Hot topics: Agonist-bound crystal structures of the CB1 cannabinoid receptor

Antagonist bound crystal structures of GPCRs are useful in giving an insight into the molecular conformation of a receptor’s inactive state whilst enabling the design of new drugs. However, they prove insufficient to understand the activation mechanism of the receptor and mediation of its physiological effects. This necessitates the study of agonist-bound structures. In this direction, Hua et al., (2017) [1] have recently reported two agonist-bound crystal structures of Cannabinoid Receptor 1 (CB1), one with a tetrahydrocannabinol derivative, AM11542 [PDB: 5XRA], and the other with a hexahydrocannabinol, AM841 [PDB: 5XR8]. Previously, two antagonist-bound crystal structures of CB1 complexed with AM6538 and MK-0364 (taranabant) were reported by Hua et al., (2016) [PDB: 5TGZ] [2] and Shao et al., (2016) [PDB: 5U09], respectively [3].

Comparing the agonist and antagonist bound structures of the CB1 receptor reveals significant details:

1. The N-terminus in 5TGZ and 5U09 is a V-shaped loop which interacts with the bound antagonists, acting like a plug to the orthosteric binding pocket. The agonist-bound versions (5XRA and 5XR8), however, have their N-terminus residing over the binding pocket without any direct involvement in ligand binding. However, the N-terminus is truncated in all the crystal structures and hence the authors do not rule out the possibility that the full-length N-terminus might assume an entirely different conformation.

2. Both the agonists adopt an L-shaped conformation in the binding pocket, in contrast to the horizontal geometry of AM6538 in 5TGZ. The helical rearrangements hence observed in TM 1 and 2 and inward movement of residues Phe1702.57 and Phe1742.64 lead to a reduction in the binding pocket volume by 53% compared to 5TGZ. This serves as a testament to the highly flexible nature of the CB1 receptor and should be considered in future structure-based drug design studies for the receptor.

3. The alkyl chain of the two agonists extends into the ‘long channel’ of the receptor formed by the transmembrane helices (TM) 3, 5 and 6. The authors point out that this orientation is similar to that of ‘arm 2′- the nitroalkyl region of AM6538 in 5TGZ and of the alkyl chain of ML056 in the previously-described structure of the S1P1 receptor [4], thus indicating that the long channel could be a conserved binding region for alkyl chains in lipid binding receptors.

4. In 5TGZ and 5U09, the residues Phe2003.36 and Trp3566.48 exhibit aromatic stacking with each other. In this report, a synergistic conformational change of the residues was observed with the rotation of TM3 and side chain flip of Phe2003.36 towards the binding pocket occurring simultaneously with the rotation of TM6 away from TM3 breaking the interaction between the residues. The authors speculate the role of this ‘twin toggle switch’ in the activation of the receptor as a previous study has already shown [5].

5. Using the crystal structure, a cholesterol molecule has been identified to bind between the cytoplasmic portions of TM 2, 3, and 4 in the agonist-bound models. This was not observed in the antagonist models. However, the possible existence of a lipid access channel proposed in the taranabant bound (5U09) structure has not been discussed in this paper. This raises questions about the influence of lipids on the receptor binding through allosteric sites.

Comments by Lahari Murali (@wavesml), Steve Alexander (@mqzspa), Steven Doughty and Abi Emtage (@AbiEmtage)

[1] Hua, T. et al. (2017). Crystal structures of agonist-bound human cannabinoid receptor CB1. Nature.doi:10.1038/nature23272. [PMID: 28678776]

[2] Hua, T. et al. (2016). Crystal Structure of the Human Cannabinoid Receptor CB1. Cell 167: 750–762.e14. doi: 10.1016/j.cell.2016.10.004. [PMID: 27768894]

[3] Shao, Z. et al. (2016). High-resolution crystal structure of the human CB1 cannabinoid receptor. Nature 540:602–606. doi: 10.1038/nature20613. [PMID: 27851727]

