Development of Nonseparation Binding and Functional Assays for G Protein-Coupled Receptors for High Throughput Screening: Pharmacological Characterization of the Immobilized CCR5 Receptor on FlashPlate(r)

Abstract

G protein-coupled receptors (GPCRs) represent a very important class of drug targets. The development of microformatted nonseparation assays constitute a key step in the process of assay development for high throughput drug screening (HTS). We have developed a microformatted nonseparation assay for membrane preparations containing the CCR5 GPCR using FlashPlate® microplates (Packard Instrument Company, Meriden, CT). The pharmacodynamic (radioligand-binding) and functional (agonist-stimulated [³⁵S]GTPγS binding) properties of this receptor observed in FlashPlate-based assays were compared with standard filtration assays. Saturation binding experiments performed using either assay platform revealed identical K_d for [¹²⁵I]-MIP-1 β (0.7 nM). Comparable signal-to-noise ratios (SNR), similar affinities (K_i), and identical order of potency (RANTES ≅ MIP-1β > MIP-1α) were observed following competition binding assays in both platforms. In functional assays, the order of potency for different agonists were similar in both platforms with RANTES ≅ MIP-1β ≥ MIP-1α, which correspond to the relative affinities determined for the three ligands in competition binding experiments. Because similar pharmacologic properties were obtained in both FlashPlate microplates and standard filtration platforms, we conclude that FlashPlate microplates could provide a valuable nonseparation platform for primary and secondary HTS for this and possibly other GPCRs.

References

Udenfriend, S. , Gerber, L. , Nelson, N. (1987). Scintillation proximity assay: a sensitive and continuous isotopic method for monitoring ligand/receptor and antigen/antibody interactions. Anal. Biochem. 161:494-500.

Patel, S. , Harris, A. , O'Beirne, G. , Cook, N.D. , Taylor, C.W. (1996). Kinetic analysis of inositol trisphosphate binding to pure inositol trisphosphate receptors using scintillation proximity assay. Biochem. Biophys. Res. Commun. 221:821-825.

Holland, J.D. , Singh, P. , Brennand, J.C. , Garman, A.J. (1994). A non separation microplate receptor binding assay. Anal. Biochem. 222:516-518.

Brown, B.A. , Cain, M. , Broadbent, J. , Tompkins, S. , Henrich, G. , Joseph, R. , Custo, S. , Harney, H. , Greene, R. , Delmondo, R. , Ng, S. (1997). FlashPlateTM technology. In High Throughput Screening. The Discovery of Bioactive Substances. J.P. Devlin (ed.), pp. 317-328, Marcel Dekker, New York.

Gorman, C. , Skinner, R.H. , Skelly, J.V. , Neidle, S. , Lowe, P.N. (1996). Equilibrium and kinetic measurements reveal rapidly reversible binding of Ras to Raf. J. Biol. Chem. 271:6713-6719.

Lemer, C.G. , Saiki, A.Y.C. (1996). Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculovirus-infected insect cells. Anal. Biochem. 240:185-196.

Watson, S. , Arkinstall, S. (1994). The G-protein Linked Receptor Facts Book. Academic Press, San Diego, CA.

Dutton, G. (1997). Genetic Engineering News, April 8, pp. 33-34.

Feng, Y. , Broder, C.C. , Kennedy, P.E. , Berger, E.A. (1996). HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 272:872-877.

10.

Alkhatib, G. , Combadiere, C. , Broder, C.C. , Feng, Y. , Kennedy, P.E. , Murphy, P.M. , Berger, E.A. (1996). CC CKR5: A RANTES, MIP-1α, MIP-1β receptor as a fusion cofactor for macrophagetropic HIV-1. Science. 272:1955-1958.

11.

Choe, H. , Farzan, M. , Sun, Y. , Sullivan, N. , Rollins, B. , Ponath, P.D. , Wu, L. , Mackay, C.R. , LaRosa, G. , Newman, W. , Gerard, N. , Gerard, C. , Sodroski, J. (1996). The β-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell. 85:1135-1148.

12.

Doranz, B.J. , Rucker, J. , Yi, Y. , Smyth, R.J. , Samson, M. , Peiper, S.C. , Parmentier, M. , Collman, R.G. , Doms, R.W. (1996). A dualtropic primary HIV-1 isolate that uses fusin and the β-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell. 85:1149-1158.

13.

Wu, L. , Gerard, N.P. , Wyatt, R. , Choe, H. , Parolin, C. , Ruffmg, N. , Borsetti, A. , Cardoso, A.A. , Desjardin, E. , Newman, W. , Gerard, C. , Sodroski, J. (1996). CD4-indiced interaction of primary HIV-1 gpl20 glycoproteins with the chemokine receptor CCR-5. Nature. 384:179-183.

14.

