Drug Discovery Biology

Background - G Protein-coupled receptors (GPCR) as drug targets

The optimum functioning of living cells, and consequently the health of the entire organism, depends on how cells respond to the multitude of physical and chemical stimuli that continually bombard them. The majority of all chemical cellular stimuli are comprised of hormones and neurotransmitters that impart their actions by binding to specific cell surface receptor proteins. G protein-coupled receptors (GPCRs) represent the largest superfamily of all receptors (approx. 2% of the human genome) and are the targets for nearly 50% of all currently used therapeutic drugs. The principal direction of our laboratory is towards understanding novel modes of regulation of GPCRs in an effort to identify novel targets or approaches for drug discovery. Our work encompasses investigation across virtually all levels of GPCR structure/function, including analysis of the functional significance of single nucleotide polymorphisms, RNA-editing and alternate mRNA splicing; signaling via G proteins and downstream messenger systems; interaction of receptors with regulatory accessory proteins; novel allosteric GPCR binding sites, and mathematical and molecular modelling of GPCR-ligand interactions.

Laboratory Heads

Major Themes for DDB

Theme 1. The structural basis of GPCR binding and activation

Within this theme we study how receptor structure is linked to ligand binding and selectivity, how structural changes allow the transmission of extracellular signals to activation of intracellular signalling proteins, and how receptors interact with other proteins to regulate their behaviour. Approaches that we use include, generation of chimeric receptors, site-directed mutagenesis, photoaffinity labelling, resonance energy transfer and three dimensional molecular modelling.

Theme 2. Allosteric modulation of GPCRs

Our laboratory is an international leader in the study of mechanisms of allosteric modulation of GPCRs by small molecules. Allosteric ligands bind to the receptor at a site topographically separate from the orthosteric site used by the endogenous ligand . We have developed methods for assaying, validating and quantifying the properties of such ligands in a manner that can facilitate understanding of structure-activity relationships and assay development - an essential prerequisite for rational drug discovery and development. Our work elucidates sites of allosteric ligand binding on GPCRs and provides understanding of: interactions that govern both affinity and cooperative properties, modes of ligand binding, the potential for allosteric ligands to engender biased signalling and/or altered receptor regulation, and the identification of novel allosteric compounds as potential drug development leads.

Theme 3. Ligand- directed signalling bias andGPCR regulation

For over half a century, pharmacological theory has held that "intrinsic efficacy," i.e., the inherent ability of a ligand to impart (or reduce) stimulus to a cell once that ligand is bound to its receptor, is a system-independent parameter that is constant for each ligand at a given receptor, irrespective of where that receptor is expressed. It is now recognised that this classical notion of intrinsic efficacy as an invariant constant cannot be correct. The most compelling data that have driven this conceptual shift involve drugs that, despite acting at the same receptor and in the same cellular background, differentially activate certain subsets of intracellular signalling pathways to the relative exclusion of others. Importantly, the actual pathways that are activated vary in a drug-specific manner, a phenomenon that has been dubbed "ligand-directed signalling bias"; this may also be extended to drug specific modulation of receptor regulatory processes (e.g. desensitisation, phosphorylation, internalization), thus opening new vistas for achieving selectivity in "sculpting" cellular responses for therapeutic benefit. We are actively working to understand the broad spectrum of drug behaviour, how differential signalling profiles are engendered at a structural level and also whether we can predict ligand-receptor behaviour based on knowledge of specific, molecular, ligand-receptor interactions.

Theme 4. Applied drug discovery

There are two major arms to our work on drug discovery. The first is the use of novel methods that we have developed that enable the prediction of small molecule binding pockets for both activators (agonists) and inhibitors of the receptor. In benchmark testing we have shown that both agonist and inhibitor models are strongly predictive of small molecule binding, and therefore suitable for application of virtual library screening to identify novel lead compounds that will be assayed for activity within the laboratory. The second area of work is screening of small scale libraries (up to ~20,000 compounds) against selected targets of high potential therapeutic value using recently installed robotics and liquid handling facilities. Work in this theme is vertically integrated with the wider MIPS themes of medicinal chemistry, drug candidate optimisation and drug delivery.

