion between fragment A and target protein (A:TP) is constructed and a second gene encoding a fusion between fragment B and a ligand (B:L) is constructed. If L and TP interact, they serve as the molecular glue to bring fragments A and B together, allowing for the reconstitution of activity. This activity can then be used as a read out for the interaction of the ligand with the target protein. Reporter genes that have been used in this manner are DHFR (Pelletier, J.N., F.X. Campbell-Valois, and S.W. Michnick, Proc Natl Acad Sci U S A, 1998. 95(21): p. 12141-6, see WO9834120A1) , adenylate cyclase (Karimova et al., PNAS 1998), β- galactosidase (Rossi et al. PNAS 1997, Mohler et al, WO98/06648 and Proc. Nat. Acad. Sci. (USA), 93:12423-7 (October 29, 1996).

Many other proteins may be used in this way and this example is intended to show the concept of using this technique for GPCRs, not limit the scope of reporter molecules. For the rest of this example DHFR is used. DHFR activity can be easily monitored in intact cells using fluorescein methotrexate. This fluorescent molecule readily enters cells, however, it is very efficiently pumped out by a membrane pump. Fluorescein methotrexate will bind to active DHFR inside the cells and is then retained, resulting in cells that are fluorescent.

Pelletier, et al., Proc. Nat. Acad. Sci. (USA), 95:12141-6

(October 13, 1998) . First a fusion between DHFRA and G_α is made and a fusion between DHFRB and a T peptide. A preferred DHFR protein would be murine DHFR, (E.C. 1.5.1.3, accession # NP_034179) . Domain A would preferably include residues 1-107, domain B preferably from residues 108-187. A 30 amino acid linker preferably would be used as described previously (Remy, I. Et al, Science 283:990-993. 1999). The fragment DHFRA preferably would be inserted into Galpha protein as described in previous work on Galpha s (Levis and Bourne, J Cell Biol., 119(5)1297-1307. 1992), Galpha i (Richardson and Robishaw J. ^' Biol. Chem., 274(19), 13525-13533. 1999), Galpha q (Hughes et al. J. Biol. Chem. 276(6) 4227-4235. 2001), and Galpha z (Wilson and Bourne, J. Biol. Chem. 270(16) 9667-9675. 1995).

Stable or transiently transfected cell lines are then established that express both of these fusions. Ideally, the cell type chosen would not have a GPCR that would interact with the DHFR:G_α fusion, however, this would not be a requirement of the system. The cell lines would then be tested for background levels of DHFR activity. One would expect the level to be very low in the absence of a GPCR that would activate the G_α. Once a cell line with low background activity is established, a expression construct encoding a GPCR would then be introduced and additional cell lines established that now expressed all three proteins (DHFRA: G_α , DHFRB: T-peptide, and the GPCR of interest) . Addition of agonist to the culture media of these cells will result in the activation of the GPCR, which will in turn activate the DHFRA: G_α fusion causing it to release GDP and bind GTP. The conformational changes that accompany this activation and exchange of nucleoside will allow the DHFRB: T-peptide to recognize and bind to the DHFRA:G_α fusion. This will result in bringing DHFRA and DHFRB together, allowing them to reconstitute active DHFR. These cells will then selectively retain fluorescein methotrexate which is readily detectable by a number of methods, fluorimeter, fluorescence microscopy, fluorescence activated cell sorting (FACS) to mention the most widely used. This is a cell based screen for agonists of the GPCR introduced into the cell line. Appropriate controls for potential active compounds include testing the compounds on the cell lines lacking the GPCR and on cell lines lacking the DHFRB: T-peptide and DHFRA: G_α fusions (no activation should be observed in either case) . Specificity of the potential agonists can be tested on cell lines expressing a different GPCR (again, no activation should be observed) .

It is also possible to configure this system to detect antagonists. In this case, an agonist is added, preferably in subsaturating concentration, such as concentrations that will give approximately 50%-75% activation of the receptor, and hence 50%-75% maximal fluorescence. Potential antagonists are then added at high concentrations and a drop in the fluorescence intensity should be observed when an antagonist is present. Appropriate controls for these experiments would be testing these compounds on cells with a different GPCR expressed as well as cells that express full length active DHFR

(no reduction in fluorescence should be observed in either case) .

Example 205. 1 : Detection of agonists of the M2 muscarinic receptor in CHO cells

Chinese hamster ovary (CHO) cells expressing the Gi coupled Acetylcholine M2 Muscarinic receptor would be used to screen for agonists. Alternatively Gαi could be coexpressed in CHO cells with the M2 receptor to increase expression levels. These cells contain endogenous Giα which is the cognate G protein for M2 Muscarinic receptors. The signaling protein will be an engineered version of the Ras protein fused to a T-peptide. This Ras fusion would contain a single amino acid substitution mutation that makes it constitutively active, a large number of which have been extensively characterized and can be at a number of different residues. In addition, this Ras fusion would have a mutation in its CAAX box, located at the C terminus, that would remove its ability to localize to the cell membrane. Ras must be localized to the membrane in order to activate downstream signaling pathways. The T peptide-Ras fusion will be located in the cytosol and thus will not initiate a signal unless brought to the membrane. In the absence of agonist, Giα will exist in the GDP form and the T peptide-Ras fusion will remain localized in the cytosol. Upon activation of the GPCR by an agonist, which will cause Giα to exchange GDP for GTP, the GTP bound Giα subunits will attract the T peptide-Ras fusion to the membrane. Once localized to the membrane, the transforming Ras will initiate signaling through the Map Kinase pathway resulting in increased transcription on promoters containing AP-1 response elements. A reporter construct carrying multiple AP-1 response elements fused to luciferase will be used to monitor activation of this pathway. Thus in the presence of agonist, an increase in luciferase activity will be observed.

To insure that this signal is specific, a system in which the T Peptide-Ras fusion has a mutation that prevents binding to the Gia will be utilized. In this case, potential agonists should not increase the activity of the AP-1 luciferase reporter.

Example 205.2 : Detection of antagonists of the M2 muscarinic receptor in CHO cells

Utilizing the system described above, we can detect antagonists for the same receptor. The experimental system is the same. In this case an agonist will be used at a concentration that produces half maximal induction of luciferase activity. It is important that the concentration and time dependence of the agonist being used is carefully titrated to determine the time and concentration needed to produce a linear response to agonist. At 50% maximal agonist concentration, any blocking of binding to the receptor by antagonist will result in a reduction in luciferase activity.

Cells would first be incubated in the presence of high concentrations of potential antagonists. After one hour, agonist will be added and the cells will be incubated for the time required to provide a linear response to this concentration of agonist. After this time, luciferase activity will be monitored. If a compound prevents activation of the M2 Muscarinic receptor, less of the Gα will exist in the GTP form and hence less of the T-peptide Ras fusion will be localized to the membrane. This will result in less signaling through the Map Kinase pathway and less AP-1 activity, resulting in lower transcription of the luciferase reporter and lower luciferase activity.

Example 206: Cell Based "Two-Hybrid" FRET Assay Using GTP- dependent Peptides for the Detection of Agonists and Antagonists of G-Protein Coupled Receptors

Fluorescence Resonance Energy Transfer (FRET) is a method for detection of complex formation, such as ligand-receptor binding, that relies upon the through-space interactions between two fluorescent groups. A fluorescent molecule has a specific wavelength for excitation and another wavelength for emission. Pairs of fluorophores are selected that have an overlapping emission and excitation wavelength. Paired fluorophores are detected by a through-space interaction referred to as resonance energy transfer. When a donor fluorophore is excited by light, it would normally emit light at a higher wavelength; however, during FRET energy is transferred to the acceptor fluorophore allowing the excited donor to relax to the ground state without emission of a photon. The acceptor fluorophore becomes excited and release energy by emitting light at its emission wavelength. This means that when a donor and an acceptor fluorophore are held in close proximity (<100 Angstroms), when one fluorophore is attached to a ligand and one is attached to a receptor and the ligand binds to the receptor, excitation of the donor is coupled with emission from the acceptor. Conversely, if no complex is formed the excitation of the donor results in no emission from the acceptor. A common modification of this technique, sometimes referred to as fluorescence quenching, is accomplished using an acceptor group that is not fluorescent but efficiently accepts the energy from the donor fluorophore. In this case, when a complex is formed the excitation of the donor fluorophore is not accompanied by light emission at any wavelength. When this complex is dissociated the excitation of the donor results in emission of light at the wavelength of the donor. FRET is an important technique used to examine the activation state of proteins inside of cells using fluorescently-labeled proteins or peptides as probes. We can use FRET to determine the activation state of a GPCR in cells by directly monitoring the activation state of its cognate G protein.

Briefly, the presence of GTP-bound Gα subunits indicates that the cognate GPCR is in its active conformation and a GDP- bound Gα subunit indicates an inactive cognate receptor. One could use fluorescently labeled peptide probes that bind specifically to the GTP or GDP-bound forms of Gα subunits.

