WO2003023361A2

WO2003023361A2 - Distinguishing agonists, antagonists, and inverse agonists using pwr

Info

Publication number: WO2003023361A2
Application number: PCT/US2002/028766
Authority: WO
Inventors: Victor J. Hruby; Gordon Tollin; Zdzislaw Salamon; Scott Cowell
Original assignee: The Arizona Board Of Regents On Behalf Of The University Of Arizona
Priority date: 2001-09-07
Filing date: 2002-09-09
Publication date: 2003-03-20
Also published as: AU2002324944A1; WO2003023361A3

Abstract

The biophysical consequences of inverse agonist binding to membrane-bound receptors are characterized by PWR (1). An inverse agonist (TMT-Tic) produces membrane thickness, mass density and refractive index anisotropy increases which are similar to, but considerably smaller than, those generated by agonists. Thus, a third conformational state is produced by this ligand, different from those formed by agonists and antagonists, providing a method of distinguishing between agonist, antagonist, and inverse antagonist ligands of membrane-bound receptors, such as G protein-coupled receptors, utilizing PWR.

Description

DISTINGUISHING AGONISTS, ANTAGONISTS, AND INVERSE AGONISTS USING PWR

U.S. GOVERNMENT RIGHTS

This work was supported in part by grants from the National Science Foundation (MCB-9904753), the National Institutes of Health (GM59630), the National Institute of Drug Abuse (DA-06284) and a U.S. Public Health Service Postdoctoral Fellowship (DA-05787). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Statement of Related Applications The present application claims the filing date benefit of U.S. Provisional Application Serial No. 60/318,241, filed on September 7, 2001, and entitled "Binding of Agonists, Antagonists and Inverse Agonists to the Human Delta-Opioid Receptor Produces Distinctly Different Conformational States Distinguishable by Plasmon- Waveguide Resonance Spectroscropy " The present application is also a continuation-in-part of PCT Patent Application Serial No. PCT/USO 1/19871, filed on June 21, 2001, and entitled "Agonist Versus Antagonist Binding to G Protein-Coupled Receptors," which is based on U.S. Provisional Patent Application Serial No. 60/213,575, filed on June 22, 2000.

Field of the Invention This invention pertains in general to the field of coupled plasmon-waveguide resonance (CPWR) spectroscopy, and, more particularly, to the use of CPWR spectroscopy to study structural changes accompanying the binding of agonist, antagonist, and inverse agonist ligands to receptors in a solid-supported lipid bilayer.

Description of the Related Art The abbreviations used herein are: GPCR, G-protein coupled receptor; hDOR, human brain delta-opioid receptor; DPDPE, c-[D-Pen² ₃ D-Pen⁵] enkephalin; CPWR (or PWR for short), coupled plasmon-waveguide resonance; NTI, naltrindole; TIPPpsi, H-Tyr- tetrahydroisoquinoline-3-carboxylic acid [CH₂NH]-Phe-Phe-OH; TMT-Tic, (2S,3R) β- methyl^'_jό'-dimethyltyrosyl-tetrahydroisoquinoline-S-carboxylic acid; cmc, critical micelle concentration; SPR, surface plasmon resonance.

Functionally, a receptor is a molecule or complex of molecules that receives a signal and then transduces the signal to achieve some physiological effect. There are at least three groups of cell membrane-bound receptors: (1) Enzyme-type receptors, which usually span the cell membrane once, and, in response to binding of a ligand, usually increase the phosphorylation of intracellular proteins (e.g., the insulin receptor effects a tyrosine kinase activity); (2) Receptors that activate transmembrane ion channels, such as the nicotinic cholinergic receptor; and (3) G-Protein Coupled Receptors (GPCRs), which use the 'G-protein' to transduce a signal (e.g., the human brain δ-opioid receptor).

Of particular interest, the superfamily of 7-transmembrane helical GPCRs mediates a wide variety of biological signal transduction processes involving activation by hormones, neurotransmitters, phospholipids, photons, odorants, growth factors, etc. (See Watson, S.P. and Arkinstall, S. (1994) G-Protein Linked Receptor Facts Book, Academic Press, London; Gether, U. and Kobilka, B.K. (1998) J. Biol. Chem. 273, 17979-17982; Brockaert, J. and Pin, IP. (1999) EMBOJ. 18, 1723-1729; and Gunderman, T., Kalkbrenner, F. and Schultz, G. (1996) .4«w Rev. Toxicol. 36, 429- 459).

