Fiona H. Marshall

Structure of class B GPCR corticotropin-releasing factor receptor 1

Structural analysis of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-molecule antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-molecule drugs for diseases of brain and metabolism.

Heterodimerization is required for the formation of a functional GABAB receptor

GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian central nervous system, where it exerts its effects through ionotropic (GABAA/C) receptors to produce fast synaptic inhibition and metabotropic (GABAB) receptors to produce slow, prolonged inhibitory signals. The gene encoding a GABAB receptor (GABABR1) has been cloned; however, when expressed in mammalian cells this receptor is retained as an immature glycoprotein on intracellular membranes and exhibits low affinity for agonists compared with the endogenous receptor on brain membranes. Here we report the cloning of a complementary DNA encoding a new subtype of the GABAB receptor (GABABR2), which we identified by mining expressed-sequence-tag databases. Yeast two-hybrid screening showed that this new GABABR2-receptor subtype forms heterodimers with GABABR1 through an interaction at their intracellular carboxy-terminal tails. Upon expression with GABABR2 in HEK293T cells, GABABR1 is terminally glycosylated and expressed at the cell surface. Co-expression of the two receptors produces a fully functional GABAB receptor at the cell surface; this receptor binds GABA with a high affinity equivalent to that of the endogenous brain receptor. These results indicate that, in vivo, functional brain GABAB receptors may be heterodimers composed of GABABR1 and GABABR2.

GABAB receptors - the first 7TM heterodimers.

Evidence supports the existence of pharmacological subtypes of GABAB receptors24xKerr, D.I.B. and Ong, J. Pharmacol. Ther. 1995; 67: 187–246Crossref | PubMed | Scopus (192)See all References24. At our current level of understanding, however, the dimerization phenomenon does not provide an explanation for these receptor subtypes. Although there appears to be multiple splice variants of GABABR1 and GABABR2, no differences in pharmacology have so far been reported5xKaupmann, K. et al. Nature. 1998; 396: 683–687Crossref | PubMed | Scopus (854)See all References5. It is possible that monomeric forms of GABABR1 and GABABR2 represent subtypes distinct from each other and the heterodimer. Detailed immunohistochemical studies will be required to provide support for this idea. Alternatively, other receptor partners or interacting proteins might exist that modify the pharmacology or signalling of the individual receptors. Such proteins would have the potential to interact both with the C-terminal domains of the receptors, which contain various protein-binding motifs25xBettler, B. et al. Curr. Opin. Neurobiol. 1998; 8: 345–350Crossref | PubMed | Scopus (129)See all References25, or with Sushi- or complement-binding motifs in the N-terminal domain of GABABR1 (Ref. 26xHawrot, E. et al. FEBS Lett. 1998; 432: 103–108Abstract | Full Text | Full Text PDF | PubMed | Scopus (55)See all ReferencesRef. 26). Interestingly, both of these domains are altered in the different GABABR1 splice variants. A novel possibility for the GABABR1a splice variant is an interaction with extracellular proteins via the complement-binding domain, which produces a change in pharmacology. Finally, there is always the possibility that there are still more distantly related GABAB receptors yet to be identified.

Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain

Metabotropic glutamate receptors are class C G-protein-coupled receptors which respond to the neurotransmitter glutamate. Structural studies have been restricted to the amino-terminal extracellular domain, providing little understanding of the membrane-spanning signal transduction domain. Metabotropic glutamate receptor 5 is of considerable interest as a drug target in the treatment of fragile X syndrome, autism, depression, anxiety, addiction and movement disorders. Here we report the crystal structure of the transmembrane domain of the human receptor in complex with the negative allosteric modulator, mavoglurant. The structure provides detailed insight into the architecture of the transmembrane domain of class C receptors including the precise location of the allosteric binding site within the transmembrane domain and key micro-switches which regulate receptor signalling. This structure also provides a model for all class C G-protein-coupled receptors and may aid in the design of new small-molecule drugs for the treatment of brain disorders.

Discovery of 1,2,4-Triazine Derivatives as Adenosine A(2A) Antagonists using Structure Based Drug Design

Potent, ligand efficient, selective, and orally efficacious 1,2,4-triazine derivatives have been identified using structure based drug design approaches as antagonists of the adenosine A2A receptor. The X-ray crystal structures of compounds 4e and 4g bound to the GPCR illustrate that the molecules bind deeply inside the orthosteric binding cavity. In vivo pharmacokinetic and efficacy data for compound 4k are presented, demonstrating the potential of this series of compounds for the treatment of Parkinson’s disease.