We have identified peptides that bind specifically to GTP and GDP-bound forms of Gα subunits (T-peptides and D-peptides, respectively) as well as peptides that bind independent of bound GDP or GTP (I-peptides) . To examine the effects that a compound may have on a GPCR, one may use (1) an I-peptide, and

(2) a T-peptide or D-peptide, with fluorophores that are matched for FRET. Two specific examples of fluorescent probes that are matched for FRET include the green fluorescent protein (GFP) and the blue fluorescent protein (BFP) . -The GFP or BFP probes could be fused to the D-, T- or I-peptides to monitor the activation state of the GPCR by FRET. Labeled peptides could be forced into cells by electroporation, liposomes, internalization sequences or by any other method that allows peptides to enter cells. The I- peptides will bind specifically to the Gα subunit that it was isolated from .during phage display, independent of G protein activation state, and the T-peptides will only bind the same Gα subunit upon receptor activation and subsequent G protein activation (GTP-bound Gα conformation) . D-peptides will be bound to inactive heterotrimeric G proteins (cognate GPCR is inactive) . In a cell-based system where a GPCR is functionally coupled to its cognate G protein and both fluorescently-labeled I- and T-peptides are inside of the cells, addition of a receptor agonist will result in an increase in intensity of the emission wavelength signal of the acceptor. On the other hand, if I- and D-peptide probes are loaded into cells, addition of an agonist will result in a decrease in emission signal compared to the signal before agonist activation. Antagonists to a specific receptor could be identified in a cell containing fluorescently-labeled I-peptides and T- peptides by adding Kd concentrations of an agonist after the putative antagonist has bound the receptor. In this instance, FRET will not occur if the compound is an antagonist. As an agonist is added to antagonist bound receptor, the intensity of the emission wavelength signal of the acceptor may increase. This increase in intensity occurs as the agonist displaces the antagonist at the ligand-binding site. However, high concentrations of an antagonist will suppress this signal. Another way to examine antagonists would be to load the cells with I-peptide and D-peptide probes then bind antagonist and agonist as described above.- As an agonist is added to antagonist-bound receptor, the intensity of the emission wavelength signal of the acceptor will decrease. Another method for examining the activation state of a GPCR using FRET would be to use a fluorescently-labeled T- peptide or D-peptide and a fluorescently-labeled Gα subunit. Again GFP and BFP fusions matched for FRET could be used as the fluorescent probes in these assays. If T-peptide fusions were forced into cells, activation of the GPCR by an agonist would allow the T-peptide fusion to bind to the activated Gα subunit fusion resulting in FRET. This receptor activation would result in an increase in signal intensity as described previously. The presence of a bound agonist would also result in a decrease in FRET signal intensity if a D-peptide fusion and the Gα subunit fusion are present in cells. Antagonist binding to the receptor could be determined using these two fluorescently-labeled fusions as previously described. Example 207: Cell Based "One Hybrid" Assay Using GDP-dependent, GTP-dependent and/or GDP/GTP-independent Peptides for the Detection of Agonists and Antagonists of G-Protein Coupled Receptors

The traditional yeast two hybrid system monitors protein/protein interactions in the nucleus by identifying proteins that bring together the DNA binding domain and the transcriptional activation domain of a transcription factor. Although this system is useful for examining some types of interactions, it is not applicable to interactions that involve transcriptional regulators, proteins modified in the cytoplasm, membrane associated proteins or integral membrane proteins.

To monitor interactions between proteins localized to the cytoplasmic membrane, a system which detects protein/protein interactions using engineered components of cell signaling pathways could be used.- In the case of a G protein-coupled receptor (GPCR) , this system would be set up in a cell line expressing the GPCR of interest, its cognate heterotrimeric G protein and a modified signaling protein. To monitor the activation state of the GPCR, we will take advantage of the fact that the alpha subunit of the associated heterotrimeric G protein (G_α) exists in either a GTP or GDP-bound form which correlates to the active and inactive forms of the receptor. The peptides from the previous examples have been shown to bind specifically to either the GTP form (T peptides), GDP form (D peptides) or either form (I peptides) of G_α. One of these peptides would be expressed fused to a mutant of. signaling protein which lacks a functional membrane activator. A signaling protein is normally a membrane bound protein which is active substantially only at the membrane, whereupon it may, possibly in conjunction with other substances, cause the production of a detectable signal. When the GPCR is in the inactive state (i.e. no agonist bound) , a T peptide-membrane anchor-free signaling protein mutant fusion would be in the cytoplasm. When the GPCR is activated upon agonist binding, the Gα subunit binds GTP and thus the T peptide-signaling protein fusion would be localized to the membrane by binding to the G_α-GTP. Once at the membrane, the fusion protein would activate a signaling pathway. In this way the activity of the T peptide-signaling protein is dependent on its localization to the membrane. A reporter system would be set up so that the readout of the T peptide-signaling protein's activity could be measured.

For detection of agonists and antagonists of other membrane receptors, we would develop biokeys specific to the active or inactive forms of these receptors, or of a second molecule (e.g., a second messenger) which changes form as a result of activation with these receptors.

The Biokey would then be fused to a signaling protein (or signaling protein activator) . In the case of an active form- specific Biokey, activation of the receptor would lead to binding of the Biokey to it's binding partner (the activated receptor itself, or the activated accessory molecule) and hence to localization of the fused signaling protein moiety to the membrane, and consequent signal production.

An example of a signaling protein would be a Ras protein mutated at the CAAX box so that it is not anchored at the membrane in a normal manner. Hence, T fused to a T-peptide, the T-peptide 's movement to and from the cell membrane would control signaling through the Ras pathway. The mutant would also preferably be constitutive, so that when the Ras was brought to the cell membrane, it would be active without the need for any further inducer, and produce a signal. Any signaling protein which needs to be at the cell membrane to be active can be modified in a similar manner by removing its membrane anchor and preferably making it constitutively active at the membrane. The Biokey is acting in place of the normal anchor.

Example 300: The Use of Gα Chimeras to Screen for agonists or antagonists of GPCRs with GTP and GDP Specific BioKeys

The purpose of this example is to outline a basic screening strategy. It has been shown that individual G protein-coupled receptors interact with and signal through specific Gα protein subunits. For example, the M2 muscarinic receptor is coupled to Giα, resulting in inhibition of AC and decreased intracellular AMP levels. This receptor does not, however, interact with G_zα or G_sα. In this way the hundreds of GPCRs that exist are linked to the relatively few Gas and the appropriate signaling pathway and biological response. The specificity for this interaction is mostly contained in the C- terminal portion of the Gα subunits. By switching as few as three C-terminal amino acids in a Gα protein one can switch the receptor specificity of that G alpha without changing the downstream effectors that the Gα or βγ interact with. In one specific example a G_qα had three residues from a G_±α exchanged at the C-terminus. This chimeric protein, which was mostly G_q, then could interact with G protein-coupled receptors (GPCRs) normally associated G_t . See Conklin, et al . , Nature, 363:274- 276 (1993) . Upon stimulation with agonist the G_x associated GPCR now can recruit the chimeric Gα, interacting with the G_x portion and signaling through the G_q portion. Upon stimulation of a receptor normally coupled to Gi with an agonist, G_q specific downstream effectors such as inositol phosphate or calcium flux were measured.

Besides this Gqα/Gi2α chimera, Conklin describes others in which the C-terminal is obtained from G₀α, G_i2α and G_sα. Fong et al., Molecul. Pharmacol., 54:249-57 (1998) constructed a chimera of G_±1α and G_sα, in which the last six amino acids of the former were replaced with the corresponding residues of the latter. This construct was used to monitor G_as coupled receptors with the reagents described in Example 204, Protocol 3.1, which are specific for G_aι (see below for figures and description) .

A different permutation of this idea could be used to enhance screening for GPCRs using Gα subunit specific BioKeys. One Gα could be used to create a set of Gα chimeras with the same backbone but different C terminal regions allowing the chimeras to be coupled to all classes of receptors. For example, to use the Gαq as the backbone we could take AA's 1 through 355 (accession number AAB64301.1) and fuse it to the last five amino acids of Gαi, (residues 350-354, accession number P04898) G s (residues 390-394, accession number P04895) , or Gαl3 (residues 373-377, accession number NP 306563.1) . (Conklin et al. Nature 363:274-276 (1993). It is not necessary to use exactly 5 AA. You can use more or less in the range from 3-13, most preferably 4-9. In addition although every fusion has not been made or shown to work, due to the similar structure of Gα's it is suspected that this scheme will be successful. In this way, one set of BioKeys could be used to screen all G protein coupled receptors using one set of well characterized BioKeys. The portion of the Gα that the BioKeys bind to would be held constant in the chimeric proteins while the portion of the Gα binding to the receptor would be varied. A set of cell lines expressing such a battery of chimeric proteins could be made and used to screen receptors without knowing the proper cognate Gα. The chimeras would be expressed individually or in groups as some GPCRs interact with several Gα's, see Guderman, et al., Ann. Rev. Neurosci, 20:399-427 (1997). The screens could be done with any of the techniques described in the other prophetic examples including fluorescence based assays, enzyme activity reconstitution assays, and those assays using cell signaling components. Specific examples

Example 300a : The Use of a G_sa-G_ia Chimera to screen for Agonists or Antagonists of a GPCR tha t is not Normally Coupled to Giα Using PCR primers encoding the C-terminal 6 amino acids from G_as fused to amino acid 348 of G_ail a chimeric cDNA molecule was amplified with an open reading frame (ORF) identical to the one described by Fong et al. (see above), except that the G_ail sequence used was from human G_ail (differs in 1 amino acid residue from the rat sequence in the work cited) . This construct was produced as a baculovirus and Sf9 cells were infected with this "G_ai6s" virus along with the Gs coupled b2- Adrenergic Receptor (b2AR ) and G_bl and G_g2. Membranes prepared from these cells were used in assays analogous to the ones described in Example 204. When these membranes were tested with the specific adrenergic agonist isoproterenol there was a clear signal over background which could be inhibited in a dose-dependent manner by the inverse agonist ICI 118,551 (Fig. 14) . Also tested in this experiment was the influence of the presence of DTT. DTT has been described as pre-activating the b2AR and possibly enhancing signaling (Pedersen and Ross, 1985 J Biol Chem 260 (26) : 14150-7) , even though there are also reports which indicate interference with ligand binding (Lin et al., 1996, Biochemistry 35 (46) : 14445-51) . The above results would indicate that DTT does not increase the signal, but could lead to higher background. Because of that, it is omitted from most subsequent assays.

In order to test whether the assays designed in this way reflect the pharmacological properties of GPCRs which had been established in other test systems, a selection of partial agonists of the β2AR were chosen as activators. This selection had been described by Lee et al., Biochemistry 38: (42)13801. The results (Fig. 15) were in perfect accordance with Lee at al. with respect .to both relative efficacies of the agonists as well as the detected rank order of potencies. All signals could be inhibited by the inverse agonist ICI 118,551, again demonstrating that they are truly receptor dependent. To demonstrate that the assay is modular in the sense that other Gs coupled receptors could be used in place of the b2AR, DI dopamine receptor was coexpressed with G_αi5s, G_β and G_γ and tested in an analogous way. Dose response curves were produced with the specific agonist CY 208-243. As was the case with the b2AR, the results proved that pharmacological profiles procured by this assay are in good agreement with literature values.

