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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 modeling of GPCR-ligand interactions.

Personnel

Laboratory heads

Personal assistant to Laboratory heads

  • Jessica Richarson

Senior Lecturer

  • Dr Richard Loiacono

NHMRC Career Development Award Postdoctoral Fellow

  • Dr Dana Hutchinson

Senior Research Fellow

  • Dr Bronwyn Evans

Senior Research Officers

  • Dr Nathan Hall

Research Officers

  • Dr Katie Leach
  • Dr Celine Valant
  • Dr Sebastian Furness 
  • Dr John Simms
  • Dr Caroline Hick
  • Dr Masa Sato
  • Dr Denise Wooten
  • Dr Martina Kocan

CJ Martin Fellows

(currently overseas)
  • Dr Lauren May
  • Dr Michelle Halls

Senor Research Assistant

  • George Christopoulos

Technical Assistants

  • Christine Yeo

PhD Students

  • Karen Gregory
  • Gregory Stewart
  • Vindhya Nawaratne
  • Cassandra Koole
  • Maria Morfis
  • Stephanie Robinson
  • Stephanie Catus
  • Leigh Brown
  • Nur (Sue) Suratman
  • Anna Davey

Honours Students

  • Peter Keov
  • Daniel Stepanenko
  • Rohan Sridhar
  • Ema Stancic
Drug Discovery Biology Lab

Current research projects

1. Orthosteric and Allosteric Binding Sites on GPCRs

Traditionally, GPCR-based drug discovery has focused on targeting drugs to the orthosteric site of the receptor, that is, the binding site for the endogenous agonist. However, it is now recognized that GPCRs can possess allosteric sites that modulate receptor activity; targeting such sites can potentially lead to greater selectivity for GPCRs that exhibit high sequence homology within the orthosteric site across subtypes. However, the detection, quantification and validation of allosteric drug effects represent a significant challenge for drug discovery. This is because allosteric modulators can affect orthosteric binding affinity and/or signaling efficacy in a manner that is totally orthosteric-ligand-dependent; some modulators can also demonstrate agonist activity in their own right. We are currently investigating the structural basis and pharmacological consequences of ligand interaction with both orthosteric and allosteric sites on a variety of GPCRs.

  1. Analysis of the calcitonin receptor ligand-binding interface
  2. Allosteric modulators of muscarinic acetylcholine receptors
  3. Mechanisms of action of allosteric modulators of adenosine A1 receptors

2. Mechanisms of GPCR Signal Transduction

Despite the clear success of GPCRs as a therapeutic target class, there are many instances of where we do not fully understand why some GPCR drugs yield better therapeutic outcomes than others, despite similar apparent activity at their target receptors. Similarly, there are many examples where the utility of drugs is limited because activation of secondary pathways leads to unwanted side effects. In part, these situations reflect an incomplete understanding of the mechanisms underlying GPCR signal transduction and the important role that the ligand exposure time and the cellular environment, including an ever-expanding list of novel GPCR-interacting proteins, play in sculpting the final cellular response.

  1. Novel effects of allosteric modulators on GPCR trafficking and regulation.
  2. Characterization of RAMP-receptor interactions.
  3. Characterization of Family B GPCRs.
  4. Biophysical studies of GPCR-G protein coupling
  5. GPCR signaling promiscuity and trafficking of receptor stimulus.

3. Isoform-specific GPCR Functions

Like many other proteins, GPCRs can exhibit remarkable diversity in their functionality via the generation of different isoforms of the same protein. There are a number of different processes that lead to different receptor isoforms, and these can be impacted by genetic factors, disease and drug treatment. We are currently exploring the functional role of various isoforms of specific GPCRs.

  1. Functional consequences of RNA editing of the 5HT2C receptor.
  2. Polymorphic variants of the human calcitonin receptor.

4. Structure-Function Studies of GPCRs

Despite the obvious relevance of GPCRs to drug discovery, a number of fundamental issues remain unresolved with respect to mechanisms underlying GPCR activation and the basis of drug selectivity. Many of these issues relate to limitations in structural analysis; to date, the only high-resolution crystal structure of a GPCR that has been published is that of bovine rhodopsin. Clearly, a better understanding of the structure of GPCRs, and the critical residues that contribute to ligand binding pockets, can lead to the design of more selective drugs that target these receptors. Given the paucity of crystal structures available, our structure-function studies of GPCRs utilize a combination of computerized molecular modeling, receptor mutagenesis and experimental testing of model predictions.

  1. Mutational analysis of allosteric binding sites on GPCRs.
  2. Molecular modeling of the orthosteric binding site on Family B GPCRs.

Collaborators

  • Professor Peter Scammells, Dept. of Medicinal Chemistry, Victorian College of Pharmacy, Monash University
  • Prof. Lawrence Miller, Mayo Clinic, Scottsdale, USA
  • Prof. Ruben Abagyan, Scripps Research Institute, USA
  • Prof. Andrew Tobin, University of Leicester, UK
  • Dr. Debbie Hay, University of Auckland, NZ
  • Prof. 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

Recent key references

  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.