Collaborators

• Professor Peter Scammells, Dept. of Medicinal Chemistry, MIPS, Monash University
• Professor Lawrence Miller, Mayo Clinic, Scottsdale, USA
• Professor Ruben Abagyan, Scripps Research Institute, USA
• Professor Andrew Tobin, University of Leicester, UK
• Dr Debbie Hay, University of Auckland, NZ
• Professor Michel Bouvier, University of Montreal, Canada
• Dr Christian Felder, Eli Lilly, Indianapolis, USA
• Dr Michael Crouch, TGR Biosciences, Australia
• Professor Arthur Conigrave, University of Sydney, Australia

Funding

• National Health and Medical Research Council of Australia
• Australian Research Council
• National Institutes of Health (USA)
• GlaxoSmithKline Australia
• Pfizer Australia

DDB lab personnel

Recent key publications

1. Antony, J., Kellershohn, K., Mohr-Andrä, M., Kebig, A., Prilla, S., Muth, M., Heller, E., Disingrini, T., Dallanoce, C., Bertoni, S., Schrobang, J., Tränkle, C., Kostenis, E., Christopoulos, A., Höltje, H.-D., Barocelli, E., De Amici, M., Holzgrabe, U. and K. Mohr (2009) Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity, FASEB J., In Press.
2. Conn, P.J., Christopoulos, A. and C.W. Lindsley (2009) Allosteric modulators of GPCRs: A novel approach for the treatment of CNS disorders. Nature Rev. Drug Discover. 8: 41-54.
3. Morfis, M., Tilakaratne, N., Furness, S.G.B., Christopoulos, G., Werry, T.D., Christopoulos, A. and P M. Sexton (2008) Receptor activity modifying proteins differentially modulate the G protein-coupling efficiency of amylin receptors. Endocrinology, 149: 5423-5431.
4. Werry, T.D., Stewart, G.D., Crouch, M.F., Watts, A., Sexton, P.M. and A. Christopoulos (2008) Pharmacology of 5HT2C receptor-mediated ERK1/2 phosphorylation: Agonist-specific activation pathways and the impact of RNA editing, Biochem. Pharmacol., 76: 1276-1287.
5. Valant, C., Gregory, K.J., Hall, N.E., Scammells, P.J., Lew, M.J., Sexton, P.M. and A. Christopoulos (2008) A novel mechanism of G protein-coupled receptor functional selectivity:  muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand, J. Biol. Chem, 283: 29312-29321. (See also “Research Highlights”, Nature Rev. Drug Discover. (2008) 7:  976).
6. Werry, T.D., Loiacono, R.L., Sexton, P.M. and A. Christopoulos (2008) RNA editing of the serotonin 5HT2C receptor and its effects on cell signalling, pharmacology and brain function. Pharmacol. Ther. 119: 7-23.
7. Ferguson, G., Valant, C., Horne, J. Figler, H., Flynn, B., Linden, J., Chalmers, D.,  Sexton, P., Christopoulos, . and P.J.  Scammells (2008) 2-aminothienopyridazines as novel adenosine A1 receptor allosteric modulators and antagonists, J. Med. Chem., 51: 6165-6172.
8. Nawaratne, V.,  Leach, K., Suratman, N., Loiacono, Felder, C.C., Armbruster, B.N., Roth, B.L., Sexton, P.M. and A. Christopoulos (2008) New insights into the function of M4 muscarinic acetylcholine receptors gained using a novel allosteric modulator and a DREADD (Designer Receptor Exclusively Activated by a Designer Drug), Mol. Pharmacol., 74: 1119-1131.
9. Qi, T., Christopoulos, G., Bailey, R. L., Christopoulos, A., Sexton, P.M. and D. L. Hay (2008) Identification of N-terminal receptor activity-modifying protein residues important for CGRP, adrenomedullin and amylin receptor function, Mol. Pharmacol., 74: 1059-1071.
10. Chan W.Y., McKinzie D., Bose S., Mitchell S., Witkin J., Thompson R.C., Christopoulos, A., Lazareno S., Birdsall N.J.M., Bymaster F.P. and C.C. Felder (2008) Allosteric modulation of the muscarinic M4 receptor as an approach to treating schizophrenia. Proc. Natl. Acad. Sci. (USA), 105: 10978-10983. (See also “Research Highlights”, Nature Rev. Drug Discover. (2008) 7:  806)
11. Gao, F., Sexton, P.M., Christopoulos, A. and L.J. Miller (2008) Benzodiazepine ligands can act as allosteric modulators of the Type 1 cholecystokinin receptor. Bioorg. Med. Chem. Lett., 18: 4401-4404.