This assay was also used to test the influence of small variations in pH in the activation buffer. The dose response curves obtained with buffers between pH7.2 and 7.8 did not differ significantly, further advancing the view that the robustness of the assay makes it suitable for a high throughput setting. The use of the G_aies chimera was extended to the FRET based detection system in a way analogous to what has been described above: The internally His-tagged G_ai construct was modified at its C-terminus by replacing the residues KDCGLF (SEQ ID NO: 134) with the RQYELL (SEQ ID NO: 135) motif present in G_as. Coexpression of this chimera together with the Beta-2 Adrenergic Receptor was tested and there was a signal over background of 3.9 (Fig. 17)

Example 300b: The Use of a set of G_qα-Giα Chimeras to screen for Agonists or Antagonists of a GPCR tha t is not Normally Coupled to Giα

There is ample evidence for the important role which the C-terminus of the G protein has in receptor coupling. Several examples of G protein chimeras which couple a receptor to none- native downstream effectors exist which are only mutated within the C-terminal 6 amino acids. Based on the remark by Milligan and Rees (TIPS, 20:118-124 March 1999) that they had only "limited success" in engrafting Gq coupling onto a Gi backbone and the arguments made by Hamm et al. for an involvement of the N-terminal domain (Proc. Nat. Acad. Sci., 98:4819-21 April 24, 2001) in receptor - G protein coupling we designed a set of chimeras between these two which also involved manipulation of the N-terminus. Depicted in a diagram in Fig. 18 are constructs which have been expressed in the baculovirus system. Expression of all constructs has been demonstrated by Western Blot analysis.

When constructs 175 and 176 were coexpressed with the Ml AChR there was no detectable signal over background. Coexpression of the receptor with construct 211 resulted in a maximal signal to background of 2.1:1 (data not shown). This signal was not sufficient to develop an assay comparable to the M2 AChR/ G_ai assay described above and will require modifications.

More extensive domains from G_aq were chosen to graft onto a G_ai backbone (see constructs 450-453) . When tested in the context of the Ml AChR, these constructs gave very similar results to what is described for construct 211 above (data not shown), with a maximal signal to background of 2:1.

These data show that the property of specific coupling to a receptor does not exclusively reside in either the N- or C- terminal domain but rather is dependent on a specific structural context, which in some cases, like G alpha i6s, is interchangeable between G_a subclasses while it is not in others .

Example 300c: The Use of G_qα-G_sα Chimeras to screen for Agonists or Antagonists of a GPCR that is not Normally Coupled to G_sa

In this prophetic example a GPCR specific for G_aq coupling is coexpressed with a chimeric G protein consisting of G_as amino acids 1-390 (numbering in gi2144866) linked to 355-359 from G_aq (numbering in qil2620875) . Exchanging these amino acids has been described by Conklin et al. (Mol. Pharm. 50:885, 1996) , who demonstrated coupling of some Gq specific receptors to adenylyl cyclase by this type of chimera. Using G_as specific peptides an assay is designed which can monitor G_aq coupled receptors. Alternatively, the number of amino acids swapped between G_as and G_aq can be varied, between 1 and 21, ideally between 3 and 8.

Example 300d: Use of further chimeric constructs to refine specificity of the assay

Further chimeric constructs can be made to analyze the specificity of individual receptors. The C-terminal amino acids of G_ai3, G_aθ G_a2, G_al2, G_al3, G_al4 and G_al5 could be grafted onto either G_ai, G_as or G_aq backbones in order to "redirect" receptor coupling and obtain more detailed information about specific interactions. In all these examples the number of amino acids exchanged would lie between 1 and 21, most preferably 5 or 6.

The invention, as contemplated by applicant (s) , includes but is not limited to the . subject matter set forth in the appended claims, and presently unclaimed combinations thereof. It further includes such subject matter further limited, if not already such, to that which overcomes one or more of the disclosed deficiencies in the prior art . To the extent that any claims encroach on subject matter disclosed or suggested by the prior art, applicant (s) contemplate the invention (s) corresponding to such claims with the encroaching subject matter excised.

The term "comprising" , as used herein, signifies that the comprised element (s) is/are required, but that other elements may be added, if they do not violate some explicit exclusion . The term "consisting" , as used herein, signifies that the consisted element (s) is/are required, and that other elements cannot be added. The term "consisting essentially of" is of intermedia te scope, implying that unreci ted elements can be added if they do not change the basic nature of the thing in question . All references cited anywhere in this specification are hereby incorpora ted by reference, as are any references cited by said references .

171

Table A: List of Proteins for Analysis:

Target Modulators of Activity

GPCRs β-adrenergic receptor Isopreterenol (agon) , alprenolol (antag)

Rhodopsin Dopamine D2 Dopamine (agon) , haloperidol (antag) opioid Leu-enkephalin (agon) , Naltrindole (antag)

Endothelin Endothelin l(agon), BQ-123 (antag)

Erythropoietin receptor Erythropoietin FAS ligand receptor FAS ligand Interleukin receptor Interferon (agon) IL-6 (agon)

Table A: GPCRs and their respective ligands

(This list is not comprehensive for the receptors shown and it is just intended as an example)

Sources: The G-protein linked receptor Factsbook (Academic Press); "Pharmacology" (Churchill-Livingstone); Tocris website on adrenoceptors http://www.tocris.com/adrenorev.htm

TABLE G: HUMAN G PROTEIN-COUPLED SEVEN TRANSMEMBRANE RECEPTORS: REFERENCES FOR CLONING

Receptor Reference oiji-adrenergic receptor Bruno et al. (1991) α_1B-adrenergic receptor Ramarao et al. (1992) α₂- drenergic receptor Lomasney et al. (1990) α_2B-adrenergic receptor Weinshank et al. (1990) β_x-adrenergic receptor Frielle et al. (1987) β₂-adrenergic receptor Kobilka et al . (1987) β₃-adrenergic receptor Regan et al. (1988) itij AChR, m2 AChR, m₃ Bonner et al . (1987) AChR, m₄ AChR Peralta et al. (1987) m5 AChR Bonner et al. (1988)

D_x dopamine Dearry et al. (1990) Zhou et al. (1990) Sunahara et al. (1990) Weinshank et al. (1991)

D₂ dopamine Grandy et al . (1989) D₃ dopamine Sokoloff et al. (1990) D₄ dopamine Van Tol et al. (1991) D₅ dopamine M. Caron (unpub.) Weinshank et al. (1991)

Al adenosine Libert et al. (1992) adenosine A2b Pierce et al . (1992) 5-HTla Kobilka et al. (1987) Fargin et al . (1988)

5-HTlb Hamblin et al. (1992) Mochizuki et al. (1992)

5HTl~like Levy et al . (1992a)

5-HTld Levy et al . (1992b)

5HTld-like Hamblin and Metcalf (1991) 5HTld beta Demchyshyn et al. (1992) substance K (neurokinin A) Gerard et al. (1990) substance P (NK1) Gerard, et al. (1991); Takeda et al. (1991) f-Met-Le -Phe Boulay et al. (1990) Murphy & McDermott (1991) DeNardin et al. (1992) angiotensin II type 1 Furuta et al. (1992) mas proto-oncogene Young et al . (1986) endothelin ETA Hayzer et al. (1992) Hosoda et al. (1991) endothelin ETB Nakamuta et al. (1991) Ogawa et al. (1991) thro bin Vu et al. (1991) growth hormone-releasing Mayo (1992) hormone (GHRH) vasoactive intestinal Sreedharan et al . (1991) peptide (VIP) oxytocin Ki ura et al., (1992) so atostatin SSTR1 and Yamada et al. (1992a) SSTR2

Yamada et al. (1992b)

SSTR3 cannabinoid Gerard et al. (1991) follicle stimulating Minegish et al. (1991) hormone (FSH)

LH/CG Minegish et al. (1990) thyroid stimulating Nagayama et al. (1989) hormone (TSH) Libert et al. (1989) Misrahi et al. (1990) thromboxane A2 Hirata et al . (1991) platelet-activating factor Kunz et al. (1992) (PAF)

C5a anaphylatoxin Boulay et al . (1991) Gerard and Gerard (1991)

Interleukin 8 (IL-8) IL- Holmes et al. (1991) 8RA

Murphy and Tiffany (1991)

IL-8RB

Delta Opioid Evans et al. (1992) Kappa Opioid Xie et al. (1992) mip-1/RANTES Neote et al. (1993) Murphy et al . , in press

Rhodopsin Nathans and Hogness (1984)

Red opsin, Green opsin, Nathans, et al . (1986) Blue opsin metabotropic glutamate Tanabe et al . (1992) mGluRl-6 histamine H2 Gantz et al . (1991)

ATP Julius, David (unpub.) neurope tide Y Herzog et al . (1992) Larhammar et al . (1992) amyloid protein precursor Kang et al . (1987) Mita, et al . (1988) Lemaire et al. (1989) insulin-like growth factor Kiess et al . (1988) II bradykinin Hess et al. (1992) gonadotropin-releasing Chi et al. (1993) hormone cholecystokinin Pisegna et al . (1992) melanocyte stimulating Chhajlane et al. (1992) hormone receptor Mountjoy et al. (1992) antidiuretic hormone Birnbaumer et al. (1992) receptor glucagon receptor Sprecher et al. (1993) adrenocorticotropic Mountjoy et al. (1992) hormone II

For complete citations to references, see references following Table 2 of Fowlkes, et al., USP 5,789,184. For additional GPCRs, and/or their ligands, see http://www.gpcr.org/7tm/ http://tinygrap.uit.no/. (mutant GPCR information) http://pdsp.cwru.edu/PDSP.asp (list of many receptors and ligands including binding affinity data)

Table H GPCR/G Protein Coupling

Receptor Subtype

Coupling to: Not to

(Muscarinic) Ml Gq Gi, Gs

Acetylcholine M2 Gi Gq, Gs

M3 Gq Gi, Gs

M4 Gi Gq, Gs

M5 Gq Gi, Gs

Adenosine Al Gi Gq,Gs

A2A Gs Gi,Gq

A2B Gs Gi.Gq

A3 Gi Gq,Gs

Adrenergics αla Gq Gi,Gs αlb Gq Gi,Gs αlc Gq Gi, Gs aid Gq Gi, Gs a2a Gi Gq, Gs α2b Gi Gq,Gs α2c Gi Gq,Gs βl Gs Gi, Gq β2 Gs Gi, Gq β3 Gs Gi, Gq

Adrenomedullin Gs Gi, Gq

Angiotensin Ala Gq,Gi Gs

Alb Gq,Gi Gs

A2 unclear, not Gas Gs (?)