One member of this group of integral membrane proteins, the human brain δ-opioid receptor (See Evans, C.J., Keith, Jr., D.E., Morrison, H., Magendzo, K. and Edwards, R.H. (1992) Science 258, 1952-1955 and Knapp, R.J., Malatynska, E., Fang, L., Li, X., Babin, E., Nguyen, M., Santoro, G., Varga, E.V., Hruby, V.J., Roeske, W.R. and Yamamura, H.I. (1994) Life Sciences (Pharm. Letts.) 54, PL463-469) mediates pain responses by binding endogenous agonist ligands such as enkephalins and synthetic peptide analogs such as DPDPE. In a previous work (Salamon, Z., Cowell, S., Varga, E., Yamamura, H.I., Hruby, V.J., and Tollin, G. (2000) Biophys. J. 79, 2463-2474), the inventors have utilized coupled plasmon-waveguide resonance (CPWR, or PWR for short) spectroscopy to demonstrate that the binding of DPDPE and the selective synthetic non-peptide antagonist NTI produced distinctly different conformational states of a purified preparation of hDOR incorporated into a solid-supported lipid bilayer membrane.

However, until the work described herein, the biophysical effects of inverse agonist ligand/membrane-bound receptor interactions has remained largely unknown. Thus, there continues to be a need in the art for a method of distinguishing agonist, antagonist, and inverse agonist ligands of membrane-bound receptors.

SUMMARY OF THE INVENTION

In general, the invention involves a method of characterizing the biophysical properties of membrane-bound receptor/ligand interactions utilizing PWR. More particularly, a novel method of characterizing the changes in membrane thickness, mass density, and refractive index anisotropy that characterize inverse agonist binding to membrane bound receptors, such as GPCRs, is disclosed.

In the work described herein, we have extended our previous studies to include an additional agonist/antagonist pair: deltorphin II (See Erspanier, V., Melchiorri, P., Falconieri-Erspanier, G., Negri, L., Corsi, R., Sererini, C, Barra, D., Simmaco, M. and Kreil, G. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5180-5191) and TlPPpsi (See Schiller, P.W., Weltrowska, G, Nguyen, T.M.-D., Wilkes, B.C., Chung, N.M., and Lemieux, C. (1993) J. Med. Chem. 36, 3182-3187), as well as a member of a third class of ligands known as inverse agonists: TMT-Tic (See Hosohata, K., Burkey, T.H., Alfaro-Lopez, j., Hruby, V.J., Roeske, W.R. and Yamamura, H.I. (1999) Eur. J. Pharmacol. 380, R9-R10).

As is well known, the inverse agonist category of ligand is able to inhibit signal transduction due to the endogenous G-protein activation that exists in constitutively active GPCRs in the absence of agonist binding (Spiegel, A.M., Shenker, A. and Weinstein, L.E. (1992) Endocrine Reviews 13, 536-584). In contrast, ordinary antagonists (so-called "neutral antagonists") are unable to inhibit this constitutive receptor activity, but rather act only to competitively prevent agonist activation. We have obtained evidence that all three types of ligands (agonists, antagonists, and inverse agonists) produce different receptor conformational states upon binding to the hDOR, which are clearly distinguishable by PWR spectroscopy, and whose properties correlate well with their different biological activities. In other words, structural changes induced by the binding of agonists, antagonists and inverse agonists to the cloned δ-opioid receptor from human brain immobilized in a solid-supported lipid bilayer can be determined by plasmon-waveguide resonance (PWR) spectroscopy.

As a specific illustration of this method for distinguishing agonists, antagonists, and inverse agonist ligands of membrane-bound receptors, a member of the GPCR family was treated as described in detail below. Briefly, the agonist (deltorphin II) binding caused an increase in membrane thickness due to receptor elongation, a mass density increase due to an influx of lipid molecules, and an increase in refractive index anisotropy due to transmembrane helix and fatty acyl chain ordering. In contrast, antagonist (TIPPpsi) binding produced no measurable change in either membrane thickness or mass density, and a significantly larger increase in refractive index anisotropy, the latter presumably due to a greater extent of helix and acyl chain ordering within the membrane interior. These results are closely similar to those reported earlier for another agonist (DPDPE) and antagonist (naltrindol) (See Salamon et al., (2000) Biophys.J. 79, 2463-2474).

