The state of GPCR research in 2004 (20 Questions) Nat Rev Drug Discov. 2004 Jul;3(7):575, 577-626. PMID: 15272499 CONTENTS What new technologies are having the greatest impact on GPCR research, 578 1 and why? What tools and technologies for GPCR research would you most like to be available 583 2 in the future, and why? Tamas Bartfai 578 Jeffrey L. Benovic 582 What are the main pitfalls and advantages of currently used cell-based assay 585 3 systems for GPCRs? How will changing views of agonist and antagonist behaviour at GPCRs change the 588 4 way we view these receptors as therapeutic targets? Joël Bockaert 584 Richard A. Bond 590 Many predictions for heptahelical receptors have been based on using the 592 5 rhodopsin crystal structure as a template. How successful have these been? How close are we to having further GPCR structures, and what are the main 594 6 barriers to obtaining these? Michel Bouvier 591 Arthur Christopoulos 595 7 What big questions remain for GPCRs at the structure/function interface? 597 How widespread do you think GPCR heteromerization will turn out to be, 599 8 and how great a functional significance will it have? Olivier Civelli 598 Lakshmi A. Devi 601 Almost two-thirds of drugs on the market are thought to interact with GPCRs, 603 9 by far the largest family of targets, and yet new drugs targeting GPCRs are few and far between. Why? 10 What are the barriers to achieving specificity using GPCR ligands? 606 How will our increasing knowledge of interacting protein partners for GPCRs 607 Susan R. George 602 Akio Inui 603 11 change the way that we think about achieving selective GPCR modulation? Could there be an approach to GPCR pharmacology based around monoclonal 609 12 antibodies or proteins, rather than small molecules? How close are we to being able to integrate understanding of how individual 612 Brian Kobilka 608 Rob Leurs 610 13 GPCRs function with their role in disease states? How successful are the animal models that seek to recapitulate diseases linked 614 14 to the aberrant function of peptide-activated GPCRs? In your opinion, which diseases are most strongly associated with aberrant 615 Rick Neubig 612 Jean-Philippe Pin 614 15 function of peptide-activated GPCRs? What are the main hurdles that will need to be overcome to create further 617 16 successful therapeutic approaches based around targeting GPCRs? What would you describe as the major concerns surrounding the continued 619 Rémi Quirion 615 Bernard P. Roques 618 17 development of GPCR-targeted therapies? 18 What effect is the deorphanizing of GPCRs likely to have on the field? 620 What outstanding questions in GPCR research are likely to be clarified in 622 19 the next few years? Thomas P. Sakmar 618 Roland Seifert 619 20 What areas are unlikely to be clarified by research over the next few years? 623 Glossary 611 Ronald E. Stenkamp 622 Philip G. Strange 623 References 624 NATURE REVIEWS | DRUG DISCOVERY VOLUME 3 | JULY 2004 | 577 T WENTY QUESTIONS Olivier Civelli. A technology that has had a major interaction interface3,4.Refining these techniques will impact is the use of fluorescence detection as a tool to help in structure-based rational drug design. In addition, monitor G-protein-coupled receptor (GPCR) reac- proteomics is being used to identify and quantitate 1 tivity.This has led to studies of receptor–receptor and proteins that associate with GPCRs, thereby delineating What new receptor–ligand interactions, and to real-time mea- the signal-transducing complexes5.Finally,a variation technologies surement of signal transduction. Practically every of knockout technology (that is, targeted inducible are having report dealing with GPCR expression at the mem- deletion of individual GPCRs) is increasingly being the greatest brane, or with GPCR interactions with intracellular used to define the spatio-temporal relationships of impact on GPCR proteins, relies on fluorescence detection, including receptor activities6,7. research, techniques such as fluorescence resonance energy trans- and why? fer (FRET) or bioluminescence resonance energy Tamas Bartfai. The steady introduction of non- transfer (BRET) (FIG. 1; page 579). peptide ligands to neuropeptide GPCR receptors has enhanced research, and proven that these GPCRs are Michel Bouvier. Many new technologies are turning valuable drug targets in many important diseases. out to be extremely useful for analysing GPCR func- Such new ligands have arisen from industrial tions. Among those, I would cite fluorescence-based research, using binding, FLIPR and other cell-based techniques, such as INTERNAL FLUORESCENCE REFLECTION assays in ultra-HTS formats, and chemical libraries and single-molecule fluorescence, and resonance enhanced with β-turn mimics, which often turned energy