Bombesin Bb Gq, (?)

GRP Gq Gi,Gs

Neuromedin ? ?

Bradykinin BK1 Gq, Gi(?) Gs

BK2 Gq, Gi(?) Gs

C5a Gq Gi, Gs

Calcitonin 1 Gs Gi, Gq

Calcitonin Gene Related CGRP1 Gs? ?

Peptide (CGRP) CGRP 2 Gs? ?

Amylin Gs Gi, Gq

Cannabinoid CB1 Go, Gi Gq, Gs

CB2 Go,Gi Gq, Gs

Chemokines CCR1 GJ Gs

CCR2 G Gs

CCR3 Gi Gs

CCR4 G Gs

CCR5 G Gs

CCR6 Gi Gs

CCR7 G_ Gs 173

CCR8 Gi Gs

CCR9 Gi,Gq(?) Gs

CCR10 Gi,Gq(?) Gs

CCR11 Gi,Gq(?) Gs

CXCR1 Gi_;Gq(?) Gs

CXCR2 Gi Gs

CXCR3 Gi Gs

CXCR4 Gi Gs

CXCR5 Gi,Gq(?) Gs

CX3CR1 Gi Gs

XCR1 Gi Gs

Cholecystokinin CCK_A Gq Gi,Gs

CCK_B Gq Gi,Gs

Corticotropin Releasing Gs Gi,Gq

Factor (CRF)

Dopamine DI, Gs Gi, Gq

D2 Gi Gq,Gs

D3 Gi Gq, Gs

D4 Gi Gq,Gs

D5 Gs Gi, Gq

Endothelin ET_AR Gq, (Gs) Gi

ET_BR Gq, (Gi) Gs

£ LP Gi? Gq,Gs

GABA_B Gi?

Galanin Gi, Gq Gs

Glucagon and Glucagon GR Gs Gi, Gq

Like Peptide GLP-1 Gs, also Gq and Gil&2?

GLP-2 Gs Gi,Gq

Metabotropic Glutamate mGlul Gq Gi, Gs

(mGlu) mGlu2 Gi Gq,Gs mGlu3 Gi Gq, Gs mGlu4 Gi Gq, Gs mGlu5 Gq Gi, Gs mGluό Gi Gq, Gs mGlu7 Gi Gq, Gs mGluδ Gi Gq, Gs

Glycoproteins FSH Gs Gi, Gq

LH Gs Gi, Gq^'

TSH Gs Gi,Gq

Gonadotrophin Releasing Gq Gi, Gs

Hormone (GnRH)

Histamine HI Gq Gi,Gs

H2 Gs Gi,Gq

H3 Gi Gs, Gq

H4 Gi Gs 174

5-Hydroxytryptamine 5HT1A Gi , Gq, Gs

(5-HT) 5HT1B Gi ' Gq, Gs

5HT1D ^■ Gi Gq, Gs

5HT1E Gi Gq, Gs

5HT1F Gi Gq, Gs

5HT2A Gq Gi, Gs

5HT2B Gq Gi, Gs

5HT2C Gq Gi, Gs

5HT4 Gs Gi, Gq

5HT5A Gi Gq, Gs

5HT6 Gs Gi, Gq

5HT7 Gs Gi, Gq

Latrotoxin Neurexin signalling unclear

CL signalling unclear

Leukotriene LTB4 Gi Gq, Gs

LTD4 Gq Gi, Gs

Melanin Concentrating MCH1 Gq? Gi, Gs?

Hormone, MCH MCH2 Gq? Gi, Gs?

Melanocortins ACTH (MC2- Gs Gi, Gq

(Melanotropins, MSH) R)

MSH (MC1-R) Gs Gi, Gq

MC3-R Gs Gi, Gq

MC4-R Gs Gi, Gq

MC5-R Gs Gi, Gq

MC6-R cloned?

Melatonin Mella (MTl) Gi Gq, Gs

Mellb (MT2) Gi

Motilin Gq?

Neuropeptide Y (NPY) Gi Gq, Gs

Neurotensin Gq Gi, Gs

Nucleotides (P2Y) P2Y1 Gq Gi, Gs

P2Y2 Gq (Gi*), Gs

P2Y4 Gq Gi, Gs

P2Y6 Gq Gi, Gs

P2Y11 Gs Gi, Gq

P2Y12 Gi Gq, Gs

Opioid mu Gi Gq, Gs kappa Gi Gq, Gs delta Gi Gq, Gs

Sphingosine- 1 -Phosphate EDG-1 Gi Gs

(SIP) EDG-3 Gi, (Gq) Gs

EDG-5 Gi, (Gq) Gs

EDG-6 Gi Gs

EDG-8 Gi, Gq Gs

Lysophosphatidic Acid (LPA) EDG-2 Gi Gs

EDG-4 Gi Gs

EDG-7 Gi Gs

Parathyroid Hormone (PTH) PTH1 Gs Gi, Gq

PTH2 Gs Gi, Gq

Platelet Activating Factor Gi or Gq Gs

(PAF)

Prostanoids DP Gs Gi, Gq

EP1 Gq Gi, Gs

EP2 Gs Gi, Gq

EP3 Gi Gq, Gs

EP4 Gs Gi, Gq FP Gq Gi, Gs

IP Gs Gi, Gq

TP Gq Gi, Gs

Somatostatin SSI Gi Gq, Gs

SS2 Gi Gq, Gs

SS3 Gi Gq, Gs

SS4 Gi Gq, Gs

SS5 Gi Gq, Gs

Tachykinin NK1 Gq (?) Gi, Gs

NK2 Gq Gi, Gs

NK3 Gq Gi, Gs

Thrombin PAR1 Gq, Gi Gs

PAR2 Gq Gs, Gi

PAR3 Gq?

PAR4 Gq?

Thyrotrophin Releasing TRH-R1 Gq Gi, Gs

Hormone (TRH) TRH-R2 Gq Gi, Gs

Vasoactive Intestinal Peptide GHRHR Gs Gi, Gq

(VIP) and Pituitary Adenylate VIP Gs Gi, Gq

Cyclase Activating PACAP Gs Gi, Gq

Polypeptide (PACAP) Secretin Gs Gi, Gq

Vasopressin VIA Gq Gi, Gs

V2 Gs Gi, Gq

Oxyt. Gq Gi, Gs

There are numerous lists in reviews and websites which offer databases aimed at a comprehensive inventory of GPCRs. Two problems make it difficult to name one such site or list as the best one: 1) there is a constant stream of information on newly discovered receptors or receptors for which the ligand has been identified ("deorphanization") . 2) There is a growing appreciation for the fact that many receptors that had been discovered as coupling to a particular signal transduction machinery in a particular tissue have slightly different roles in other tissues or cells. This is compounded by the fact that a lot of analysis of GPCRs occurs by expression in an exogenous system, which doesn't always reflect the actions of the receptor in its native environment. For that reason, any list of receptors and their coupling pathways can only be seen as temporary. A good example because it seems to be updated regularly is a database hosted at the CMBI (Centre for Molecular and Biomolecular Informatics in Nijmegen , The Netherlands, http://www. gpcr. org/7tm/) .

The list above is updated with respect to most receptors to the best of our knowledge but some assignments, especially for recently discovered receptors, may be controversial .

Table NC: N-terminus and C-terminus of selected G-protein alpha (a) subunits N-terminus

5713315 1 MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIV 50 ail

1181671 7 .A.C..E.A.E.RRINDE.E.HV.R.KRD.R.. L T IF 56 aq

386748 4 .E..QR.AN. .EKQ.QK.K VYRATHR 46 as

4504041 1 V A K 50 ai2

5729850 1 K 50 ai3

232134 1 ER..L A.EK..K.. -IS..KD 50 ao2

13649022 1 ..AGA...E. H.RELEKK.K..A..D..T 46 at

6680033 33 ERE.RR..RD..AL .ARERRAVR. .. I FL 74 al2

5729848 17 .. - .. G . -AEQQRK.. E .. KG . SREKTYVK. L .. I FR 69 al3

4758444 5 .C....E.ESQRI.AE.E. ..R.KKD.R..L T FI 52 al4

4504039 14 .TEDE...ARVDQE.N.I.L.QKKQDRG.L P FI 59 al5

C-terminus I

317 KEIYTHFTCATDTKWQFVFDAVTl^IIKNNLKDCGLF 354 ail

323 .I..S E.IR...A..K.T.LQL...EYN.V 358 aq

334 HYC.P V..E.IRR.. NDCR. I . QRMH . RQYE . L 384 as

318 355 ai2

319 E...Y 354 ai3

320 S.V N.I AK..RG...Y 354 ao2

313 S.M Q..K I...E 350 at

344 .PLFH...T.I..E..R...H..K.T.LQE IM.Q 381 al2

340 .PL.H...T.IN.E.IRL..RD.K.T.LHD...QLM.Q 377 al3

318 V..S D.IR...A..K.T.LQL..REFN.V 355 al4

325 RRLFS.Y Q. IRK.. KD. R. SVLARY. DEIN. L 375 al5

Table 202A: Giαl GDP/GTPyS-Independent Phage (I-Peptides)

1-1 Group

Sequence/Motif Aligned a Library Seq ID

99 AHLLTWSEFLDSHTK BUF E 1

103 (743) GELITWYEFLGDLNP BUF E 2

107 GELTTWYEFLSHGRP BUF E 3

361 DELTWWEFISD GTP W 4

388,391 VT YDFLMEDTK GTP C L 5

45 GL T REFLQE BUF R 6

397,401,412 NLMT EY ADGERL GTP Y 7

15r2,301,394 ADRL TWOEFLY BUF PHD12 8

380,381,140 KTYSLYEFLEL GTP (1) 9

16 QLLTLHEFLNS BUF H 10

360 RGEY EFLGY 11

101 (779) ADGIFWWEYAREAGE BUF E 12

387 (801) KWWESDWFVNFG GTP CWL 16

386 (816) EEGMD FMRVVE GTP CWL 17

1-2 group 375,123,125,247 (782) LGRGTTDMPPWAWWS GTP M 13 331,334 (786) NYTERPWVWYH GDP P 14 37 (782) LYSMEPWKWYT BUF P 15

Mote 1: Library is unknown.