Progress in Structure Based Drug Design for G Protein-Coupled Receptors

In 1998, Bikker, Trumpp-Kallmeyer, and Humblet published a Perspective in this journal entitled “G-Protein Coupled Receptors: Models, Mutagenesis and Drug Design” and reviewed the state of the art at that time.1 No high resolution structure of a G protein-coupled receptor (GPCR) had been solved, and researchers were working with models generated with only the structure of bacteriorhodopsin,2 which had been published 8 years earlier and solved using high resolution electron cryomicroscopy and the low resolution electron density footprint of bovine rhodopsin.3 These models, despite greatly improving understanding of GPCR structure and function, posed as many questions as they answered and were not able to clearly rationalize how ligands bound to their target receptor. The authors stated “The principal limitation of the current generation of models when used for rational drug design is that the resolution of the binding cavity is too low to predict specific ligand–receptor interactions. Attempts to dock ligands into various GPCR models are further complicated by difficulty in identifying unique, sensible modes of binding, especially when dealing with molecules of the size of the neurotransmitter ligands.” How things have changed. Today, there are six GPCRs for which medium to high resolution crystal structures have been solved, in most cases with multiple small molecules ligands. The six receptors are rhodopsin, the β1 and β2 adrenergic receptors, adenosine A2A receptor, chemokine CXCR4 receptor, and dopamine D3 receptor (Table ​(Table11 and references therein). In addition, rhodopsin, the β1 and β2 adrenergic receptors (ARs), and the adenosine A2A receptor have been solved with both antagonists and agonists bound (Table ​(Table1).1). Much current research is now engaged in using this new body of structural information for hit identification and drug design purposes, and we will review the state of the art of both structures and the impact they are now having on structure based drug design (SBDD) for GPCR targets in this article. Table 1 List of Published GPCR Crystal Structures

RAMPs: accessory proteins for seven transmembrane domain receptors

. TheCT receptor was cloned in 1991(Ref.5) but the others have proven diffi-cult to identify. When the calcitoninreceptor-like receptor (CRLR) wasdiscovered (Fig. 1), its 55% identitywith the CT receptor suggestedmembers of the CT peptide family as candidate ligands but the onlyreports to appear in the literaturereported negative findings

New insights from structural biology into the druggability of G protein-coupled receptors

The recent availability of X-ray structures for diverse ligand-bound Family A G protein-coupled receptors (GPCRs) in multiple conformations (inactive form with an antagonist/inverse agonist bound and active form with an agonist bound) now enables rational drug design efforts that have historically been applied to soluble enzyme targets. Here, we review properties of these GPCR binding sites, using a unique combination of calculated physicochemical properties and water energetics (GRID, WaterMap and SZMAP) to provide a new perspective and rational assessment of druggability for each GPCR target binding site. Examples are described from several well-studied enzyme systems to support this advanced structure-based approach to assessing druggability and to contrast their properties with those of GPCRs. Changes in receptor conformations between the GPCR inactive and active forms evident from the protein structures are discussed, yielding important pointers for rational drug design of antagonists and agonists and a better understanding of GPCR activation.

The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery.

G protein-coupled receptors (GPCRs) are one of the most important target classes in the central nervous system (CNS) drug discovery, however the fact they are integral membrane proteins and are unstable when purified out of the cell precludes them from a wide range of structural and biophysical techniques that are used for soluble proteins. In this study we demonstrate how protein engineering methods can be used to identify mutations which can both increase the thermostability of receptors, when purified in detergent, as well as biasing the receptor towards a specific physiologically relevant conformational state. We demonstrate this method for the adenosine A(2A) receptor and muscarinic M(1) receptor. The resultant stabilised receptors (known as StaRs) have a pharmacological profile consistent with the inverse agonist conformation. The stabilised receptors can be purified in large quantities, whilst retaining correct folding, thus generating reagents suitable for a broad range of structural and biophysical studies. In the case of the A(2A)-StaR we demonstrate that surface plasmon resonance can be used to profile the association and dissociation rates of a range of antagonists, a technique that can be used to improve the in vivo efficacy of receptor antagonists.

The impact of GPCR structures on pharmacology and structure-based drug design

After many years of effort, recent technical breakthroughs have enabled the X‐ray crystal structures of three G‐protein‐coupled receptors (GPCRs) (β1 and β2 adrenergic and adenosine A2a) to be solved in addition to rhodopsin. GPCRs, like other membrane proteins, have lagged behind soluble drug targets such as kinases and proteases in the number of structures available and the level of understanding of these targets and their interaction with drugs. The availability of increasing numbers of structures of GPCRs is set to greatly increase our understanding of some of the key issues in GPCR biology. In particular, what constitutes the different receptor conformations that are involved in signalling and the molecular changes which occur upon receptor activation. How future GPCR structures might alter our views on areas such as agonist‐directed signalling and allosteric regulation as well as dimerization is discussed. Knowledge of crystal structures in complex with small molecules will enable techniques in drug discovery and design, which have previously only been applied to soluble targets, to now be used for GPCR targets. These methods include structure‐based drug design, virtual screening and fragment screening. This review considers how these methods have been used to address problems in drug discovery for kinase and protease targets and therefore how such methods are likely to impact GPCR drug discovery in the future.