However, we now find that an inverse agonist (TMT-Tic) produces membrane thickness, mass density and refractive index anisotropy increases which are similar to, but considerably smaller than, those generated by agonists. Thus, a third conformational state is produced by this ligand, different from those formed by agonists and antagonists. These results shed new light on the mechanisms of ligand-induced receptor functioning. An object of the invention is to provide a method of characterizing the effects of inverse agonists on membrane-bound receptors that is more rapid and direct then existing methodologies.

A second object of the invention is to provide a highly sensitive method of characterizing membrane-bound receptors and their interactions with inverse agonist ligands.

A third object of the invention is to provide a method of characterizing the biophysical properties of membrane-bound receptors, and their interactions with inverse agonist ligands, that does not produce toxic or radioactive waste products.

A fourth object of the invention is to provide a method of characterizing the biophysical properties of membrane-bound receptors, and their interactions with inverse agonist ligands, that does not modify the physical or chemical properties of the molecules being characterized.

A fifth object is to provide a method of distinguishing between agonist, antagonist, and inverse agonist ligands of G protein-coupled receptors.

In accordance with these and other objects, the inventive method has the unique capability of independently examining real-time changes in the structure of a membrane- bound receptor, both parallel and perpendicular to the lipid membrane plane, in response to receptor-ligand interactions. The method also provides greatly enhanced sensitivity and spectral resolution compared to conventional SPR. For example, only femtomole amounts of receptor (and ligand) are needed for complete spectral determination and analysis. Furthermore, since radioactivity measurements do not need to be performed, the methodology is much more rapid and direct in the determination of critical binding parameters. The invention thus provides a novel general procedure that can replace previous methods in characterizing inverse agonist ligand/membrane-bound receptor interactions by elucidating structural transitions that are not obtainable using other methodologies.

Various other purposes and advantages of the invention will become clear from its description in the specification that follows and from the novel features particularly pointed out in the appended claims. Therefore, to the accomplishment of the objectives described above, this invention consists of the features hereinafter illustrated in the drawings, fully described in the detailed description of the preferred embodiments, and particularly pointed out in the claims. However, such drawings and description disclose only some of the various ways in which the invention may be practiced.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig.1 shows the effect of changes in ligand concentration on the relative position of the PWR resonance minimum obtained using either ^-(closed circle) or ^-(closed triangle) polarization. Resonance position displacement towards higher values represents shifts to larger angles of incidence. Aliquots of solutions of the first ligand were added to the sample cell after receptor incorporation into a supported lipid bilayer membrane containing 75% egg phosphatidylcholine and 25% phosphatidylglycerol. Aliquots of solutions of the second ligand were added subsequently. The octylglucoside concentration in the initial receptor solution was 30 mM; after dilution into the PWR sample cell the detergent concentration never exceeded 5 mM, which is below the critical micelle concentration. The buffer also contained 10 mM Tris (pH 7.3), 0.5 mM EDTA, and l0 mM KCI..

Fig. 2 shows the changes in optical parameters (thickness [upper panel], refractive index anisotropy [middle panel], and average refractive index squared [lower panel] of a proteolipid membrane containing human δ-opioid receptor as a function of the added ligand concentration (deltorphin II [closed circle]; TIPP-psi [closed triangle]; TMT-Tic [closed square]. Values were obtained from theoretical fits to the experimental spectra (see insert for an example). Solid lines through the data points represent nonlinear least- squares fits to a hyperbolic function. The following binding constant values were obtained from either theA_n or n² plots: deltorphin II:K_D = 13 nM; TIPPpsi:K_D = 0.06 nM; TMT-Tic:K_D = 0.85 nM.

Fig. 2 Insert: Representative PWR spectra obtained with s-polarized exciting light for the lipid bilayer system described in Fig. 1, before (curve 1) and after incorporation of human δ-opioid receptor (curve 2: bulk receptor concentration approximately 10 nM), and after subsequent additions of deltorphin II (curve 3: bulk ligand concentration = 50 nM), and TIPP-psi (curve 4: bulk ligand concentration = 45 nM). Open circles in curve 1 correspond to a theoretical fit to the observed spectrum (solid line).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates in general to a method for distinguishing inverse agonist ligands from agonist and antagonist ligands of membrane-bound receptors. More specifically, the invention provides a method for characterizing biophysical properties of inverse agonist ligand/membrane-bound receptor complexes using PWR. Prior to the invention described herein, there was no spectroscopic methodology available in the art to distinguish inverse agonists from other types of ligands in membrane-bound receptor systems. Thus, the present invention also provides a novel ligand screening method.