Table 202B: Giαl GTP-Specifiσ Phage (T-Pept3Ldes)

ID Seguence/Motif Aliqned Liq Lib Seq ID

Group T-l

370,377,378 (792) SVLSSSEMCFGWACY GTP M 18

244 SEMCFGWACY GDP (3) 19

366, G12 FNEVCLGWQCY GTP K 20

G33,G34 NARPCQGWHCYLPSQ W<P> 21

Group T-2

353 WDGGVWMGPAS GTP w 22

408 (796) MGDSVLPYGGVWLGP GTP Y 23

G22,G25 YGGVWLGPEGN L 24

Gll,G26-29 WDGGVWWGQYG W 25

G9,G10 NLDGCFTSGGVWSGC X14C<W> 26

382 LGYDINGVWIG GTP N 27

Group T-3

384 (806) ICDIIPWEESC GTP j? 28

413 (817) ACGPAICPWDF PQL GTP PARO 29

Note 1: clone 244, which was identified in a screen for peptide which bound GDP.G-alpha, is suspected to havi increased the affinity of the G-alpha for GTP through a confor ational change.

Note 2: flanking SS or SR not shown.

Note 3: unknown library

Table 202C : Giαl GDP-Specific Phage (D-Peptides)

Sequence/Motif Aligned Buf Lib Seq Id

Group D-l

G4 GPQLTWQEFLTGAAS E 30

314 ( 757 ) NWTWWEFLGP GDP W 31

73 EFVTWKEFLGS BUF K 32

343 SQLTWREFLFG GDP R 33

217 HLMTWHEFISD GDP H 34

93 ( 740 ) DGFETWAEFLGASGS BUF E 35

62 LTWSEYLSEIDP BUF CWL 36

193 TVTWVDFLKET GDP D 37

324 MSWYEFMTEESM GDP CWL 38

400 AKHDLSWYE LQLPI GTP V 39

281 LSWWEFLGASDCGTC GDP X14C<W> 40

359 , 161 PLLS KEFIAT GTP K 41

380 , 381 , 140 KTYS YEFEL GTP N 43

176 PNLLTLEEFLS GDP L 42

409 , 24r2 SNRYTIYEFI-NLHS GTP Y 44

320 LHWWEVLAEK GDP CWL 45

230 POPLLHWWEM TEPP GDP KNK 46

213 AGESVHWWEVL GDP H 47

266 RAGPSEHWWEYIATL GDP W<P> 48

237 EMISWHQYLLSIENN GDP Y 49

126, 128 , 133 , 242 , 24 ! LRWDEFLMELGGGVA BUF M 50

379 VP WVWLAEGD GTP W 51

196 EIYWWDWLTDT GDP D 52

117 FGSNMLDLPTFLDWL BUF PARO 53

92 ITFWEL LEGG BUF L 54

179 TPYEWLGYWGA GDP L 55

289 YDMCTWLEFLDGGEC GDP X14C<W> 56 265 SPLCTWAEYLMEPSC GDP X14C<W> 57 273 TQWCTWAEFLSSTDC GDP X14C<W> 58 272, 282 , 6R2 DGCTWQEFLAGHGPC GDP X14C<W> 59

Group D-2

337,339 PFNNPPWMWWS GDP P 60

268 PTVHENLPPWLWWSP GDP PARO 61

330 LIHVPPWAWYD GDP P 62

329 GFDVPPWYWDF GDP P 63

280 YSQVFGDAPVWAWYSSR GDP PARO 64

319 WTPSDWQWWRSK GDP CWL 65

No Group 115 HWSSDSIFPGFWYSG BUF PARO 66 197 GGVDLDIGNSA GDP D 67 347 EGEDVRTRIAN GDP R 68

Note 1 : unknown library

Note 2: In general, flanking SS or SR are not shown. However, an exception was made in the case of clone 280, where a flanking SR was close to the conserved Trp (W) and clones 319 and 115 featured S in the same or similar position. Table 203

Correlation of First Peptides to Second Generation Libraries

Peptide Second Generation Library

Table 202C peptide 213 gα oligo 1 (D) AGESVHWWEVL (SEQ ID NO: 47) etc.

Table 202A peptide 397,401,412* gα oligo 2 (D) NLMTWYEYLADGERL (SEQ ID NO: 7)

Table 202 B peptide 370(792) gα oligo 3 (T) SVLSSEMCFGWACY (SEQ ID NO: 18) etc.

Table 202B peptide 353 (796) gα oligo 4(T) WDGGVW GPAS (SEQ ID NO: 22) etc.

Table 202B peptide 384 (806) gα oligo 5(T) ICDIIPWEESC (SEQ ID NO: 28)

*This I-peptide was a very good binder to GDP-loaded Gαi when analyzed as an AP fusion construct.

Table 204

Design of Second Generation Gαi Libraries

When AAs are bracketed, it means that one of the bracketed AAs is randomly chosen to appear at that position i a given member of the library.

Table 205

Peptides from Second Generation Gαi Libraries (cp Table 204)

LIBRARY #5 (T-3 motif) constrained residues shown in bold

(other positions could be any of 20 amino acids)

(See SEQ ID NO:_, Table 20

Phage ID tt SEQ ID NO galphai gtpgs 5-1, 2, 5 1755 SSRLCPEWICPWEWPASSR 74 galphai gtpgs 1-1 1756 SSERCPRWVCPWDYSSDSR 75 galphai gdp 5-2, 5-6 1757 SSGRCPRTICPWDYMGDSR 76

Gi24 1758 SRTYCPQWICPWEYQEFSR 77

Gi22, 23, 25 1759 SRWSCPPFICPWESDGISR 78

Peptide 5 806 (w/SS) SSICDIIPWEESCSR 79 Cluster Pal 807 (w/SS) SSACGPAICPWDFMPQLSR 80

LIBRARY #4 (T-2 motif) galphai gtpgs 4-3 1753 SSRGYYHGIWVGEEGRLSR 81 galphai gdp 4-6 1754 SSXHRXEGVWWGDRXGKSR 82

Peptide6 796 (w/SR) SRMGDSVLPYGGVWLGPSR 83

(X here means unknown, i.e. , the sequencer data was ambiguous)

SEQ ID

LIBRARY #3 (T-l motif)

Phaqe ID # galphai gtpgs 2-1, 2- -3 1744 SSKGPEICRGWGCYTRESR 84 galphai gtpgs 4-6 1745 SSVLDDVCIGWGCYNYSSR 85 galphai gtpgs 1-6 1746 SSSYSEHCQGWGCYARLSR 86 galphai gtpgs 3-2, 3- -5 1747 SRHDYEVCKGWGCYLGQSR 87 galphai gtpgs 2-6 1748 SSXEXEXCVGWGCYLRXSR galphai gdp 3-6 1749 SREGPEFCKGWGCYATGSR galphai gdp 3-3 1750 SRGEMEYCLGWQCYLGQSR 90 galphai gdp 3-2 1751 SSTRLDVCKGWECYVPRSR 91 galphai gtpgs 3-6 1752 SSAGPDSCFGWSCYVGGSR 92

Other Cluster 792 (w/SS ) SSSVLSSSEMCFGWACYSR 93

Table 206

Binding Affinities

Peptides 1746, 1753 and 1755 have comparable affinities. All have higher affinities than the four peptides in Table 206, but this has not been quantified.

Table 210

First Generation G s:GTP Binding peptides

Note

1686 a k.a. Gs-22/23 :S2-38(10), -39(11), -40(12)

1687 a k.a. Gs-21

1688 a k.a. Gs-20

1689 a k.a. Gs-17/18

1690 a k.a. Gs-19

1691 a k.a. Gs2-34(6 -35(7) -37(9)

(1) Peptide 2123 nominally came from the W<P> library, but has a sequence incompatible with the design of that library; the W is at the wrong position. It is believed to have occurred through contamination from another library, most likely the AroXAro library. These libraries were in adjacent wells. Hence, we believe that peptide 2123 should be considered to have been derived from the AroXAro library.

Table 211

Second Generation Gas : GTP Libraries

Table 212

Second Generation G s : GTP Peptides

ID

Parent 1687 SSIWGLASPWQSPRSPESR 108

Core X7 R2 H.23 2116 SSIWGLAHRVDVEMWARSR 109 Core X7 R2 L.3 2117 SSI GLATPVYRGDNNYSR 110 Core X7 R3 H.14 2121 SSI GLARPWRGGDDEVSR 111 Core X7 R3 H.17 2122 SSIWGLADPWSMWDRDSSR 112 Core X7 R3 H.ll 2120 SSIWGLATPWRVDDDEWSR 113 Core X7 R2 L.5 2118 SSI GLAQPWAPVHALNSR 114 Core X7 R3 H.l 2119 SSIWGLATPWSAEKRGESR 115 Core X7 R2 H.6 2115 SSIWGLANQYTPWGGGFSR 116

Fix WG/PW R4 H.4, 9, 18 2109 SSIWGLATPWPAREGPMSR 117

Fix WG/PW R4 H.l, 17 2108 SSIWGLATPWERTESRPSR 118

Fix R3H19, 23; R4L12, H23 2107 SSIWGLAEPWKPKSAMESR 119

Fix WG/PW R4 L.2 2113 SSIWGLAVPWOPGSSEWSR 120

Fix WG/PW R4 H.19 2112 SSLWGLAVPWMPTPSTGSR 121

Fix WG/PW R4 H.6 2110 SSLWGLASPWEPRQGTLSR 122

Fix WG/PW R4 L.19 2114 SSLWGLALP EPTVRTASR 123

Fix WG/PW R4 H.10 2111 SRLWGGIWPWTEEELALSR 124

The X4 core X3 library did not yield in any phages selected.