[4] Hanson, M. A. et al. (2012). Crystal structure of a lipid G protein-coupled receptor. Science 335:851–855. doi: 10.1126/science.1215904. [PMID: 22344443]

[5] Singh, R., Hurst, D.P., Barnett-Norris, J., Lynch, D.L., Reggio, P.H., and Guarnieri, F. (2002). Activation of the cannabinoid CB1 receptor may involve a W6 48/F3 36 rotamer toggle switch. J. Pept. Res. 60: 357–370. [PMID: 12464114]

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Hot topics: Identifiers for the 21st century

While identifiers are not a traditional “hot topic” in pharmacology the subject is becoming increasingly important. One of the reasons is that for mechanistic pharmacology the community needs to define (and communicate) identifiers for the key entities of model organism species and strains, proteins, protein complexes, genes, sequences, sequence variants, as well as the explicit molecular structures of chemicals, peptides and therapeutic biologicals (including antibodies) used for experimentation. Indeed one of the roles of IUPHAR (as NC-IUPHAR) is to review and recommend protein target nomenclature, in collaboration with the Human Gene Nomenclature Committee (HGNC) [1]. The paper featured here is a technical review [2] of identifier qualities and best practices that facilitate large-scale data integration. It also goes into problems related to persistence and web-accessibility/resolvability. As a database provider, the relevance of this article for GtoPdb is clear (since we are largely about identifiers and their relationships). We are carefully considering its implications and possible consequent changes in our practice. The GtoPdb team has already engaged with this theme some time ago in a blog post [3] that provided an introduction to resolving bioactive ligands and their protein targets from the literature to standardised molecular identifiers.

Comments by Chris Southan (@cdsouthan).

[1] International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification

[2] McMurray et al. (2017). Identifiers for the 21st century: How to design, provision, and reuse persistent identifiers to maximize utility and impact of life science data. PLoS Biol. 29;15(6). [PMID:28662064].

[3] A Pharmacologists’ Guide to Resolving Chemical Structures and their Protein Targets from the Literature

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Hot topics: X-ray crystal of the apelin receptor

Activation of the apelin receptor by the peptides apelin or Elabela/Toddler mediates vasodilatation and positive inotropic effects in the adult cardiovascular system and knocking out the receptors results in failure of the heart to develop in developing embryos. To date, only a limited number of Family A structures (including opioid, endothelin ETB, and orexin OX1 and OX2) have been deduced using X-ray crystallography. Ma et al., (2017) have recently reported the 2.6-Å resolution crystal structure of human apelin (APJ) receptor in complex with a synthetic 17-amino-acid apelin analogue agonist. The authors identify a two-site ligand binding mode that has not been seen in previous solved Family A receptor structures. The structure is in reasonable agreement with studies using NMR (Langelaan et al 2013) and molecular dynamics simulations (Macaluso and Glen 2010, Yang et al, 2017) In addition, many of the key interfacial receptor:agonist residues identified from the crystal structure are in agreement with mutation data on apelin binding. However, it is worth noting the following caveats that make interpretation of the data difficult:

1. Residues were removed from the N-terminus (residues 1–6) and C terminus (residues 331–380).

2. A putative glycosylation motif at N175 was eliminated from ECL2. It is an open question as to whether glycosylation affects agonist binding in the apelin receptor.

3. Two putative palmitoylation sites were removed from TM8. Again, it is an open question as to whether palmitoylation affects agonist binding in the apelin receptor.

4. To achieve crystallization, mutations V117A and W261K were introduced. These force the intracellular portion of the receptor into the inactive state. In fact, these mutations seem to render the receptor unable to bind apelin-13.

5. The synthetic 17-amino-acid apelin analogue agonist is significantly different from the apelin sequence. In particular, a macrocycle and significant mutations have been introduced, which may alter the peptide conformation and its interactions.

6. The crystal structure is unable to explain the importance of the Arg2 and Leu5 residues of apelin 13, which are known to be key binding elements from mutation data.