The invention, having been described above, may be better understood by reference to examples. The following example is intended for illustration purposes only, and should not be construed as limiting the scope of the invention in any way.

Materials and Methods

The peptides tfPDPE (Mosberg, H.I., Hurst, R., Hruby, V.J., Gee, K., Yamamura, H.I., Gaffigan, JJ. and Burks, T.F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5871-5874) and (2S,3R) TMT-Tic (Liao, S., Lin, J., Shenderovich, M.D., Han, Y., Hosohata, K., Davis P., Qiu, W., Porreca, F., Yamamura, H.I. and Hruby, V.J. (1997) Bioorg. and Medicinal Chem. Letts. 7, 3049-3062) were prepared by standard methods (Hruby, V.J. and Meyer, J.-P. (1998) in Bioorganic Chemistry: Peptides and Proteins (Hecht. S.M. ed.) pp. 27-64, Oxford Univ. Press) of solid phase and solution phase peptide synthesis, respectively. Deltorphin II and TJJPPpsi were obtained from the National Institute of 5 Drug Abuse as part of the drug distribution to NTDA grantees (Victor. J. Hruby). Naltrindol was purchased from Sigma (St. Louis, MO).

The hDOR was prepared, modified at the C-terminal region with myc and His tags, and stably transfected into a CHO cell line as previously reported (Salamon, Z., Cowell, S., Varga, E., Yamamura, H.I., Hruby, V.J., and Tollin, G. (2000) Biophys. J. 79, 2463- 10 2474). Cells containing the hDOR were grown to confluency, harvested and homogenized, and the membrane fraction was solubilized in an octylglucoside containing buffer and purified by affinity chromatography as previously described (Salamon, Z., Cowell, S., Varga, E., Yamamura, H.I., Hruby, V.J., and Tollin, G. (2000) Biophys. J. 79, 2463-2474).

15 Bilayer Deposition and Incorporation of hDOR

The methods used for depositing a lipid bilayer onto the surface of a PWR resonator and for incorporation of an integral membrane protein into such a bilayer have been described previously (Salamon, Z., Wang, Y., Brown, M.F., Macleod, H.A., and Tollin, G. (1994) Biochemistry 33, 13706-13711; Salamon, Z. and Tollin, G. (1996) Biophys. 0 J. 71, 858-867; and Salamon, Z., Huang, D., Cramer, W.A., and Tollin, G. (1998) Biophys. J. 75, 1874-1885). Briefly, this involves a variation of the Mueller-Rudin method for producing a freely suspended bilayer across an orifice in a hydrophobic barrier (Teflon) separating two aqueous compartments. Such a bilayer is anchored to the barrier by a plateau-Gibbs border of lipid solution (in the present case 75 mol% egg 5 phophatidycholine (PC), 25 mol% l-palnτitoyl-2-oleoyl-s«-glycero-3-[phopho-rac-(l- glycerol) (sodium salt) (POPG) in squalene/butanol/methanol, 0.05:9.5:0.5, v/v). The lipids were purchased from Avanti Polar Lipids (Birmingham, AL). In PWR spectroscopy, one of these aqueous compartments is replaced by a hydrated silica surface. This produces a solid-supported lipid bilayer that is quite stable, and which readily accommodates insertion of integral membrane proteins by allowing displaced lipid molecules to flow into the plateau-Gibbs border. Lipid molecules can also move between the Gibbs border and the bilayer in response to conformational changes occurring in an incorporated protein. Such incorporation is accomplished by introducing a detergent-solubilized membrane protein into the aqueous compartment under conditions that dilute the detergent to below the critical micelle concentration. This allows mass transfer of protein from the detergent micelle into the lipid bilayer. In these studies, a solution of hDOR in a lOmM tris buffer (pH 7.3), containing 0.5 mM EDTA, 10 mM KCI and 30 mM octylglucoside (cmc = 25 mM) was used.