Claims

ClaimsWe hereby claim:

1. A non-naturally occurring and/or at least partially purified peptide, which does not comprise a V-H or V-L homology unit of an antibody, which specifically binds to an activated G alpha subunit of a G protein.

2. The peptide of claim 1 where the G alpha subunit is activated by the binding of GTP thereto, the peptide ("T peptide") binding specifically to the GTP .bound G alpha subunit.

3. The peptide of claim 1 or 2 where the G alpha subunit is a G_x subunit.

4. The peptide of any one of claims 1-3 where the peptide comprises the T-l sequence Cys-Xaa-Gly-Trp-Xaa-Cys-Tyr

(residues 3-9 of SEQ ID NO: 125) where Xaa is any amino acid.

5. The peptide of claim 4 where the peptide comprises the extended T-l sequence Xaai-Xaa_a-Cys-Xaa-Gly-Trp-Xaa-Cys-Tyr (SEQ ID NO: 125) where Xaa_x is a charged residue and Xaa₂ is any amino acid.

6. The peptide of claim 5 where Xaa_x is acidic.

7. The peptide of claim 5 where Xaa_x is basic.

8. The peptide of claim 5 where Xaa₂ is hydrophobic.

9. The peptide of claim 8 where Xaa₂ is aliphatic.

10. The peptide of claim 8 where Xaa₂ is aromatic.

11. The peptide of any of claims 1-8 where Xaa₃ is Ala.

12. The peptide of any one of claims 1-3 where said peptide comprises the T-2 sequence Gly-Val-Trp-Xaa-Gly

(residues 2-6 of SEQ ID NO: 126) where Xaa is any amino acid.

13. The peptide of claim 12 which comprises the extended T-2 sequence

Gly-Gly-Val-Trp-Xaax-Gly

(SEQ ID NO: 126) where Xaa_x is any amino acid.

14. The peptide of claim 13 where Xaa_x is hydrophobic.

15. The peptide of claims 13 or 14 where Xaa_x is aliphatic.

16. The peptide of any one of claims 12-15 which comprises the sequence

Gly-Gly-Val-Trp-Xaa^Gly-Pro (SEQ ID NO: 127).

17. The peptide any one of claims 1-3 which comprises the sequence Gly-Gly-Val-Trp (residues 1-4 of SEQ ID

NO: 126) .

18. The peptide of any one of claims 1-3 which comprises the T-3 sequence

Pro-Trp-Xaa (residues 6-8 of SEQ ID NO: 128) where Xaa is a charged, acidic residue.

19. The peptide of claim 18 which comprises the extended T-3 sequence

Xaa_x-Xaa₂-Pro-Trp-Xaa where Xaal and Xaa2 are hydrophobic residues.

20. The peptide of claim 19 where Xaa_x is lie.

21. The peptide of claim 18 which comprises the T-3' sequence

Cys-Pro-Xaa₁-Xaa₂-Cys-Pro-Trp-Xaa₃-Xaa₄ (SEQ ID NO: 128) where Xaa_x and Xaa₂ are any amino acid, Xaa₃ is a charged, acidic residue, and Xaa₄ is any amino acid.

22. The peptide of claim 21 where Xaa_x is Glu, Arg, Gin or Pro.

23 The peptide of claim 21 where Xaa₂ is hydrophobic.

24 The peptide of claim 23 where Xaa₂ is Trp or Phe.

25 The peptide of claim 21 where Xaa₄ is hydrophobic.

26 The peptide of claim 21 where Xaa₄ is aromatic.

27 The peptide of any one of claims 1-26 which comprises

(a) a sequence set forth in any of Tables 202B or 205, or

(b) a sequence differing from a sequence of (a) solely by (i) a single nonconservative substitution and/or (ii) one or more conservative substitutions .

28. The peptide of claim 27 where the sequence (b) is at least 50% identical to a sequence of (a) .

29. The peptide of claim 27 where the sequence (b) differs from a sequence (a) solely by one or more conservative substitutions.

30. The peptide of claim 27 where the sequence (b) differs from a sequence (a) solely by one or more highly conservative substitutions.

31. The peptide of claim 27 where the single nonconservative substitution, if any, is semi-conservative.

32. The peptide of claim 27 where the sequence (b) differs from a sequence (a) solely by a single conservative substitution.

33. The peptide of claim 27 where the sequence (b) differs from a sequence (a) solely by a single highly conservative substitution.

34. The peptide of claim 27 which comprises a sequence of (a) .

35. The peptide of claim 27 which consists of a sequence of (a) .

36. The peptide of any one of claims 27-35 the sequence (a) is the sequence of peptide 1753.

37. The peptide of any one of claims 27-35 the sequence (a) is the sequence of peptide 1755.

38. The peptide of any one of claims 27-35 where the sequence (a) is the sequence of peptide 1746.

39. The peptide of any one of claims 1-38 which competively inhibits the binding of a peptide of Table 202B or 205 to GC_i:GTP.

40. The peptide of any of the preceding claims wherein all of the amino acid residues are genetically encoded residues.

41. The peptide of claim 1 or 2 where the G alpha subunit is a G_s subunit.

42. The peptide of claim 41, said peptide comprising the sequence

Xaa^Trp-Gly-Leu-Ala (amino acids 1-5 of SEQ ID NO:129) where Xaa_x is a hydrophobic amino acid.

43. The peptide of claim 42 where Xaa_x is lie, Leu, Val or Met.

44. The peptide of claim 43 where Xaa_x is lie.

45. The peptide of claim 43 where Xaa_x is Leu.

46. The peptide of any one of claims 42-45 which comprises the sequence Xaa^Trp-Gly-Leu-Ala-Xaag-Pro-Xaag (SEQ ID NO: 129) where Xaa₆ is any amino acid and Xaa₈ is a hydrophobic amino acid.

47. The peptide of claim 46 in which the Xaa₆ is a hydrophilic amino acid.

48. The peptide of claim 47 in which Xaa₆ is selected from the group consisting of Ser, His, Thr, Arg, Asp, Asn, and Gin.

49. The peptide of any one of claims 46-48 in which Xaa₈ is Trp.

50. The peptide of any one of claims 46-48 in which Xaa₈ is Val.

51. The peptide of any one of claims 41-50 which comprises (a) a sequence set forth in any of Tables 210 or 221, or

(b) a sequence differing from a sequence of (a) solely by (i) a single nonconservative substitution and/or (ii) one or more conservative substitutions.

52. The peptide of claim 51 where the sequence (b) is at least 50% identical to a sequence of (a) .

53. The peptide of claim 51 where the sequence (b) differs from a sequence (a) solely by one or more conservative substitutions.

54. The peptide of claim 51 where the sequence (b) differs from a sequence (a) solely by one or more highly conservative substitutions.

55. The peptide of claim 51 where the single nonconservative substitution, if any, is semi-conservative.

56. The peptide of claim 51 where the sequence (b) differs from a sequence (a) solely by a single conservative substitution.

57. The peptide of claim 51 where the sequence (b) differs from a sequence (a) solely by a single highly conservative substitution.

58. The peptide of claim 51 which comprises a sequence of (a) .

59. The peptide of claim 51 which consists of a sequence of (a) .

60. The peptide of any one of claims 41-59 which competively inhibits the binding of a reference peptide of Table 210 or 212 to Gas: GTP.

61. The peptide of any one of claims 41-60 wherein all of the amino acid residues are genetically encoded residues.

62. A peptide which binds to a GDP:bound G alpha subunit of a G protein.

63. The peptide of claim 62 which comprises the Dl-l sequence Xaa₄-Xaa₅-Xaa₆, where

Xaa₄ is a negatively charged amino acid, or is Val or Gin, Xaa₅ is Phe, Tyr, Trp, Met, Leu, He, Val or Ala and Xaa₆ is Met, Leu, He, Val or Ala.

64. The peptide of claim 63 which comprises the extended DI-1 sequence

X a^-Xaa₂—Xaa₃—Xaa—Xa ₅~Xaa_§ where Xaa_x is any amino acid,

Xaa₂ is Leu, He, Trp, Phe or Pro, and Xaa₃ is any amino acid.

65. The peptide of claim 64 where Xaa_x is Ser, Thr, Tyr, Phe, His or Pro.

66. The peptide of claim 65 in which Xaa_x is Thr.

67. The peptide of claim 65 in which Xaa_! is Ser.

68. The peptide of any one of claims 64-67 where Xaa₂ is Trp.

69. The peptide of any one of claims 64-67 where Xaa₂ is Leu.

70. The peptide of any one of claims 64-69 where Xaa₃ is Glu, Gin, Tyr, Trp, Val, Ser, Ala, His, Arg or Lys.

71. The peptide of any one of claims 64-69 where Xaa₄ is Glu.

72. The peptide of any one of claims 64-70 where Xaa₅ is Phe, Tyr or Trp.

73. The peptide of claim 72 where Xaa₅ is Phe.

74. The peptide of any one of claims 64-73 where Xaa₆ is Leu.

75. The peptide of claim 62 which comprises the DI-2 sequence

Trp-Xaa-Trp, where Xaa is any amino acid.

76. The peptide of claim 75 which comprises the sequence

Pro-Trp-Xaa-Trp.