Despite these caveats, the crystal structure provides much needed data on the apelin receptor. A key question in the apelin field is why two peptides have evolved binding to the same ligand in mammals. Although the authors did not report binding with Elabela/Toddler analogues, the initial structure will foster understanding whether these two ligands differ in signalling mechanisms. Intriguingly, Lena et al (2017) have identified a key role for Elabela/Toddler in preeclampsia but this may not be mimicked by apelin suggesting spatiotemporal differences in signalling.

Comments by Anthony Davenport, David Huggins, Janet Maguire, and Robert Glen.

Ma et al. (2017) Structural Basis for Apelin Control of the Human Apelin Receptor. Structure. 6:858-866.e4. [PMID:28528775]

Langelaan et al. (2013) Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking. Biochim Biophys Acta. 1828:1471-83. [PMID:23438363]

Macaluso NJ, Glen RC. (2010) Exploring the ‘RPRL’ motif of apelin-13 through molecular simulation and biological evaluation of cyclic peptide analogues. ChemMedChem. 8:1247-53. [PMID:20486151]

Yang P et al. (2017) Elabela/Toddler Is an Endogenous Agonist of the Apelin APJ Receptor in the Adult Cardiovascular System, and Exogenous Administration of the Peptide Compensates for the Downregulation of Its Expression in Pulmonary Arterial Hypertension. Circulation. [PMID:28137936]

Lena et al. (2017) ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice. Science DOI. 10.1126/science.aam6607


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HGNC gene links to GtoPdb

As regular users of GtoPdb will be aware, our target pages include HUGO Gene Nomenclature Committee (HGNC) gene symbols, nomenclature and links to HGNC gene pages (Fig 1).


Figure 1. GtoPdb gene information including link outs to HGNC pages by clicking on the human gene symbol.

HGNC pages in turn link back to GtoPdb target pages under the “Specialist database” link (Fig 2).


Figure 2. HGNC gene page showing the link to the IUPHAR/BPS Guide to Pharmacology (GtoPdb) under “Specialist Databases”.

In GtoPdb release 2017.4 there were 2823 HGNC target links. In June 2017 we also added a further 430 HGNC links to GtoPdb peptide ligand pages (Figs 3 and 4). These are either small proteins such as cytokines, or endogenous peptides derived from Swiss-Prot proteins that have an HGNC entry. Implicit in our classification as ligands these will have (in most cases quantitative) reported interactions with cognate receptors.  The peptides will usually correspond to sequences processed from their precursor proteins in HGNC (e.g. the eight angiotensin peptides from Angiotensinogen, AGT).  Note that some of these peptides are cross-linked to other ligand entries which are similar synthetic bioactive sequences that do not exactly match the cleavage cross-references in UniProt.


Figure 3. GtoPdb peptide ligand page which links to the HGNC gene page by clicking on the gene symbol.


Figure 4. HGNC gene page for a chemokine ligand showing a link to the GtoPdb ligand page.

For over 50 genes which encode multiple mature peptides, the HGNC page links to the longest peptide sequence included in GtoPdb. This is because HGNC currently only supports one external link per resource, and they are working on providing support for multiple links by the end of this year.

This expansion was driven by importance of these reciprocal links for HGNC users to navigate across to pharmacolgical information. In addtion, going the other way, they enable GtoPdb users to find out more about gene nomenclature and other genetic information via HGNC.

The full list of HGNC in-links to GtoPdb can be downloaded here (as a CSV file as illustrated in Fig 5). Ligand links can be distinguished from target links by the URL.


Figure 5. Part of the CSV file containing all the HGNC links to GtoPdb showing some of the new ligand links.