PWR Spectroscopy

This technique has been described extensively in previous publications (Salamon, Z. and

Tollin, G. (1999) in Encyclopedia of Spectroscopy and Spectromety, Vol. 3, Lindon, J.C, Tranter, G.E. and Holmes, J.L., eds., pp. 2311-2319, Academic Press, New York and Salamon, Z. and Tollin, G. (1999) in Encyclopedia of Spectroscopy and Spectrometry, Vol. 3, Lindon, J.C, Tranter, G.E. and Holmes, J.L., eds., pp. 2294- 2302, Academic Press, New York.). Briefly, it involves using a polarized continuous- wave laser (He-Ne; λ = 632.8 nm) to excite plasmon and waveguide modes in a resonator consisting of a thin silver film coated by a thicker silica layer deposited onto the front surface of a glass prism. Such excitation generates an evanescent electromagnetic field that localizes at the outer surface of the silica layer, and decays exponentially on both sides of that surface.

Evanescent field generation is a resonance process that depends on the angle of incidence of the' exciting light that passes through the prism and impinges upon the interface between the prism and the silver film at slightly above the critical angle for total internal reflection. Molecules (a proteolipid membrane in this work) that are immobilized at the outer silica surface interact with the evanescent field and change the resonance characteristics, thereby influencing the amplitude, position and width of the resulting PWR spectrum. The latter corresponds to the curve generated by plotting reflected laser light intensity from the prism-silver interface as a function of incident angle (see insert in Figure 2).

A major advantage of PWR spectroscopy over the closely related SPR methodology is that the former allows resonances to be excited by light that is both >-polarized (electric field perpendicular to the plane of the metal film) and s-polarized (electric field parallel to the plane of the metal film); for details see Salamon, Z., Brown, M.F., and Tollin, G. (1999) Trends Biochem. Sci. 24, 213-219; Salamon, Z. and Tollin, G. (1999) in Encyclopedia of Spectroscopy and Spectromety, Vol. 3, Lindon, J.C, Tranter, G.E. and Holmes, J.L., eds., pp. 2311-2319, Academic Press, New York; and Salamon, Z. and Tollin, G. (1999) in Encyclopedia of Spectroscopy and Spectrometry, Vol. 3, Lindon, J.C, Tranter, G.E. and Holmes, J.L., eds., pp. 2294-2302, Academic Press, New York. This allows the anisotropic optical properties of oriented immobilized molecules, such as those found in a proteolipid membrane, to be evaluated by this methodology. This is accomplished by theoretical fitting of the experimental resonance curves. The optical properties that control resonance excitation correspond to the refractive index (n) and the optical absorption coefficient (k) at the exciting wavelength, as well as the thickness (I) of the immobilized layer. In the present case, none of the immobilized molecules have specific absorption bands at the laser wavelength, and so the k parameter has a negligible influence on the PWR spectral properties.

In PWR spectroscopy, two values of the refractive index (n_p and n_s) and a single value oft are obtained by curve fitting procedures. The two refractive index parameters can be used to calculate the anisotropy of n, thereby describing the degree of molecular order in the membrane, using the following relationships:

n² _^ = (n² _p + 2 n² _s) / 3 = average value of the refractive index squared (1)

A„ — (n² - n² _s) I (n² _m + 2) = refractive index anisotropy (2) Such information, taken together with the film thickness, provides insights into the microscopic structure of the film. Furthermore, the optical parameters can also be employed to calculate the mass density of a deposited thin layer, using the Lorentz- Lorenz relation for a film containing a mixture of substances:

d = M L = (M I A) [(n² _^ - 1) / (μ²„ + 2)] = mass density (3)

where M= molecular weight, L = number of moles per unit volume and A = molar refractivity.

Results and Discussion

As we have demonstrated previously (Salamon, Z., Cowell, S., Varga, E., Yamamura, H.I., Hruby, V.J., and Tollin, G. (2000) Biophys. J. 79, 2463-2474), the binding of a ligand to hDOR molecules incorporated into a lipid bilayer results in changes in the position, depth and width of the PWR spectrum. For the ligands used in the present work, this can be conveniently illustrated by plotting the resonance position shifts as a function of the bulk concentration of the added ligand. Typical results are shown in Figure 1 for experiments in which two ligands are successively added to the aqueous compartment of a PWR cell containing membrane-bound hDOR. It should be noted that control experiments in which these ligands were added to a PWR cell containing a membrane in the absence of hDOR showed no significant changes in the PWR spectra, indicating that non-specific binding was not observable under these conditions. It is also important to note that the results obtained for the agonist (deltorphin II) and antagonist (TIPPpsi) used in the present work are quite similar to those obtained previously for the agonist DPDPE and the antagonist NTI (Salamon, Z., Cowell, S., Varga, E., Yamamura, H.I., Hruby, V.J., and Tollin, G. (2000) Biophys. J. 79, 2463-2474).