77. The peptide of claim 76 which comprises the sequence

Pro-Pro-Trp-Xaa-Trp (SEQ ID NO: 130).

78. The peptide of any one of claims 75-77 which comprises the sequence

Trp-Xaa-Trp-Trp/Tyr .

79. The peptide of claim 78 which comprises the sequence

Pro-Trp-Xaa-Trp-Trp/Tyr .

80. The peptide of claim 79 which comprises the sequence Pro-Pro-Trp-Xaa-Trp-Trp/Tyr (SEQ ID NO: 131).

81. The peptide of claim 22 which comprises the DI supermotif sequence

Trp-Asp/Glu-Trp-Xaa where Xaa is any hydrophobic amino acid.

82. The peptide of claim 81 which comprises the sequence

Pro-Trp-Asp/Glu-Trp-Xaa.

83. The peptide of claim 82 which comprises the sequence Pro-Pro-Trp-Asp/Glu-Trp-Xaa (SEQ ID NO-.132).

84. The peptide of any one of claims 81-83 where Xaa is Trp or Tyr.

85. The peptide of any one of claims 62-84 where the G alpha subunit is a Gi subunit.

86. The peptide if any one of claims 62-85 which binds substantially more strongly to Gαi: GDP than to Gαi: GTP.

87. The peptide of any one of claims 62-85 which binds substantially equally to Gαi: GDP and Gαi: GTP.

88. The peptide of any one of claims 62-87 which competively inhibits the binding of a peptide of Table 202A to Gαi: GDP.

89. The peptide of any one of claims 62-87 which competitively inhibits the binding of a peptide of Table 202C to Gαi:GDP.

90. The peptide of any one of claims 62-88 which comprises

(a) a sequence set forth in table 202A, or (b) a sequence differing from a sequence of (a) solely by (i) a single nonconservative substitution and/or (ii) one or more conservative substitutions.

91. The peptide of any one of claims 62-87 or 89 which comprises

(a) a sequence set forth in table 202B, or

(b) a sequence differing from a sequence of (a) solely by (i) a single nonconservative substitution and/or (ii) one or more conservative substitutions.

92. The peptide of claims 90 or 91 where the sequence (b) is at least 50% identical to a sequence of (a) .

93. The peptide of claims 90 or 91 where the sequence (b) differs from a sequence (a) solely by one or more conservative substitutions.

94. The peptide of claims 90 or 91 where the sequence (b) differs from a sequence (a) solely by one or more highly conservative substitutions.

95. The peptide of claims 90 or 91 where the single nonconservative substitution, if any, is semi-conservative.

96. The peptide of claims 90 or 91 where the sequence (b) differs from a sequence (a) solely by a single conservative substitution.

97. The peptide of claims 90 or 91 where the sequence (b) differs from a sequence (a) solely by a single highly conservative substitution.

98. The peptide of claims 90 or 91 which comprises a sequence of (a) .

99. The peptide of claims 90 or 91 which consists of a sequence of (a) .

100. The peptide of claim 62-99 wherein all of the amino acid residues are genetically encoded residues.

101. The peptide of claim 41, said peptide comprising the sequence

Asp-Tyr-Phe-Glu-Trp-Asp-Gly-Trp (amino acids 1 to 8 of SEQ ID NO:133) .

102. The peptide of claim 101 which comprises the sequence

Asp-Tyr-Phe-Glu-Trp-Asp-Gly-Trp-Xaa-Gly (SEQ ID NO:133).

103. The peptide of claim 102 where Xaa is Ala or Pro.

104. The peptide of claim 101 which comprises the sequence of amino acids 3-17 of peptide 1686 or 3-17 of peptide 2123.

105. The peptide of claim 104 which comprises the sequence of peptide 1686 or peptide 2123.

106. The peptide of claim 105 which is peptide 1686 or peptide 1623.

107. The peptide of claim 60 where the reference peptide is peptide 1686 or peptide 2123.

108. The peptide of claim 60 where the reference peptide is peptide 1690 or 1691.

109. The peptide of claim 108 which is peptide 1690 or 1691.

110. The peptide of claim 60 where the reference peptide is peptide 1677.

111. The peptide of claim 110 which is peptide 1677.

112. The peptide of any one of claims 41-61 or 90-111 which binds said alpha subunit substantially more strongly when the subunit is complexed to GTP, than when it is complexed to GDP.

113. An assay kit for the identification of the activation state of a G protein coupled receptor (GPCR) , said kit comprising a first peptide according to claim 1, labeled with a first label.

114. The kit of claim 113, where said GPCR, when activated, causing the binding of GTP to a G alpha subunit of a protein coupled to said GPCR, where said first peptide binds specifically to the GTP bound G alpha subunit.

115. The kit of claim 114 wherein said first peptide is a peptide according to any of one of claims 1-61 or 101- 112, in labeled form.

116. The kit of claim 113, where said GPCR, when inactivated, causing the binding of GDP to a G alpha subunit of a G protein coupled to said GPCR, kit where said first peptide binds specifically to the GDP bound G alpha subunit.

117. The kit of claim 111 wherein said first peptide is a peptide according to any one of claims 62-85, 88, 90 in labeled form.

118. The kit of any one of claims 113-117 where said kit further comprises a second peptide, labeled with a second label, which binds to said G alpha subunit regardless of whether it is GDP or GTP bound.

118a. The kit of claim 118 where said second peptide is a peptide according to any one of claims 62-85, 87, 89, 91.

119. The kit of claim 118 where said first and second labels interact when said peptides both bind to said G alpha subunit .

120. The kit of any one of claims 113-117 where said kit further comprises a G alpha subunit labeled with a second label.

121. The kit of claim 120 where said first and second labels interact when said first peptide binds to said labeled G alpha subunit.

122. The kit of^' any one of claims 113-121 where said kit comprises said GPCR and a G protein to which said GPCR couples, and said G protein comprising said G alpha subunit to which said peptide binds.

123. The kit of claim 122 where said kit provides a membrane comprising said GPCR and said G protein.

124. The kit of claim 123 where said membrane is a mammalian cell membrane.

125. The kit of claim 123 where said membrane is an insect cell membrane.

126. The kit of claim 123 where said membrane is a yeast cell membrane.

127. The kit of claim 123 where said kit comprises a whole cell providing said GPCR and said G protein.

128. The kit of any one of claims 113-127 which comprises a plurality of different GPCRs.

129. The kit of claim 128 which comprises a first GPCR which couples to a first G protein and not to a second G protein, a second GPCR which couples to the second G protein but not to the first G protein, and said first and second G proteins .

130. The kit of any one of claims 113-129 where said kit comprises a chimeric G alpha subunit.

131. A method of identifying the activation state of a G-protein coupled receptor which comprises

(a) providing a G protein-coupled receptor in a potentially modulating environment comprising a Gα subunit which couples to an active conformation of said receptor when the Gα subunit is bound by GTP, and to an inactive conformation of said receptor when the Gα subunit is bound by GDP, and a potential or known modulator of the activation state of said receptor

(b) contacting said G-alpha subunit with a first reagent comprising an activation-state specific G-alpha binding peptide, and comprising a first label, (c) detecting a signal resulting from the specific binding of said peptide to the activated or inactive Gα subunit and (d) correlating said signal with the activation state of said receptor.

132. A method of identifying a modulator of a G protein coupled receptor which comprises identifying the activation state of a G protein-coupled receptor by the method of claim 131, said environment comprising a potential modulator, and thereby determining whether the activation state of said receptor was modulated by said potential modulator.

133. A method of determining the presence or amount of a modulator, of a G protein coupled receptor, in a sample which comprises identifying the activation state of a G protein coupled receptor, in an environment comprising said sample and said receptor, by the method of claim 131, and correlating the activation state of said receptor with the presence or amount of said modulator in said sample.

134. The method of any one of claims 131-133 where said modulator is an antagonist, and said environment comprises a known amount of a known agonist of said receptor.

135. The method of any one of claims 131-133 where said activation state-specific peptide is a T-peptide.

136. The method of claim 135 where said T-peptide is a peptide according to any one of claims 1-61, 101-112.

137. The method of any one of claims 131-133 where said activation-specific peptide is a D-peptide.

138. The method of claim 137 where said D-peptide is a peptide according to any one of claims 62-100.

139. The method of claim 135 or 136 where said environment comprises GTP or an analogue thereof.

140. The method of claim 139 where said environment comprises GTP.

141. The method of claim 137 or 138 where said environment comprises GDP or an analogue thereof.

142. The method of claim 140 where said environment comprises GDP.

143. The method of any one of claims 131-133 which further comprises providing a second reagent comprising G- alpha binding peptide which is not activation-specific, and a second label, said first and second labels or products thereof interacting to provide a signal correlatable with the activation state of the receptor.

144. The method of any one of claims 131-143 where the signal is a fluorescent signal.

145. The method of any one of claims 131-143 where one label is an enzymatic label.

146. The method of claim 145 where the enzymatic label acts upon a substrate to generate a substantially more luminescent product.

147. The method of any one of claims 131-146 where said GPCR couples to Gi.

148. The method of any one of claims 131-146 where said GPCR couples to Gs .

149. The method of any one of claims 141-148 where said environment comprises a chimeric Gα subunit.

150. The method of claim 143 where, as a result of the binding of the first and second peptides, first and second fluorophores which are matched for fluorescence resonance energy transfer are brought into proximity, providing a signal.

151. The method of claim 143 where as a result of the binding of first and second peptides, a donor fluorophore is brought into proximity with a nonfluorescent acceptor, quenching the fluorescence of the donor fluorophore, and thereby providing a signal.

152. The method of any one of claims 131-151 where said environment comprises a cell membrane providing said GPCR and said G-alpha.

153. The method of any one of claims 131-151 where said environment comprises a whole cell providing said GPCR and said G-alpha.

-154. The method of claim 143 in which the second peptide is an antibody.

155. The method of claim 154 in which the G-alpha subunit is a mutant subunit comprising a foreign epitope, and said antibody binds said epitope.