12. Avlani, V.A., McLoughlin, D.J., Sexton, P.M. and A. Christopoulos (2008) The impact of orthosteric radioligand depletion on the quantification of allosteric modulator interactions. J. Pharmacol. Exp. Ther. 36: 1637-1640.
13. Sexton, P.M., Christopoulos, G., Christopoulos, A., Nylen, E.S., Snider Jr., R.H. and K.L. Becker (2008) Procalcitonin has bioactivity at calcitonin receptor family complexes: Potential mediator in sepsis. Crit. Care. Med. 325: 927-934.
14. Udawela, M., Christopoulos, G., Morfis, M., Tilakaratne, N., Christopoulos, A. and P. M. Sexton (2008) The effects of C-terminal truncation of receptor activity modifying proteins on the induction of amylin receptor phenotype from human CTb receptors. Reg. Pep., 145: 65-71.
15. Gregory, K.J., Sexton, P.M. and A. Christopoulos (2007) Allosteric modulation of muscarinic acetylcholine receptors. Current Neuropharmacol.  5: 157-167.
    Avlani, V.A., Gregory, K.J., Morton, C.J., Parker, M.W., Sexton, P.M. and Christopoulos, A. (2007) Critical role for the second extracellular loop in the binding of orthosteric and allosteric G protein-coupled receptor ligands.  J. Biol. Chem., 282: 25677-25686.
16. May, L.T., Avlani, V.A., Langmead, C.J., Herdon, H.J., Wood, M. D., Sexton, P.M. and Christopoulos, A. (2007) Structure-function studies of allosteric agonism at M₂ muscarinic acetylcholine receptors.  Mol. Pharmacol., 72: 463-476.
17. Bisson, W.H., Cheltsov, A.V., Bruey-Sedano, N., Chen, J., Goldberger, N., May, L.T., Christopoulos, A.,Dalton, J.T., Sexton, P.M., X.-K. Zhang and R. Abagyan (2007), Discovery of antiandrogen activity of non-steroidal scaffolds of marketed drugs,  Proc. Natl. Acad. Sci. (USA), 104: 11927-11932.
18. Leach, K., Sexton, P.M. and Christopoulos, A. (2007) Allosteric modulators of GPCRs: Taking advantage of permissive receptor pharmacology. Trends Pharmacol. Sci. 28: 382-389.
19. May, L.T., Leach, K., Sexton, P.M. and Christopoulos, A. (2007) Allosteric modulation of G protein-coupled receptors, Ann. Rev. Pharmacol. Toxicol., 47: 1-51.
20. Langmead, C. and Christopoulos, A. (2006) Allosteric agonists of 7TM receptors: Expanding the pharmacological toolbox. Trends Pharmacol. Sci. 27: 475-481.
21. van der Westhuizen, E.T., Summers, R.J., Halls, M.L., Bathgate, R.A.D. and P.M. Sexton. Relaxin receptors – new drug targets for multiple disease states. Current Drug Targets, In Press.
22. Urban, J.D., Clarke, W.P., von Zastrow, M., Nichols, D.E., Kobilka, B., Weinstein, H., Javitch, J.A., Roth, B.L., Christopoulos, A., Sexton, P.M., Miller, K.J., Spedding, M. and R B. Mailman (2007) Functional selectivity and classical concepts of quantitative pharmacology, J. Pharmacol. Exp. Ther., 320: 1-13.
23. Lanzafame, A.A., Sexton, P.M. and Christopoulos, A. (2006) Interaction studies of multiple binding sites on M4 muscarinic acetylcholine receptors. Mol. Pharmacol., 70: 736-746
24. Udawela, M. Christopoulos G, Tilakaratne, N, Christopoulos, A., Albiston, A. and P. M. Sexton (2006) Distinct receptor activity modifying protein (RAMP) domains differentially modulate interaction with calcitonin receptors. Mol. Pharmacol. 69: 1984-1989.
25. Hay, D.L., Poyner, D.R. and P.M. Sexton (2006) GPCR modulation by RAMPs. Pharmacol. Ther. 109, 173-197.
26. Harikumar, K.G., Morfis, M., Lisenbee, C.S., Sexton, P.M. and L.J. Miller (2006) Constitutive formation of oligomeric complexes between Family B G Protein-coupled vasoactive intestinal polypeptide and secretin receptors. Mol. Pharmacol. 69, 363-373, 2006
27. Langmead, C.L., Fry V.A.H., Forbes, I.T., Branch, C.A., Christopoulos, A., Wood, M.D. and H. J. Herdon (2006) Probing the molecular mechanism of interaction between AC-42 and the muscarinic M1 receptor:  Direct pharmacological evidence that AC-42 is an allosteric agonist. Mol. Pharmacol., 69: 236-246
28. Pham, V., Dong, M., Wade, J.D., Miller, L.J., Morton, C.J., Ng, H.L., Parker, M.W. and P.M. Sexton (2005) Insights into interactions between the alpha -helical region of the salmon calcitonin antagonists and the human calcitonin receptor using photoaffinity labelling. J. Biol. Chem. 280, 28610-28622.