Several conclusions can be derived from these data, again similar to those derived from the previous experiments (Salamon, Z., Cowell, S., Varga, E., Yamamura, H.I., Hruby, V.J., and Tollin, G. (2000) Biophys. J. 79, 2463-2474): First, ligand binding is saturable (i.e. hyperbolic) and occurs within concentration ranges consistent with the known affinity constants for these systems; binding constants obtained in these experiments are given in the legend to Figure 2.

Second, the amplitude and direction of the spectral shifts produced by a given ligand added either first or second is a characteristic of the ligand type. For example, agonists and inverse agonists cause shifts of both ?-polarized and s-polarized resonances to larger incident angles, whereas antagonists cause shifts of ?-polarized resonances to larger and s-polarized resonances to smaller incident angles. Adding an antagonist after an agonist, or an agonist after an antagonist, causes a different pattern of spectral shifts than those produced by addition of either an inverse agonist followed by an agonist or an agonist followed by an inverse agonist. Thus, these patterns provide a simple, rapid, convenient and non-radioactive method for distinguishing between these classes of ligand, which has important implications for drug screening protocols.

Third, a bound ligand can be displaced by addition of a second ligand, causing the spectral shifts to revert to those that are characteristic of the second ligand, either partially or completely.

It is important to point out that these spectral shifts are predominantly a consequence of changes in the proteolipid membrane structure produced by ligand binding, rather than being due to the added mass of the ligand. The evidence for this is as follows:

(1) all of the ligands used here have quite similar masses, yet they produce very different spectral changes. On the other hand, structurally different ligands within the same functional class produce very similar spectral changes (i.e. the agonists DPDPE and deltorphin II vs. the antagonists NTI and TEPPpsi).

(2) the refractive index changes that are induced by ligand binding are too large to be accounted for by the added ligand mass. In order to understand the physical basis for these spectral shifts, it is necessary to include the changes in spectral amplitudes and line widths that are also caused by ligand binding. This can be done by carrying out a more complete analysis via fitting theoretical spectra to the experimental ones. An example of such a fit is shown in the insert in Figure 2. Figure 2 also shows plots of the changes in membrane thickness (upper panel), mass density (expressed as n²; middle panel) and refractive index anisotropy (lower panel) as a function of the concentration of added ligand, obtained from these fits for experiments in which an agonist, an antagonist and an inverse agonist are added to the membrane-bound hDOR.

The results for deltorphin II and TIPPpsi and their interpretation are quite similar to those reported previously for the agonist DPDPE and the antagonist NTI (Salamon, Z., Cowell, S., Varga, E., Yamamura, H.I., Hruby, V.J., and Tollin, G. (2000) Biophys. J. 19, 2463-2474). As is evident, agonist addition causes an increase in he average proteolipid membrane thickness of approximately 4 angstroms. This is a consequence of structural changes in both the receptor and in the arrangement of the surrounding lipid molecules. In contrast, the antagonist induces no membrane thickness change. The inverse agonist (TMT-Tic) produces a significant thickness increase (approximately 1 angstroms), albeit considerably smaller than produced by the agonists. As before (Salamon, Z., Cowell, S., Varga, E., Yamamura, H.I., Hruby, V.J., and Tollin, G. (2000) Biophys. J. 19, 2463-2474), we attribute these thickness changes to an elongation of the receptor molecule in the membrane. This induces a corresponding change in the associated lipids that causes positive curvature in the membrane surface.

Similarly, agonist binding produces a mass density increase, whereas the antagonist causes no mass increase, and the inverse agonist produces a significantly smaller increase. Inasmuch as the ligand masses are small and all quite similar and the mass changes are large, we attribute this increase to an influx of lipid molecules from the plateau-Gibbs border caused by the receptor elongation noted above. The refractive index anistropy changes are somewhat less distinctive, although antagonist binding clearly produces a larger increase, and inverse agonist binding a significantly smaller increase, than that generated by agonist binding. In the present experiment, the relative increase in_^4„ produced by TIPP is not quite as large as that produced by Nti in the earlier work (Salamon, Z., Cowell, S., Varga, E., Yamamura, H.I., Hruby, V.J., and Tollin, G. (2000) Biophys. J. 79, 2463-2474), although in both cases the increase is larger than that produced by the agonists. Part of the reason for the variability in this parameter is that antagonists having differing structures may bind somewhat differently to a given receptor, thereby creating a range of conformations. It is important to note that this may also be true of agonists and inverse agonists, although perhaps to a lesser extent.