Oberlin, E. , Amara, A. , Bachelerie, F. , Bessia, C. , Virelizier, J.-L. , Arenzana-Seisdedos, F. , Schwartz, O. , Heard, J.-M. , Clark-Lewis, I. , Legler, D.F. , Loetscher, M. , Baggiolini, M. , Moser, B. (1996). The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1. Nature. 382:833-835.

15.

Combadiere, C. , Ahuja, S.K. , Tiffany, H.L. , Murphy, P.M. (1996). Cloning and functional expression of CC CKR5, a human monocyte CC chemokine receptor selective for MIP-1(alpha), MIP-1(beta), and RANTES. J. Leukoc. Biol. 60:147-152.

16.

Liu, R. , Paxton, W.A. , Choe, S. , Ceradini, D. , Martin, S.R. , Horuk, R. , MacDonald, M.E. , Stuhlmann, H. , Koup, R. , Landau, N.R. (1996). Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell. 86:367-377.

17.

Paxton, W.A. , Martin, S.R. , Tse, D. , O'Brien, T.R. , Skurnick, J. , VanDevanter, N.L. , Padian, N. , Braun, J.F. , Kotler, D.P. , Wolinsky, S.M. , Koup, R.A. (1996). Relative resistance to HIV-1 infection of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual exposure. Nat. Med. 2:412-417.

18.

Simmons, G. , Clapham, P.R. , Picard, L. , Offord, R.E. , Rosenkilde, M.M. , Schwartz, T.W. , Buser, R. , Wells, T.N.C. , Proudfoot, A.E. (1997). Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science. 276:276-279.

19.

Arenzana-Seisdedos, F. , Virelizier, J.L. , Rousset, D. , Clark-Lewis, I. , Loetscher, P. , Moser, B. , Baggiolini, M. (1996). HIV blocked by chemokine antagonist. Nature. 383 :400.

20.

Cullen, B.R. (1987). Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enzymol. 152:684-704.

21.

Samson, M. , LaRosa, G. , Libert, F. , Paindavoine, P. , Detheux, M. , Vassart, G. , Parmentier, M. (1997). The second extracellular loop of CCR5 is the major determinant of ligand specificity. J. Biol. Chem. 272:24934-24941.

22.

Raport, C.J. , Gosling, J. , Schweickart, V.L. , Gray, P.W. , Charo, I.F. (1996). Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-lbeta, and MIP-lalpha. J. Biol. Chem. 271:17161-17166.

23.

Aramori, I. , Zhang, J. , Ferguson, S.S.G. , Bieniasz, P.D. , Cullen, B.R. , Caron, M.G. (1997). Molecular mechanism of desensitization of the chemokine receptor CCR-5: receptor signaling and internalization are dissociable from its role as an HIV-1 co-receptor. EMBO J. 16:4606-4616.

24.

Costa, T. , Ogino, Y. , Munson, P.J. , Onaran, H.O. , Rodbard, D. (1992). Drug efficacy at guanine nucleotide-binding regulatory protein-linked receptors: thermodynamic interpretation of negative antagonism and of receptor activity in the absence of ligand. Mol. Pharmacol. 41:549-560.

25.

Kenakin, T. (1996). The classification of seven transmembrane receptors in recombinant expression systems. Pharmacol. Rev. 48:413-463.

26.

Lorenzen, A. , Fuss, M. , Vogt, H. , Schwabe, U. (1993). Measurement of guanine nucleotide-binding protein activation by Al adenosine receptor agonists in bovine brain membranes: stimulation of guanosine-5′-O-(3-[35S]thio)triphosphate binding. Mol. Pharmacol. 44:115-123.

27.

Hilf, G. , Gierschik, P. , Jakobs, K.H. (1989). Muscarinic acetylcholine receptor-stimulated binding of guanosine 5′-O-(3-thiotriphosphate) to guanine-nucleotide-binding proteins in cardiac membranes. Eur. J. Biochem. 186:725-731.

28.

Milligan, G. , Bond, R.A. , Lee, M. (1995). Inverse agonism: pharmacological curiosity or potential therapeutic strategy. Trends Biochem. Sci. 10:10-13.

29.

Kosterlitz, H.W. , Paterson, S.J. , Robson, L.E. (1988). Modulation of binding at opioid receptors by mono- and divalent cations and by cholinergic compounds. J. Recept. Res. 8:363-373.

30.

Stiles, G.L. (1988). Al adenosine receptor-G protein coupling in bovine brain membranes: effects of guanine nucleotides, salt, and solubilization. J. Neurochem. 51:1592-1598.

31.

Dairaghi, D.J. , Oldham, E.R. , Bacon, K.B. , Schall, T.J. (1997). Chemokine receptor CCR3 function is highly dependent on local pH and ionic strength. J. Biol. Chem. 272:28206-28209.