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Hot topic: Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474

The historical context for this commentary can be found in this blogpost. This latest report, based on activity-based profiling (ABPP), constitutes the first open biochemical investigation of BIA 10-2474 (1). The ABPP results show it inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. In addition BIA 10-2474 (but not PF04457845) produced substantial alterations in lipid networks in human cortical neurons. The authors are appropriately cautious in not over-extrapolating their findings to causality of pathology recorded in the unfortunate patients (see clinical report in PMID 27806235). However, biochemical and pharmacological questions still remain. One of these is that, given the initial binding interaction is no less than three orders of magnitude lower that PF004457845, it’s not entirely clear why 10-2474 was chosen as the lead. Another question is the basic kinetic parameters for purified enzymes (not just crude cell extracts in vitro) are still not available. This should include at least two methods for confirming irreversibility (e.g. IC50 vs pre-incubation or using a 10-2474 radiolabeled derivative). Word has it that a BIAL paper is in preparation so this aspect might be addressed by new results. Note also there are now two compound suppliers in PubChem offering BIA 10-2474 so more experimental reports could be expected.

The GtoPdb entries below have been updated with key interactions from this paper and will go live at the next release.

BIA 10-2474




(1) van Esbroeck ACM et al. (2017). Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474. Science, 356(6342): 1084-1087 [PMID:28596366]

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Hot topic: GPR3 and GPR6, novel molecular targets for cannabidiol

Cannabidiol is a major metabolite from the Cannabis plant, although levels vary dependent on genetic, regional, cultivation and other factors.  It lacks the psychotropic nature of THC, but has been reported to have many biological effects, to the extent that clinical trials for infantile intractable epilepsy are currently ongoing in the US. GPR3 and GPR6 are orphan GPCRs, which have previously been reported to elevate cAMP levels constitutively when expressed in recombinant systems.  Although there was some evidence for activation by sphingosine 1-phosphate, this was not reproduced.  In this report, a number of endogenous and Cannabis-derived metabolites were examined for their effects on β-arrestin2 recruitment in cells expressing either GPR3 or GPR6.  Of these agents, only CBD caused a reduction in β-arrestin2 recruitment in a concentration-dependent manner, with pIC50 values of 5.9 and 6.7 at GPR3 and GPR6, respectively.  The authors suggest that the inverse agonist nature of CBD at these receptors might be of relevance for neurodegenerative disorders, such as Parkinson’s and Alzheimer’s Disease.

Comments by Steve Alexander (@mqzspa)

(1) Laun AS, Song ZH. (2017) GPR3 and GPR6, novel molecular targets for cannabidiol. Biochem Biophys Res Commun. 2017 May 29. pii: S0006-291X(17)31074-4. doi: 10.1016/j.bbrc.2017.05.165. [Epub ahead of print] [PMID:28571738]

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Hot topic: Selectivity determinants of GPCR–G-protein binding

As a detailed comparative sequence/structure/evolution analysis it is relatively unusual (in a good sense) to see such a bioinformatics article in Nature. This tour de force was a collaboration between MRC Laboratory of Molecular Biology, Cambridge UK and the Department of Drug Design and Pharmacology, University of Copenhagen (home of the GPCRdb team). As we know, GPCR signal transduction involves the binding of ligand-activated receptors to their appropriate Gα proteins. In this work selectivity-determining positions for signal transduction (as structural “barcodes”) were inferred by comprehensively comparing the sequence conservation between paralogues and orthologues, incorporating information from recent structures. The residue positions for the interaction interfaces are collated and presented at (tab ‘Signal Proteins’) for all human receptors and their 16 Gα proteins. This will be updated (including data from new structures) as a guide to interface determinants of coupling selectivity. Many applications of this resource can be envisaged. These could include: exploring options to target GPCR-G protein interfaces with agents that block coupling between the receptor and G protein intracellularly, protein engineering, structural studies and understanding the consequences of natural variation or rare disease associated mutations occurring in the vicinity of the barcode positions.

Note that all GtoPdb GPCRs have cross-references to GPCRdb (who we collaborate with) so users can navigate structural data (including the barcode positions) via GPCRdb, but also exploit ligand-centric navigation via GtoPdb and links out to genomic variants via the Ensembl links.

[1] Flock et al. (2017). Selectivity determinants of GPCR-G-protein binding. Nature, 545: 317-322. [PMID:28489817].

Comments by by Chris Southan (@cdsouthan)


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