29. Price, M.R., Baillie, G., Thomas, A., Stevenson, L.A., Easson, M., Goodwin, R., McLean, A., McIntosh, L., Goodwin, G., Walker, G., Westwood, P., Marrs, J., Thomson, F., Cowley, P., Christopoulos, A., Pertwee, R.G. and Ruth A. Ross (2005) Allosteric modulation of the cannabinoid CB1 receptor. Mol. Pharmacol., 67: 1484-1495.
30. Werry, T.D., Gregory, K.J., Sexton, P.M. and Christopoulos, A. (2005) Characterization of serotonin 5-HT2C receptor signaling to extracellular signal-regulated kinases 1 and 2.  J. Neurochem.  93, 1603-1615.
31. Werry, T.D., Sexton, P.M. and Christopoulos, A. (2005) “Ins and Outs” of seven-transmembrane receptor signalling to ERK, Trends Endocrinol. Metab., 16: 26-33.
32. Hay, D.L., Christopoulos, G., Christopoulos, A., Poyner D.R. and P.M. Sexton (2005) Pharmacological discrimination of calcitonin receptor-receptor activity modifying protein complexes. Mol. Pharmacol. 67, 1655-1665.
33. Kostenis, E., Milligan, G., Christopoulos, A., Sanchez-Ferrer, C., Heringer-Walther, S., Sexton, P.M., Gembardt, F., Kellett, E., Martini, L. Vanderheyden, P., Schultheiss, H-P. and T. Walther (2005). The G protein-coupled receptor Mas is a physiological antagonist of the angiotensin II AT1 receptor. Circulation, 111, 1806-1813.
34. Pham, V., Wade, J.D., Purdue, B.W. and P.M. Sexton (2004) Spatial proximity between a photolabile residue in position 19 of salmon calcitonin and the amino terminus of the human calcitonin receptor. J. Biol. Chem. 279, 6720-6729.
35. Pham, V. and P.M. Sexton (2004) Photoaffinity scanning in the mapping of the peptide receptor interface of class II G protein-coupled receptors. J. Peptide Sci. 10, 179-203.
36. Lanzafame, A. and Christopoulos, A. (2004), Investigation of the interaction of a putative allosteric modulator, N-(2,3-diphenyl-1,2,4-thiadiazole-5-(2H)-ylidene) methanamine hydrobromide (SCH-202676), with M1 muscarinic acetylcholine receptors, J. Pharmacol. Exp. Ther., 308, 830-837.
37. Avlani, V. May, L.T., Sexton, P.M. and Christopoulos, A. (2004), Application of a kinetic model to the apparently complex behavior of negative and positive allosteric modulators of muscarinic acetylcholine receptors, J. Pharmacol. Exp. Ther., 308, 1062-1072.
38. Motulsky, H. J. and Christopoulos, A. (2004) Fitting models to biological data using linear and nonlinear regression. Oxford University Press, NY, USA ISBN 0 19 517180 2.
39. Christopoulos, A., Christopoulos, G., Morfis M., Udawela, M., Laburthe, M., Couvineau A., Kuwasako, K., Tilakaratne, N. and P. M. Sexton (2003), Novel partners and function of receptor activity modifying proteins, J. Biol. Chem., 278, 3293-3297.
40. Morfis, M., Christopoulos, A. and P. M. Sexton (2003) RAMPs: 5 years on, where to now? Trends Pharmacol. Sci. 24, 596 - 601.
41. Lanzafame, A., Christopoulos, A. and F. Mitchelson (2003) Cellular signalling mechanisms for muscarinic acetylcholine receptors, Receptors and Channels, 9, 241-260.
42. Devlin, M.G. and A. Christopoulos (2002), Modulation of cannabinoid agonist binding by 5-HT in the rat cerebellum, J. Neurochem. 80, 1095-1102.
43. Lyon, G., Wright, J., Christopoulos, A., Novick, R.P. and T.W. Muir (2002), Reversible and specific extracellular antagonism of receptor-histidine-kinase signaling, J. Biol. Chem. 227,6247-6253.
44. Christopoulos, A. and T. Kenakin (2002) G protein-coupled receptor allosterism and complexing, Pharmacol. Rev. 54, 323-374.
45. Christopoulos, A. (2002) Allosteric binding sites on cell-surface receptors: Novel targets for drug discovery. Nature Rev. Drug Discovery 1, 198-210.
46. Poyner, D.R., Sexton, P.M., Marshall, I., Quirion, R., Kangawa, K., Born, W., Fischer, J.A., Muff, R., Smith, D.M. and S.M. Foord (2002) The mammalian CGRP, adrenomedullin, amylin and calcitonin receptors. Pharmacol. Rev. 54, 233-246.