Another possible contribution relates to the fact that these anistropy changes are due to an increase in the ordering of both the receptor transmembrane helices and the lipid acyl chains in the bilayer. The latter contribution in particular will be very sensitive to the initial anistropy of the bilayer generated on the resonator surface. In our experience, we have noted that this can vary considerably from experiment to experiment due to variability in the packing density of the lipid molecules in the bilayer. Despite these qualifications, the anisotropy changes also indicate that the structural states induced in the proteolipid membrane differ between the three classes of ligands.

The present study clearly shows the capability of PWR spectroscopy to provide insights into the structural changes that accompany the binding of molecules to ordered molecular arrays such as occur in biomembrane systems. This is a direct consequence of being able to probe the array both perpendicular and parallel to the film plane. Conventional SPR, by virtue of being limited to using onlyjø-polarized excitation, is unable to directly measure either mass changes or anisotropy changes due to structural transitions. This restricts its applicability to optically isotropic systems, and precludes the possibility of providing structural information. In the work presented here, we have shown that only agonist and inverse agonist binding to the hDOR causes increases in the dimensions of the receptor molecule perpendicular to the plane of the bilayer. This suggests that these ligands influence the extramembrane portions of the receptor, which correlates quite well with the ability of these two types of ligand to cause changes in the activity of a G-protein located on the opposite side of the membrane from the ligand binding site. Based on the inability of antagonists to generate such thickness changes, it would appear that the structural transitions caused by the binding of this class of inhibitor must be limited to the transmembrane region of the receptor, without any influence on the extramembrane loops involved in G-protein activation. This provides some important new insights into GPCR function.

As would be understood by those skilled in the art, any number of functional equivalents may exist in lieu of the preferred embodiment described above. Thus, as will be apparent to those skilled in the art, changes in the details and materials that have been described may be within the principles and scope of the invention illustrated herein and defined in the appended claims.

Accordingly, while the present invention has been shown and described in what is believed to be the most practical and preferred embodiment, it is recognized that departures can be made therefrom within the scope of the invention, which is therefore not to be limited to the details disclosed herein but is to be accorded the full scope of the claims so as to embrace any and all equivalent products and methods.

Claims

We claim:

1. A method for distinguishing agonist, antagonist, and inverse agonist ligands of membrane-bound receptors, comprising the steps of:

(a) providing a receptor bound by a preformed lipid membrane, (b) incubating the receptor of step (a) with a ligand, thereby forming a ligand/receptor complex,

(c) acquiring a coupled plasmon-waveguide resonance spectrum of the ligand/receptor complex of step (b); and

(d) characterizing the ligand of step (b) as an agonist, antagonist, or inverse agonist of the receptor based on the coupled plasmon-waveguide resonance spectrum of step (c).

2. The method of claim 1, wherein step (d) comprises measuring a dimension of the receptor of step (a) perpendicular to the plane of the preformed lipid membrane using coupled plasmon-waveguide resonance spectrum.

3. The method of claim 1, wherein the receptor is a G protein-coupled receptor.

4. The method of claim 3, wherein the G protein-coupled receptor is a human delta- opioid receptor.

5. A method for screening ligands of G protein-coupled receptors, comprising the steps of: (a) incorporating a G protein-coupled receptor into a preformed lipid membrane,

(b) incubating the receptor of step (a) with a ligand, thereby forming a ligand/receptor complex,

(c) acquiring a coupled plasmon-waveguide resonance spectrum of the ligand/receptor complex of step (b); and (d) characterizing the ligand of step (b) as an agonist, antagonist, or inverse agonist of the receptor based on the coupled plasmon-waveguide resonance spectrum of step (c).

6. The method of claim 5, wherein the G protein-coupled receptor is the human delta- opioid receptor.

7. The method of claim 5, wherein the step (d) comprises measuring a dimension of the receptor of step (a) perpendicular to the plane of the preformed lipid membrane using coupled plasmon-waveguide resonance spectrum.