transfer approaches, including FRET, BRET out to be recognized by GPCRs and were therefore and FLUORESCENCE LIFETIME IMAGING (FLIM). These new good starting points for medicinal chemistry. approaches should allow the real-time probing of Transgenic techniques have been important for vali- conformational changes, and of the dynamic protein– dating the involvement of GPCRs in physiological protein interactions that are involved in GPCR activa- and pathophysiological processes. No pharma project tion and regulation in the environment in which they to generate GPCR ligands proceeds today without normally occur; that is, living cells. high-quality hits from screening and access to null- mutant animals and, if possible, access to animals Lakshmi A. Devi. In recent years, high-throughput with a mutation generating a constitutively active screening (HTS) for functional activity (using methods receptor. Academic GPCR research has benefited such as the FLUOROMETRIC IMAGING PLATE READER (FLIPR) most from embracing more spectroscopic techniques, and SECRETED ALKALINE PHOSPHATASE (SEAP) ASSAYS) has been often using FLUOROPHORE-labelled ligands and/or recep- useful for deorphanizing receptors, screening for tors to follow interactions in real time, and to follow mutant receptors (to understand structure/function the trafficking of receptors during DESENSITIZATION and the relationships)1 and screening ligand libraries (to identify development of TOLERANCE. receptor-selective ligands)2.In addition, X-ray crystal- lography and modelling-based analyses of GPCRs Philip G. Strange. Industry has been quick to use have provided information on the ligand–receptor fluorescence-based technologies, in some cases in a single-molecule-detection mode — for example, fluo- rescence polarization, fluorescence intensity distribution Tamas Bartfai analysis, FLUORESCENCE CORRELATION SPECTROSCOPY and FRET — for drug screening at GPCRs. This has been stimu- Director and Professor of Neuropharmacology lated by the need for high-throughput systems and less Harold L. Dorris Neurological Research Center, 10550 North Torrey Pines Road, SR-307, La Jolla, reliance on radioactivity. Reporter-gene assays linked to California 92037, USA fluorescence or luminescence readouts, and cell-based e-mail: [email protected] assay systems for examining the movement of proteins, have also been used. These technologies have had a big Tamas Bartfai completed undergraduate studies in Physics impact on industrial research into GPCRs. and Chemistry at Eötvös Loránd University, Budapest, Hungary. He completed a Ph.D. in Biochemistry under the The academic community has been rather slow to supervision of Bengt Mannervik at Stockholm University, pick up on these techniques, perhaps owing to financial Sweden, where he was appointed Associate Professor and then Professor of Biochemistry, constraints, and much academic GPCR research and subsequently Chairman of the Department of Neurochemistry and Neurotoxicology. remains rooted in older technologies. Nevertheless, Bartfai left Stockholm University in 1997 to take up the position of Head of Central Nervous some of these techniques are likely to be very important System Research at F. Hoffman-La Roche, Basel, Switzerland. He was appointed Director of in pushing forward research on GPCRs into new areas. The Harold L. Dorris Neurological Research Center at The Scripps Research Institute, La Jolla, California, USA, in 2000, where he is also Professor in the Department of A few academic labs are using some of these technolo- Neuropharmacology and holds the Harold L. Dorris Chair in Neuroscience. Bartfai’s research gies to study GPCRs, and I would single out the work interests have spanned several topics in the field of physiological chemistry, including the from Brian Kobilka’s lab on the use of fluorescence to study of acetylcholine, glutamate, dopamine, noradrenaline and the neuropeptides VIP, NPY probe conformational changes in GPCRs8, and the use and galanin. While at Hoffman-La Roche, he was involved in research on the metabotropic by one or two labs of cyan fluorescent protein glutamate receptor, monoamine receptors and additional neuropeptide receptors from the GPCR class. His group developed several ligands that have been used as research tools or (CFP)/yellow fluorescent protein (YFP) FRET for study- 9–11 advanced into the clinic. Bartfai has been the recipient of several awards, including the ing protein–protein interactions .This latter tech- Eötvös Prize in Chemistry, the Svedberg Prize and the Eriksson Prize. nique may be of general use for examining the internal dynamics of receptors, and receptor–G-PROTEIN dynamics. 578 | JULY 2004 | VOLUME 3 www.nature.com/reviews/drugdisc T WENTY QUESTIONS Arthur Christopoulos. There