156. The method of claim 155 in which the epitope is a His tag.

157. The method of claim 132 which comprises identifying agonists by permitting a labeled D-peptide to interact with an inactive G-alpha/GDP complex, exposing the GPCR to a potential agonist, whose agonist activity will activate the GPCR, and cause the conversion GDP: G-alpha to GTP:G-alpha, whereupon the labeled D-peptide dissociates, and detecting the decrease in the signal in close proximity to the GPCR.

158. The method of claim 132 which comprises identifying antagonists by first activating a G protein coupled receptor, thereby causing formation of the active G- alpha/GTP complex, and then _.permitting the potential antagonist to deactivate the GPCR and detecting the binding of a labeled D-peptide to the resulting inactive G-alpha/GDP complex.

159. The method of claim 132 which comprises (a) providing a cell which co-expresses (i) a GPCR,

(ii) a G-alpha which binds said GPCR, (iii) a first fusion protein comprising a donor fluorophore, and a first peptide which binds said G-alpha, and (iv) a second fusion protein comprising either an acceptor fluorophore which is matched with said donor fluorophore for fluorescence resonance energy transfer, or a nonfluorescent acceptor which accepts fluorescence energy and thereby quenches from said donor fluorophore, and a second peptide which binds said G-alpha; where one of said first and second peptides binds the G-alpha subunit in a substantially activation state- sensitive manner, and the other in a substantially activation state-insensitive manner; (b) exposing said cell to said substance, thereby changing the level of activation of said G-alpha subunit, where said first and second peptides bind said G-alpha subunit simultaneously if it is in the appropriate activation state; said simultaneous binding resulting in a signal which is a fluorescence resonance energy transfer if the second fusion protein comprises an acceptor fluorophore, and is fluorescence quenching if said second fusion protein comprises a nonfluorescent acceptor, and

(c) detecting the change in the level of the signal.

160. The method of claim 159 in which the acceptor is a fluorophore.

161. The method of claim 159 in which the acceptor is not fluorescent.

162. The method of claim 159 in which the activation state-sensitive peptide is a T-peptide, so that if the substance is an agonist, it causes an increase in the signal, and if it is an antagonist, in a decrease in the signal .

163. The method of claim 159 in which the activation state-sensitive peptide is a D peptide so that if the substance is an agonist, it causes a decrease in the signal, and if it is an antagonist, in an increase in the signal.

164. A method of identifying a substance as an agonist or antagonist of GPCR which comprises

(a) providing a cell which co-expresses (i) a GPCR, (ii) a G-alpha which binds said GPCR,

(b) preparing said cell in such a way that allows access to both the receptor and the G protein (c) exposing said preparation to said substance, so said substance can interact with said GPCR and thereby change the level of activation of said G alpha subunit moiety,

(d) combining this preparation with (i) a first fusion protein comprising a donor fluorophore, and first peptide which binds said G- alpha, and

(ii) a second fusion protein comprising either an acceptor fluorophore which is matched with said donor fluorophore for fluorescence resonance energy transfer, or a nonfluorescent acceptor which accepts fluorescence energy and thereby quenches from said donor fluorophore; and a second moiety which binds said G-alpha; where said first peptide binds the G-alpha subunit in a substantially activation state-sensitive manner; whereby, if the G-alpha subunit is in the appropriate activation state, said second fusion protein and said second moiety bind simultaneously to said G protein, resulting in the interaction of said donor and acceptor to produce a signal, and

(e) detecting the change in the level of the signal as a result of said exposure of step (c) .

165. The method of claim 132 which comprises (a) providing a cell which co-expresses (i) a GPCR,

(ii) a first fusion protein comprising a G-alpha subunit which binds said GPCR,

(iii) a second fusion protein comprises an activation state-specific peptide which binds said G-alpha subunit in the appropriate activation state, where one of said fusion proteins comprises a donor fluorophore and the other comprises an acceptor, and where said acceptor is either an acceptor fluorophore matched to the donor fluorophore for FRET, or a nonfluorescent acceptor which quenches the fluorescence of the donor fluorophore, (b) exposing said cell to said substance, so said substance can interact with said GPCR and thereby change the level of activation of said G alpha subunit moiety, whereby, if the G-alpha subunit is in the appropriate activation state, said second fusion protein binds said first fusion protein, resulting in the interaction of said donor and acceptor to produce a signal, and (c) detecting the change in the level of the signal as a result of said exposure of step (b) .

166. A method of identifying a substance as an agonist or an antagonist of a G protein coupled receptor which comprises (a) providing a cell which co-expresses (i) said GPCR receptor, (ii) a G-alpha subunit which binds with said receptor, (iii) a fusion protein comprising a peptide which binds said G-alpha subunit in an activation state-specific manner, and a signalling protein moiety,

(b) exposing said cell to said substance, so it may interact with said receptor and thereby alter the level of activation of said G-alpha subunit, and thereby the level of recruitment of said fusion protein to the membrane, said fusion protein, upon recruitment to the membrane, either activating a second, membrane-bound signalling protein, or becoming an active signalling protein itself, thereby resulting in a detectable signal,

(c) detecting the change in level of said signal as a result of said exposure.

167. A method of identifying a G-alpha subunit which interacts with a G-protein coupled receptor which comprises

(a) providing a library of cells, each cell coexpressing (i) GPCR and (ii) a chimeric G alpha subunit, said chimeric subunits having different receptor-binding C-terminal but otherwise being substantially identical so as to participate in the same signalling pathway in said cell, said library collectively providing a plurality of different chimeric subunits representative of G alpha subunits with differing receptor specificities, (b) exposing the cells to a known agonist of said GPCR, (c) detecting the activation of the signaling pathway in one or more positive cells, and (d) determining which chimeric G alpha subunit had been expressed in one or more of the positive cells .

168. A method of identifying a substance as an agonist or antagonist of a G-protein coupled receptor which comprises

(a) providing a cell which co-expresses (i) G-protein- coupled receptor (GPCR) , (ii) a first fusion protein comprising a G-alpha subunit which interacts with said GPCR, and a first reporter protein moiety, and (iii) a second fusion protein comprising a G-alpha subunit activation-specific binding peptide according to any one of claims 1- 112, and a second reporter protein moiety,

(b) exposing said cell to said substance,

(c) where said agonist or antagonist activity results in a change in the level of activation of said GPCR, and hence a chance in the level of binding of said second fusion protein to said first fusion protein, said binding bringing said reporter protein moieties into sufficient proximity so as to reconstitute reporter protein activity, and (d) detecting the signal produced by said reporter protein.

169. The method of claim 168 where the method is used to detect agonists of the GPCR, and the activation-specific peptide is a T-peptide which binds the GTP-bound (active) form of the G-alpha subunit substantially more strongly than the GDP-bound form.

170. The method of claim 168 where the method is used to detect antagonists of the GPCR, the activation-specific peptide is a D-peptide which substantially more strongly binds the GDP-bound (inactive) form of the subunit than the GTP bound form, and the cell is also exposed to a subsaturating amount of a known antagonist for said GPCR.

171. The method of claim 168 in which the reporter protein is DHFR, adenylate cyclase, or B-galactosidase.

172. The method of claim 168 in which the signal is a fluorescent signal.

173. The method of claim 168 in which the reporter protein is DHFR and the signal is detected by exposing the cells to fluorescence methotrexate, which can enter the cell and there bind to DHFR, rendering the cells fluorescent.

174. The method of claim 168 where the cells are subsequently subjected to fluorescence activated cell sorting.

175. The method of claim 168 where the receptor is the acetylcholine M2 muscarinic receptor.

176. The method of claim 168 where the reporter protein is a constituitively active Ras protein mutant which is unable to localize to the cell membrane constituted there by the interaction of said first and second fusion proteins, and the signal is initially increased transcription on promoters containing AP-1 response elements.

177. The method of claim 176 where said cell further comprises a second reporter construct comprising a promoter with at least one AP-1 response element, said promoter operably linked to a second reporter gene.

178. The method of claim 168 in which the G alpha subunit is a Giα subunit.

179. The method of claim 168 where said reporter gene encodes a second reporter protein which is at least substantially identical to luciferase.

180. The method of claim 170 in which the cell is exposed to the substance before it is exposed to the known agonist.

181. The method of claim 170 in which the cell is exposed to the substance after it is exposed to the known agonist.

182. A method of identifying the activation state of a G-protein coupled receptor which comprises

(a) providing a G protein-coupled receptor in a potentially modulating environment comprising a Gα subunit which couples to an active conformation of said receptor when the Gα subunit is bound by GTP, and to an inactive conformation of said receptor when the Gα subunit is bound by GDP, and a potential or known modulator of the activation state of said receptor, and further comprising a Gβ subunit and a Gγ subunit, said three subunits forming a heterotrimeric complex when the receptor is not activated, and dissociating to yield the Gα subunit and a heterodimeric Gβy complex when the receptor is activated,

(b) contacting said G-alpha subunit with a first reagent comprising an activation-state specific G protein subunit binding peptide, and comprising a first label,

(c) detecting a signal resulting from the specific binding of said peptide to the activated or inactive Gα subunit and (d) correlating said signal with the activation state of said receptor.

183. A method of identifying the activation state of a G-protein coupled receptor which comprises

(a) providing a G protein-coupled receptor in a potentially modulating environment comprising a Gα subunit which couples to an active conformation of said receptor when the Gα subunit is bound by GTP, and to an inactive conformation of said receptor when the Gα subunit is bound by GDP, and a potential or known modulator of the activation state of said receptor, and further comprising a Gβ subunit and a Gγ subunit, said three subunits forming a heterotrimeric complex when the receptor is not activated, and dissociating to yield the Gα subunit and a heterodimeric Gβγ complex when the receptor is activated, (b) contacting said G-alpha subunit with a first reagent comprising an activation-state specific G protein subunit binding molecule, and comprising a first label, (c) detecting a signal resulting from the specific binding of said molecule to the activated or inactive Gα subunit and (d) correlating said signal with the activation state of said receptor.